Mechanistic Studies of Taxadiene and Abietadiene Synthases.

摘要

Investigation aimed at understanding the enzymatic mechanism of the two diterpene cyclases, taxadiene and abietadiene synthase, may yield an alternative approach to combinatorial chemistry in the search for new and improved chemotherapeutic and antibiotic drugs. Taxadiene synthase catalyses the rate limiting step in the conversion of gerapylgeranyl diphosphate to taxol. Taxol (Paclitaxel Brystol Myers Squibb) and the related cyclic diterpene, taxotere (Docetaxel Rhone-Poulenc) are successfully used as front line chemotherapeutics in the treatment of several types of cancer including breast cancer. Progress was made in establishing assay conditions for the diterpene cyclase and synthesizing novel substrate analogs to be used in investigation of this class of enzymes. The analogs 2,3-dihydro-geranylgeranyl diphosphate and 5- geranylgeranyl diphosphorothiolate have been synthesized. Synthesis of verticillene and copalyl diphosphate were not attempted due to potential availability from collaborators. Attempts at synthesizing both substrate analogs 6,7-dihydro-geranylgeranyl diphosphate and geranylgeranyl methylphosphonophosphate were unsuccessful. Several technical hurdles in establishing a reliable enzyme assay were overcome. Transfer of this assay to the rapid quench machine was difficult due to lack of product signal. Lack of signal may be caused by adsorption of the hydrophobic hydrocarbon product to the plastic tubing within the rapid quench machine. This project remains uncompleted but has potential to ultimately yield progress towards its original goals.