Biosynthetic studies on taxol

The biosynthesis of the plant antitumor agent, taxol, was studied by feeding radioactive or stable isotope-labeled precursors to cut stems or to inner bark tissue of Taxus brevifolia. The labeled taxol was purified to radiochemical purity and subjected to chemical degradation or was analyzed by electrospray tandem mass spectrometry. It was demonstrated that in the plant taxol is synthesized from baccatin-111, containing the fully functionalized diterpene moiety, and a precursor of the phenylpropanoid side chain. The latter arises from phenylalanine not via cinnamic acid, but via B-phenylalanine and phenylisoserine. Benzoylation of the side chain occurs only after its attachment to the diterpene moiety. The benzoate moiety is also formed from phenylalanine via B-phenylalanine and phenylisoserine, not via cinnamic acid. Due to its unique mode of action and promising early clinical results, taxol(1) commands considerable interest as a new plant anticancer agent (2). The complexity of its structure presents a considerable challenge to synthetic organic chemistry and raises the question how the plant assembles this formidable molecule (3-5). We have studied this latter issue by feeding experiments with potential precursors labeled strategically with radioactive or stable isotopes (6). Our studies focused initially on the origin of the phenylpropanoid side chain and its mode of attachment to the diterpene moiety. Feeding experiments with radiolabeled precursors were carried out with cut twigs of Taxus brevifolia to which the radioactive material was administered through the cut stem in a small volume of water. Extensive purification of the resulting taxol, first by repeated HPLC and then by co-crystallization with non-labeled carrier material was found necessary to achieve constant specific radioactivity or, in other cases, to remove traces of radioactive impurities. Under these conditions incorporation of [7-14C]benzoic acid, as its N-acetylcysteamine thioester (0.09%), and [ 13-3Hlbaccatin-III (0.12%) was obtained. Degradation of the radioactive taxol from the benzoate feeding experiment showed that 88% of the radioactivity was recovered in the side chain fragment and only 1 1 % in the baccatin-111, suggesting that under the conditions of the experiment relatively little of the diterpene moiety was synthesized de novo. Upon degradation of the taxol from the baccatin-I11 feeding experiments, all the radioactivity was recovered in the baccatin-111 moiety, none in the side chain fragment.