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.
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.
Faraldos
Juan A
JA
Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801, USA.
Zhao
Yuxin
Y
O'Maille
Paul E
PE
Noel
Joseph P
JP
Coates
Robert M
RM
D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002845:Chromatography; D003500:Cyclization; D006845:Hydrocarbons, Fluorinated / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism*; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism*; D045788:Sesquiterpenes, Germacrane / Q000737:chemistry / Q000378:metabolism*; D013057:Spectrum Analysis; D013237:Stereoisomerism; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology*
D006845:Hydrocarbons, Fluorinated; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D045788:Sesquiterpenes, Germacrane; C054277:aristolochene; C471077:germacrene A; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C078217:5-epi-aristolochene synthase