Stereoselective Interaction of Tetraconazole with 14α-Demethylase in Fungi

Tetraconazole racemate and its enantiomers were compared for their different capability to affect growth and ergosterol biosynthesis in Ustilago maydis and Saccharomyces cerevisiae. At subletal doses quantitative effects on sterols in sporidia of U. maydis, when expressed as in vivo IC 50 values, were found to coincide with in vitro IC 50 values (concentrations which inhibit net ergosterol biosynthesis in cell-free extract by 50%). Impairment of cell separation was the early symptom of fungitoxicity and the related EC 50 values (concentrations which inhibit cell multiplication by 50%) were aligned with the biochemical effects. S. cerevisiae was shown to be the less sensitive species of several fungi. Sterol biosynthesis inhibition ranked the enantiomers according to their order of fungitoxicity on both organisms tested. The enantiomeric activity ratios ( R/ S), when determined by the in vitro IC 50 values, were in the range of 10-13 in both cases, with the R-(+)-isomer being the most active one. Interaction with cytochrome P450, partially purified from microsomes of S. cerevisiae and utilized as 1.4 x 10 −7 M solution, showed type II spectra with saturation reached when approximately equimolecular amounts of the separate isomers or racemate were added. This supports the view that both tetraconazole enantiomers have very high affinity to the target site, as also shown with microsomal P450 isolated from U. maydis. The different strength of their bindings to the target site apparently rests on the CO displacement tests, by which the S-(−)-isomer is more easily removed than its optical antipode. Accordingly, the stereoselective accommodation of dichlorophenyl and tetrafluoroethyl ether moieties of tetraconazole within the active site of the target enzyme are briefly discussed.