ISOPRENOID COMPOUNDS FOR BIOLOGICAL STUDIES

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Isoprenoid compounds are synthesized in all living organisms where they play essential roles in metabolism, cellular architecture, and signalling both within and between cells. The molecules required for the fundamental building reactions in the pathway are needed in unlabeled, stable isotope labeled, and radiolabeled form for enzyme assays, drug screening protocols, metabolism studies, and cell biology experiments. Only a limited number of these compounds are available commercially. Biologists and biochemists who require all but the most common derivatives or special labeling patterns must resort to costly "custom" syntheses. Molecules with fluorescent or photoaffinity tags are not available in all but a few academic laboratories. In phase I, (a) we optimized the "large scale" synthesis of simple linear C5, C10, C15, and C20 isoprenoid diphosphates, (b)developed procedures for purification and storage of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase, and (c) synthesized photoaffinity probes consisting of photolabile benzophenone moieties appended to isoprenoid diphosphates. In phase II we will expand our product line to include a wider range of naturally occurring isoprenoid molecules not commercially available, add photoaffinity labels, and add a family of fluorescent compounds for assays, binding measurements, and tags for prenylated proteins. A set of isoprenoid enzymes, including isopentenyl diphosphate, isomerase; an engineered set of prenyltransferases selective for synthesis of geranyl (C10), farnesyl (C15), geranylgeranyl (C20), and farnesylgeranyl (C25) diphosphates from simple C5 building blocks; and protein farnesyltransferase, will be provided individually or as kits for synthesis of labeled molecules. PROPOSED COMMERCIAL APPLICATIONS: Isoprenoid compounds are increasingly used in drug discovery assays, in the areas of atherosclerosis and cancer, to study cell signalling mechanisms, and studies of cellular metabolism. ERL's line of isoprenoid products will provide researchers with chemicals and enzymes to facilitate these studies.