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1	An exploration of biochemistry including biotechnology, structural characterization, drug design, and chromatographic analyses Burns, Kristi Lee 28 September 2006 (has links) We now report an in depth analysis of the successful in vitro enzymatic synthesis of PHB utilizing the three-enzyme system from the bacteria Cupriavidus necator. Using HPLC methodology developed in this laboratory, and by adding each enzyme in a step-wise manner, we follow each individual stage in the three-enzyme route for PHB synthesis and delineate all stoichiometric relationships. We report the construction of the first metabolic model developed specifically for analyzing in vitro enzymatic PHB synthesis. We developed a hands-on student laboratory for culturing, producing, isolating, and purifying the bacterial biopolyesters PHB. We now report the first structural characterizations of iso-CoA, acetyl-iso-CoA, acetoacetyl-iso-CoA, and beta-hydroxybutyryl-iso-CoA using MS, MS/MS, and homo- and hetero-nuclear NMR analyses.We describe HPLC methodology to separate the isomers of several iso-CoA-containing compounds and report the first examples of iso-CoA-containing compounds acting as substrates in enzymatic acyl-transfer reactions. We describe a simple regioselective synthesis of iso-CoA from CoA. We also demonstrate a plausible mechanism, which accounts for the existence of iso-CoA isomers in commercial preparations of CoA-containing compounds. Herein we report that phenylaminoethyl selenide compounds protect DNA from peroxynitrite-mediated single-strand breaks. The mechanism of protection against peroxynitrite mediated DNA damage was investigated by HPLC. The chemistry of the reaction between peroxynitrite and HOMePAES was investigated using HPLC and HPLC/MS. The unique chemistry of the reaction between peroxynitrite and HOMePAES was investigated using HPLC and HPLC/MS. We report the development of novel CDB derivatives, which are selective COX-II inhibitors. A series of compounds were assayed with an in vitro colorimetric inhibitor screening and with a whole blood ELISA screening and the results indicate that MST is a selective inhibitor of COX-II. Iso-coa Iso-coenzyme A Poly-(beta)-hydroxybutyric acid PHB Phenylaminoethyl selenide compounds Biopolyesters Acetyl-iso-coa Acetoacetyl-iso-coa Regioselective synthesis Homepaes DNA DNA damage Single-strand breaks Acyl-transfer reactions Culturing Homo-nuclear NMR Purifying Producing Isolating Isomers Selective COX-II inhibitors Whole blood Structural characterizations ELISA In vitro Enzymatic synthesis Beta-hydroxybutyryl-iso-coa MS/MS MS HPLC Metabolic model Hetero-nuclear NMR Peroxynitrite HPLC/MS CDB derivatives Poly-beta-hydroxybutyrate Biosynthesis

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An exploration of biochemistry including biotechnology, structural characterization, drug design, and chromatographic analyses