There are some 15,000 known organophosphorus chemicals. Some of these OP’s, including VX and paraoxon, demonstrate an acute neurotoxicity due to the inhibition of cholinergic enzymes. Organophosphorus chemical warfare agents and pesticide neurotoxins are subject to hydrolysis by OP degrading enzymes. To be useful as a bioremediation or anti-chemical warfare agent, the enzyme must be tailored for, and integrated into, a practical application platform. Several studies have established enzyme-based countermeasures, describing such diverse applications as decontaminating foams for surface remediation, encapsulating enzyme with liposome for in vivo therapy, enzyme attachments to surfaces for biosensors and development of a corn expression system for large-scale enzyme production. The goal of the research described here is to select, investigate and improve the operational potential of organophosphate-degrading enzymes including Organophosphorus Hydrolase (OPH, 3.1.8.1) and Organophosphorus Acid Anhydrolase (OPAA, 3.1.8.2). Using saturation kinetics, the catalytic efficiencies of these two major detoxification enzymes were characterized with substrates representing each class of OP neurotoxin, phosphotriester, phosphothioate and phosphofluoridate. OPH presents superior kinetic parameters with each OP class tested. Variants of OPH were created to increase the operational effectiveness of OP hydrolytic enzymes against phosphorothioates. An H254S/H257L mutation in the active site resulted in an improvement in the kinetics (kcat/KM) for the phosphorothioate, demeton-S. To screen potential vascular protection therapies, an in vitro protocol was developed to predict enzymatic effectiveness for protection of acetylcholinesterase from acute OP-inhibition. The protection abilities of the enzymes were directly related to their second order rate constants as inhibitory levels of OP are below the KM of the enzymes. Consideration of contaminant nature concentration and enzyme kinetic parameters, kcat and KM, is critical to understanding decontamination and effective use of enzyme technology. These technologies continue to develop and provide promising new decontamination tools for OP compounds.

There are some 15,000 known organophosphorus chemicals. Some of these OP’s, including VX and paraoxon, demonstrate an acute neurotoxicity due to the inhibition of cholinergic enzymes. Organophosphorus chemical warfare agents and pesticide neurotoxins are subject to hydrolysis by OP degrading enzymes. To be useful as a bioremediation or anti-chemical warfare agent, the enzyme must be tailored for, and integrated into, a practical application platform. Several studies have established enzyme-based countermeasures, describing such diverse applications as decontaminating foams for surface remediation, encapsulating enzyme with liposome for in vivo therapy, enzyme attachments to surfaces for biosensors and development of a corn expression system for large-scale enzyme production. The goal of the research described here is to select, investigate and improve the operational potential of organophosphate-degrading enzymes including Organophosphorus Hydrolase (OPH, 3.1.8.1) and Organophosphorus Acid Anhydrolase (OPAA, 3.1.8.2). Using saturation kinetics, the catalytic efficiencies of these two major detoxification enzymes were characterized with substrates representing each class of OP neurotoxin, phosphotriester, phosphothioate and phosphofluoridate. OPH presents superior kinetic parameters with each OP class tested. Variants of OPH were created to increase the operational effectiveness of OP hydrolytic enzymes against phosphorothioates. An H254S/H257L mutation in the active site resulted in an improvement in the kinetics (kcat/KM) for the phosphorothioate, demeton-S. To screen potential vascular protection therapies, an in vitro protocol was developed to predict enzymatic effectiveness for protection of acetylcholinesterase from acute OP-inhibition. The protection abilities of the enzymes were directly related to their second order rate constants as inhibitory levels of OP are below the KM of the enzymes. Consideration of contaminant nature concentration and enzyme kinetic parameters, kcat and KM, is critical to understanding decontamination and effective use of enzyme technology. These technologies continue to develop and provide promising new decontamination tools for OP compounds.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 186-205). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 186-205). Also available on the Internet.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 107-111). Also available in electronic version. Access restricted to campus users.

