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

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

Burns, Kristi Lee ; Chemistry and Biochemistry

University:Georgia Institute of Technology

Department:Chemistry and Biochemistry

关键词: 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;   

Others  :  https://smartech.gatech.edu/bitstream/1853/29593/1/burns_kristi_l_200705_phd.pdf.pdf

美国|英语

来源: SMARTech Repository

【 摘 要 】

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.