germacrene-a and aristolochene

Trigonal iminium halides of (4aS,7S)-1,4a-dimethyl- and (4aS,7S)-4a-methyl-7-(prop-1-en-2-yl)-2,3,4,4a,5,6,7,8-octahydroquinolinium ions, aimed to mimic transition states associated with the aristolochene synthase-catalyzed cyclization of (-)-germacrene A to eudesmane cation, were evaluated under standard kinetic steady-state conditions. In the presence of inorganic diphosphate, these analogues were shown to competitively inhibit the enzyme, suggesting a stabilizing role for the diphosphate leaving group in this apparently endothermic transformation.

Topics: Aspergillus; Catalysis; Cyclization; Imines; Isomerases; Models, Molecular; Molecular Structure; Nicotiana; Penicillium; Salts; Sesquiterpenes; Sesquiterpenes, Eudesmane; Sesquiterpenes, Germacrane; Stereoisomerism

The catalytic mechanism of the enzyme aristolochene synthase from Penicillium roqueforti (PR-AS) has been probed with the farnesyl diphosphate analogues 6- and 14-fluoro farnesyl diphosphate (1a and 1c). Incubation of these analogues with PR-AS followed by analysis of the reaction products by GC-MS and NMR spectroscopy indicated that these synthetic FPP analogues were converted to the fluorinated germacrene A analogues 3b and 3c, respectively. In both cases the position of the fluorine atom prevented the formation of the eudesmane cation analogues 4b and 4c. These results highlight that germacrene A is an on-path reaction intermediate during PR-AS catalysis and shed light on the mechanism by which germacrene A is converted to eudesmane cation. They support the proposal that the role of PR-AS in the cyclisation is essentially passive in that it harnesses the inherent chemical reactivity present in the substrate by promoting the initial ionisation of farnesyl diphosphate and by acting as a productive template to steer the reaction through an effective series of cyclisations and rearrangements to (+)-aristolochene (7a).

Topics: Catalysis; Cyclization; Fluorine; Gas Chromatography-Mass Spectrometry; Isomerases; Magnetic Resonance Spectroscopy; Molecular Probe Techniques; Penicillium; Polyisoprenyl Phosphates; Sesquiterpenes; Sesquiterpenes, Germacrane

Tobacco 5-epi-aristolochene synthase (TEAS) catalyzes the Mg(II)-dependent cyclizations and rearrangements of (E,E)-farnesyl diphosphate (PP) to the bicyclic sesquiterpene hydrocarbon via a tightly bound (+)-germacrene A as a deprotonated intermediate. With the native enzyme, only a few percent of the putative germacrene A intermediate is released from the active site during the catalytic cycle. 6-Fluorofarnesyl PP was designed and synthesized with the aim of arresting the cyclization-rearrangement mechanism en route to 5-epi-aristolochene. Indeed, incubation of (2E,6Z)-6-fluorofarnesyl PP with recombinant TEAS afforded (-)-1-fluorogermacrene A as the sole product in 58% yield. Steady-state kinetic experiments with farnesyl PP and the 6-fluoro analogue showed that the overall catalytic efficiencies (k(cat)/K(m)) are essentially the same for both substrates. 1-Fluorogermacrene A was characterized by chromatographic properties (TLC, GC), MS, optical rotation, UV, IR and (1)H NMR data, and by heat-induced Cope rearrangement to (+)-1-fluoro-beta-elemene. (1)H NMR spectra at room temperature revealed that this (E,E)-configured fluorocyclodecadiene exists in solution as a 7:3 mixture of UU and UD conformers. 1-Fluorogermacrene A underwent trifluoroacetic acid-catalyzed cyclization to give three 1alpha-fluoroselinene isomers at a rate estimated to be about 1000 times slower than that of the similar cyclization of (+)-germacrene A to the parent selinenes.

Topics: Alkyl and Aryl Transferases; Binding Sites; Catalysis; Chromatography; Cyclization; Hydrocarbons, Fluorinated; Kinetics; Models, Chemical; Nicotiana; Polyisoprenyl Phosphates; Sesquiterpenes; Sesquiterpenes, Germacrane; Spectrum Analysis; Stereoisomerism; Substrate Specificity