The overall objective of the proposed research is to develop a fundamental understanding of the biologically important chemistry of prostaglandin (PG) endoperoxides, e.g. PGH 2 , and the biologically active products derived from these key intermediates in the oxidative metabolism of polyunsaturated fatty acids. Using model studies to guide investigations of these structurally complex and chemically reactive molecules, we discovered that secoprostanoic acid levulinaldehyde derivatives, which we named levuglandins (LGs), are generated along with PGs by rearrangement of PGH2 under the aqueous environment of its biosynthesis. We developed efficient total syntheses to confirm the structures of LGs and provide ample supplies for biological evaluation. Our immediate goal is to determine the extent and distribution of LG occurrence in vivo. Quantitative detection of LGs in biological systems is complicated by covalent adduct formation with proteins. LGs act like molecular glue binding covalently with proteins crosslinking them or sticking other nucleophiles to them, and thereby interfering with their biological roles. LGs also bind with DNA, cause DNA-protein crosslinking, and damage tissues, e.g., causing leakage through the blood-brain barrier. Since LG-derived protein-bound pyrroles are the most stereoisomerically simple and chemically stable product of the protein-LG reaction, they are one focus of our attention for detecting the biological occurrence of LGs. We will detect and quantify the natural occurrence these pyrrole derivatives using an immunoassay based on antibodies raised against a pyrrazole isostere. The assay will first be used to establish reaction conditions which result in optimum yields of pyrroles. The pyrrole immunoassay will then be applied to testing our hypothesis that LGs are involved in the tissue damage associated with the burst of fatty acid oxidative metabolism during oxygen reperfusion following the ischemia of stroke. Thus, the extent of LGE2-derived pyrrolization of blood proteins after ischemia and reperfusion will be measured and the distribution of LGE2-derived pyrrolization of tissues will be delineated immunocytochemically. A better understanding of the biochemistry of brain edema is essential to allow the development of rational therapeutic measures. Two recent observations suggest that it will also be possible to detect free LGs in solution in living cells. Thus, we found that the half life for sequestration of 10 micromoles LGE2 by covalent binding is several hours in mammalian cells in vitro. Furthermore, LGE2 forms a stable bis methoxime (LGM2) which is amenable to immunoassay, GC, and GC-MS analyses.
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