Microbial and reducing agents catalyze the rearrangement of taxanes.

5alpha, 7beta, 9alpha, 10beta, 13alpha-Pentahydroxy-4(20),11(12)-taxadiene derivative 1 was converted to two unprecedented 1(15-->11)abeo-taxanes and a taxane derivative with a C10-C11 double bond by Absidia coerula ATCC 10738a. A similar compound was obtained from treatment with zinc of a triacetoxy-4(20),11(12)-taxadiene derivative.

Taxus canadensis abundant taxane: conversion to paclitaxel and rearrangements.

An efficient conversion of Taxus canadensis abundant taxane, 9-dihydro-13-acetylbaccatin III to baccatin III is described. Since the synthesis of paclitaxel from baccatin III has been reported, this work can be used for additional supply of this powerful anticancer drug. In addition, new taxanes derived from skeletal rearrangements originating from oxidation reduction reactions of the Canadian yew major taxane, are reported.

Biosynthesis of trichothecenes and apotrichothecenes.

Fusarium culmorum produces two major trichothecenes, 3-acetyldeoxynivalenol and sambucinol, and some minor apotrichothecenes. It was desired to investigate if during their biosynthesis a C-11-keto intermediate was involved. To verify this postulate, trichodiene, a known precursor to trichothecenes, was synthesized with two deuteriums at C-11 and one at C-15. It was then fed to F. culmorum cultures, and the derived metabolites were purified and analyzed. The results ruled out the involvement of an 11-keto intermediate but revealed two novel apotrichothecenes. The characterization of their structures suggested that one of the 2-hydroxy-11alpha-apotrichothecene stereoisomers (2alpha or 2beta) could be converted to sambucinol. These apotrichothecenes were therefore synthesized labeled specifically with two deuteriums at C-4 and C-15 and fed to F. culmorum cultures. Indeed, the result established for the first time that 2alpha-hydroxy-11alpha-apotrichothecene was a precursor to sambucinol. A biosynthetic scheme for the production of trichothecenes and apotrichothecenes is described.

Biosynthesis of 3-acetyldeoxynivalenol and sambucinol. Identification of the two oxygenation steps after trichodiene.

The first two oxygenation steps post-trichodiene in the biosyntheses of the trichothecenes 3-acetyldeoxynivalenol and sambucinol were investigated. The plausible intermediates 2-hydroxytrichodiene (2alpha- and 2beta-) and 12,13-epoxytrichodiene and the dioxygenated compounds 12,13-epoxy-9,10-trichoene-2-ol (2alpha- and 2beta-) were prepared specifically labeled with stable isotopes. They were then fed separately and/or together to Fusarium culmorum cultures, and the derived trichothecenes were isolated, purified, and analyzed. The stable isotopes enable easy localization of the labels in the products by 2H NMR, 13C NMR, and mass spectrometry. We found that 2alpha-hydroxytrichodiene is the first oxygenated step in the biosynthesis of both 3-acetyldeoxynivalenol and sambucinol. The stereoisomer 2beta-hydroxytrichodiene and 12,13-epoxytrichodiene are not biosynthetic intermediates and have not been isolated as metabolites. We also demonstrated that the dioxygenated 12, 13-epoxy-9,10-trichoene-2alpha-ol is a biosynthetic precursor to trichothecenes as had been suggested in a preliminary work. Its stereoisomer was not found in the pathway. A further confirmation of our results was the isolation of both oxygenated trichodiene derivatives 2alpha-hydroxytrichodiene and 12,13-epoxy-9, 10-trichoene-2alpha-ol as natural metabolites in F. culmorum cultures.

Biosynthesis of the trichothecene 3-acetyldeoxynivalenol. Is isotrichodermin a biosynthetic precursor?

