Biosynthetic and mass spectrometric studies of fatty acid synthesis inhibitor platensimycin and related compounds, and monitoring lipid flux by mass spectrometry

Platensimycin and platencin are two natural products isolated from Streptomyces platensis. Both compounds have emerged as promising drug leads for antibacterial and antidiabetic therapies because they act as selective inhibitors of bacterial and mammalian fatty acid synthesis. The research reported in this dissertation focuses primarily on elucidation of biosynthetic pathways leading to platensimycin and platencin. Analysis of products isolated after feeding stable-isotope-labeled acetate, pyruvate and glycerol as precursors not only demonstrated that the ketolide portion of the platensimycin and platencin originates from a non-mevalonate terpenoid pathway, but also established that four of the carbon atoms in the 3-amino-2,4-dihydroxy benzoate moiety of Platensimycin originates from oxaloacetate generated by the tricarboxylic acid cycle (TCA). Similarly, results from the investigations of pyruvate incorporation showed that the other three carbon atoms of the 3-amino-2,4-dihydroxy benzoate moiety originate from phosphoenolpyruvate (PEP).;In addition a multiple-reaction-monitoring (MRM) protocol was developed for mass spectrometric detection, rapid dereplication, quantification and metabolite identification of platensimycin and platencin related compounds present in natural product extracts and samples from in vivo animal experiments. To determine the efficacy of platensimycin as an in vivo fatty acid synthesis inhibitor, we developed a high-resolution mass spectrometric method, which monitors the increase of [d1]palmitic acid level after the animals are fed with D2O.;Last, a mass spectrometric fragmentation pathway that is useful for structure confirmation of poly-hydroxy, or poly-methoxy benzoic acid derivatives was discovered. For example, a dramatic ortho effect is observed during gas-phase dissociation of methoxybenzoate anions. Upon activation, anions derived from ring isomers of methoxybenzoate undergo a facile CO 2 loss to form an m/z 107 ion. However, only the 1-dehydro-2-methoxybenzene anion derived from the ortho isomer showed an exclusive formaldehyde loss to generate an ion of m/z 77. The spectra from poly-methoxy benzoic acid showed that the negative charge of the anion, generated by the initial decarboxylation, moves in a circumambulatory manner when a methoxy group present adjacent to the ring carbon bearing the charge. This generalization can be used as a diagnostic tool to confirm the location of a methoxy group in poly-methoxy benzoic acids.