The P.I. notes that in the first portion of this proposal, he plans to study the biosynthesis of the potent anti-cancer drug taxol (paclitaxel) and that his short-term objectives are to synthesize, in stable and radioisotopically-labeled form, the primary biosynthetic intermediates following the cyclization of geranylgeranyl pyrophosphate via taxadiene to the fully oxygenated taxol core structure. He further states that the long-term objectives of this program are to elucidate and target the slow (rate-limiting) steps in the biosynthetic pathway; in collaboration with Prof. Rodney Croteau of Washington State University, he proposes to transform Taxus sp. cell culture cells with cloned genes (from the Croteau lab) for the rate-limiting steps with the ultimate aim of boosting the yield of taxol produced by Taxus sp. cell culture. It is indicated that the use of both total synthesis, semi-synthesis of natural taxoids concurrent with the isolation of labeled metabolites from cell-free microsomal extracts and Taxus sp. cell cultures will be utilized to map the biosynthetic pathway to taxol. It is noted that the second portion of this program is to fully elucidate the biosynthetic pathway from primary amino acids (L-isoleucine, L-proline and L-tryptophan) and mevalonic acid-derived isoprene moieties to the anthelmintic polycyclic indole alkaloids comprising the brevianamides, and the paraherquamides. The PI reports that provocative evidence has been elucidated that the brevianamide / paraherquamide class of alkaloids, are constructed by a key biosynthetic (4+2) cycloaddition reaction. Structural and stereochemical evidence is said to strongly implicate an enzyme-mediated Diels-Alder construction as the penultimate step in the biosynthetic pathway. It is indicated that stable and radioisotopically labeled proposed biosynthetic intermediates will be synthesized and utilized to establish the entire biosynthetic pathway with emphasis on the step responsible for constructing the bicyclo (2.2.2) ring nucleus of this class of natural products and that biomimetic, Lewis acid-catalyzed Diels-Alder cycloaddition reactions will be studied to interpose the intrinsic facial bias of the cycloaddition reactions with the anti-selective stereochemistry observed in the brevianamide system and the syn-selective stereochemistry observed in the paraherquamide system.
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