3-Acetyldeoxynivalenol is the major trichothecene produced by the fungus Fusarium culmorum. The first proven tricyclic intermediate in the biosynthesis of 3-acetyldeoxynivalenol has been shown by in vivo studies to be isotrichodermin, a natural metabolite of F. culmorum. Indeed, the feeding of ring-deuterated isotrichodermin resulted in ring-deuterated 3-acetyldeoxynivalenol as shown by NMR studies. In this work, we have shown that the 3-acetyl group of isotrichodermin is mostly lost in its metabolism to 3-acetyldeoxynivalenol. We have shown by two different approaches that the deacetylation occurs at an early step after the first oxygenation step at C-15. Derivatives of isotrichodermin lacking the 3-acetyl such as 3-deacetyl isotrichodermin or 3-oxo-12,13-epoxytrichothec-9-ene are not precursors to 3-acetyldeoxynivalenol. The role of this acetyl exchange mechanism is not clear presently.

Biosynthesis of the trichothecene 3-acetyldeoxynivalenol: cell-free hydroxylations of isotrichodermin.

3-Acetyldeoxynivalenol is the major trichothecene produced by the fungus Fusarium culmorum. Studies in vivo with F. culmorum have established the following biosynthetic precursors of 3-acetyldeoxynivalenol: isotrichool → isotrichodiol → isotrichodermin → 15-deacetylcalonectrin, 7α-hydroxyisotrichodermin, 8α-hydroxyisotrichodermin → calonectrin → deoxynivalenol. In this paper, we describe in vitro investigations of one of these metabolic steps. The cell-free system of F. culmorum that converts isotrichodermin into 15-deacetylcalonectrin, 7α-hydroxyisotrichodermin, and 8α-hydroxyisotrichodermin is described here. This preparation requires NADPH but not NADH for activity and is not inhibited by carbon monoxide, cyanide, or known oxygenase inhibitors, such as SKF-525-A or ancymidol.Key words: trichothecene, Fusarium culmorum, cell-free system, isotrichodermin, 15-deacetylcalonectrin.

Biosynthesis of Fusarium culmorum trichothecenes. The roles of isotrichodermin and 12,13-epoxytrichothec-9-ene.

Two different approaches enabled us to unambiguously establish the intermediacy of isotrichodermin and 12,13-epoxytrichothec-9-ene in the biosynthesis of the trichothecenes 3-acetyldeoxynivalenol and sambucinol, respectively. The kinetic pulse-labeling method enabled us to detect a plausible precursor to 3-acetyldeoxynivalenol biosynthesis. Feeding experiments using the pure 14C-labeled intermediate established that it was incorporated 27% into 3-acetyldeoxynivalenol but not to sambucinol. The 14C-precursor was subsequently used as a marker to purify the unlabeled intermediate which was shown to be isotrichodermin by spectroscopic techniques. In order to trace the enriched carbons incorporated into 3-acetylde-oxynivalenol, specifically deuteriated isotrichodermin was synthesized and fed to Fusarium culmorum. The 2H NMR spectrum of the derived 3-acetyldeoxynivalenol proved conclusively the position of the deuteriums and that isotrichodermin is a major biosynthetic precursor. The proof that isotrichodermin is converted in vivo to 3-acetyldeoxynivalenol but not to sambucinol led us to postulate that 12,13-epoxytrichothec-9-ene might have an important role in the biosynthesis of trichothecenes. We synthesized 12,13-epoxytrichothec-9-ene with tritium at C-15 or with two deuteriums at C-4 and two deuteriums at C-15. These labeled compounds enabled us to prove that 12,13-epoxytrichothec-9-ene is a major precursor to sambucinol biosynthesis but is neither converted to isotrichodermin nor to 3-acetyldeoxynivalenol. We also succeeded in isolating a biosynthetic intermediate between 12,13-epoxytrichothec-9-ene and sambucinol and characterized its structure as 3-deoxysambucinol by spectroscopic techniques (1H NMR, 2H NMR, 13C NMR, correlation spectroscopy, two-dimensional heteronuclear correlation experiments, and mass spectroscopy).