Thesis (M. Phil.)--University of Hong Kong, 1974. / Mimeographed.

Thesis (M. S.)--Earth and Atmospheric Sciences, Georgia Institute of Technology, 2010. / Committee Chair: Ellery Ingall; Committee Member: Irina Sokolik; Committee Member: Josef Dufek. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Chapter one reviews the recent progress in the synthesis of phosphonopeptides, pseudopeptides containing a phosphinic, phosphonic or phosphonamide linkage in place of an amide (peptide) linkage. It describes some of the general methods for the synthesis of these pseudopeptides; for example through couplings to the nitrogen of an α-aminophosphonic acid, or Michael addition to acrylates, as well as other methods, the scope for which are not as wide yet. It also provides a summary of the reported biological activities of this class of pseudopeptides. Chapter two contains the results and discussion for a novel method for the synthesis of α-hydroxy phenylphosphinate oligomers as well as hybrid oligomers containing α-hydroxy phenylphosphinic acid and α-amino carboxylic acids. In particular, synthesis of a series of dimeric α-hydroxy phenylphosphinates are reported. The analysis of these dimers by a combination of NMR spectroscopy, X-ray crystallography and computational methods shows intramolecular hydrogen bonding in these molecules depends on the relative configuration of the carbon and phosphorus atoms. However, although the development of the synthetic methods was successful, the separation and isolation of the diastereomers was not always possible, which hindered a more comprehensive analysis of folding patterns in these molecules. Chapter three contains the experimental procedures, preparation and spectroscopic characterisation of all the chemical compounds. Crystal data and details of crystal structures are in the Appendix.

No description available.

The chemical fate of organophosphorus pesticides is influenced by several factors, one being the chemistry of their aquatic environment. Dissolved metal ions have been shown in several instances to promote the hydrolysis of organophosphorus pesticides, with various reasons forwarded as to the intimate mechanism of metal ion promoted hydrolysis. Several postulates have suggested metal ion co-ordination of Lewis sites to be an important factor in promoting the reaction. In view of this, the study herein reports on the promoted hydrolysis of diazinon at various pH’s in the presence of several metal ions including Hg2+, Cu2+, Cd2+ and Ag+. The observed 1H and 31P NMR data indicated complexation of Cu2+ and Ag+ with diazinon through the formation of a six-membered ring (chelate), via co-ordination at both nitrogen and sulfur Lewis base sites. In contrast, NMR results with Hg2+ indicated Hg2+ ion promoted hydrolysis of diazinon through co-ordination at sulfur alone, possibly through stronger binding to the altered transition state than the reactant state. Another possibility would entail lowering of the pKa of H2O on metal complexation, thus facilitating nucleophilic attack. Electrospray ionization mass spectrometry (ESI-MS) substantiated the proposed metal ion complexation. In the case of Cu2+ and Ag+, complexation between diazinon and metal ions was observed at given m/z ratios, while subsequent MS/MS analysis of the complexed ions revealed co-ordination at both S and N sites by Cu2+ and Ag+. No complexation between diazinon and Hg2+ was observed in ESI-MS; however Hg2+ was shown to complex the hydrolytic product O,O diethyl phosphorothioic acid, presumably through sulfur. Hydrolysis of diazinon was studied at several pH’s at 22oC in the presence of Hg2+, Cu2+, Cd2+ and Ag+, by following spectrophotometrically the appearance of 2-isopropyl-6-methylpyrimidin-4-ol. Kinetic data showed Hg2+, Cu2+ and Ag+ to be extremely effective in neutral-to-mildly acidic conditions, with a reduced effect in the presence of Cd2+. Smaller rate enhancements were also observed at slightly lower pH of 4.0. Possible factors include; 1) Competing acid hydrolysis involving protonation at one heterocyclic N of diazinon (N-3). 2) The dominant nature of the HgCl2 species at pH 4.0. 3) Metal speciation. / Thesis (Master, Chemistry) -- Queen's University, 2008-01-31 13:47:24.044