Interception of the Enzymatic Conversion of Farnesyl Diphosphate to 5‐Epi‐Aristolochene by Using a Fluoro Substrate Analogue: 1‐Fluorogermacrene A from (2E,6Z)‐6‐Fluorofarnesyl Diphosphate

Abstract

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-fluororogermacrene 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-β-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 1α-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.

Identification of Sesquiterpene Synthases fromNostoc punctiformePCC 73102 andNostocsp. Strain PCC 7120

“…Crystal structures of several sesquiterpene synthases (11,32,44) have been solved to aid in the investigation of the complex cyclization mechanisms catalyzed by these enzymes (17,47). All crystallized sesquiterpene synthases share a conserved class I ␣-helical terpene synthase fold, but their primary sequences are much less conserved, and sequences often share less then 25% identity at the amino acid level.…”

Total (Bio)Synthesis: Strategies of Nature and of Chemists

“…A single enzyme, tobacco 5-epi-aristolochene synthase (TEAS), converts the biosynthetic precursor, farnesyl diphosphate ( 6 ), to 2 with a k cat / K M of 0.3 µM −1 min −1 [21, 22]. Nonetheless, this single transformation itself is complex: TEAS catalyzes two ring closures, a hydride and a methyl migration, and a proton abstraction to form a double bond (Fig.…”

Chemoenzymatic synthesis of the alarm pheromone (+)-verbenone from geranyl diphosphate

“…8 Recent work has revealed that several terpene cyclases accept analogues of prenyl diphosphates as substrates thereby opening up the possibility for the simple production of large numbers of modified terpenoids with potentially altered biological activity. [9][10][11][12][13][14][15][16][17][18] For such syntheses to be viable, downstream modifications of the terpene synthase-generated acyclic or (poly)-cyclic hydrocarbon product(s) should preferably be performed in the same reaction vessel as the enzymatic step; the hydrocarbons (in particular monoterpenes) are normally highly volatile (i.e. semiochemicals).…”