Stereochemistry and deuterium isotope effects associated with the cyclization-rearrangements catalyzed by tobacco epiaristolochene and hyoscyamus premnaspirodiene synthases, and the chimeric CH4 hybrid cyclase

Abstract

Tobacco epiaristolochene and hyoscyamus premnaspirodiene synthases (TEAS and HPS) catalyze the cyclizations and rearrangements of (E,E)-farnesyl diphosphate (FPP) to the corresponding bicyclic sesquiterpene hydrocarbons. The complex mechanism proceeds through a tightly bound (R)-germacrene A intermediate and involves partitioning of a common eudesm-5-yl carbocation either by angular methyl migration, or by C-9 methylene rearrangement, to form the respective eremophilane and spirovetivane structures. In this work, the stereochemistry and timing of the proton addition and elimination steps in the mechanism were investigated by synthesis of substrates bearing deuterium labels in one or both terminal methyl groups, and in the pro-S and pro-R methylene hydrogens at C-8. Incubations of the labeled FPPs with recombinant TEAS and HPS, and with the chimeric CH4 hybrid cyclase having catalytic activities of both TEAS and HPS, and of unlabeled FPP in D2O, together with gas chromatography-mass spectrometry (GC-MS) and/or NMR analyses of the labeled products gave the following results: (1) stereospecific CH3-->CH2 eliminations at the cis-terminal methyl in all cases; (2) similar primary kinetic isotope effects (KIE) of 4.25-4.64 for the CH3-->CH2 eliminations; (3) a significant intermolecular KIE (1.33+/-0.03) in competitive cyclizations of unlabeled FPP and FPP-d6 to premnaspirodiene by HPS; (4) stereoselective incorporation of label from D2O into the 1beta position of epiaristolochene; (5) stereoselective eliminations of the 1beta and 9beta protons in formation of epiaristolochene and its delta(1(10)) isomer epieremophilene by TEAS and CH4; and (6) predominant loss of the 1alpha proton in forming the cyclohexene double bond of premnaspirodiene by HPS and CH4. The results are explained by consideration of the conformations of individual intermediates, and by imposing the requirement of stereoelectronically favorable proton additions and eliminations.

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

“…The stereochemical outcome of the TEAS-catalyzed reaction leading to the halogen-free epiaristolochene 4 has been recently reported by Schenk et al [16] using deuterated farnesyl PPs as substrates. These studies provide strong circumstantial evidence that the active site of TEAS directs the precise folding of the natural farnesyl PP substrate (1) within the protein to guarantee and to secure the UD conformation of the enzyme-bound germacrene A intermediate 2 needed to account for the anti relationship of the CH 3 groups presentin 5-epiaristolochene (4).…”

“…Preparative-scale incubations of 6-fluorofarnesyl PP NH 4 + salt (12) with TEAS performed according to Schenk et al [16] [24] this clearly indicates a substantial effect of the fluoro substituent on the interaction of the modified sesquiterpene with polarized light. GC analyses (method A, see the Supporting Information) of the pentane/Et 2 O extracts from the incubation medium showed a single peak (t R = 20.1 min), albeit with a hump in the base line that is characteristic of (E,E)-configured germacrenes owing to their thermal Cope rearrangement to elemenes on the column.…”

“…[2b,14b] Initial ionization of the allylic PP to the corresponding allylic cation/PP·Mg anion pair, followed by electrophilic attack at C10 of the isoprenoid chain derived from the fluoro substrate analogue 12 generates the 1-fluoro analogue of the germacren-11-yl carbocation (15,Scheme 6). This carbocation in turn undergoes rapid TEAS-mediated proton abstraction, presumably from the cis-terminal methyl group, [16] to form the isopropenyl moiety present in fluorogermacrene A. The inability of the enzyme to proceed further through protonation of the 1(10)-double bond, [36] and to generate the 1-fluoro analogue (17) of 5-epi-aristolochene, or other bicyclic fluoro sesquiterpene isomers, as the final sesquiterpene product of the reaction, is attributed to the intrinsic electronegative nature of the fluorine.…”

“…[15] Considerable evidence supports the formation of an enzyme-bound germacrene A intermediate (2), which undergoes proton-induced cyclization and a 5→4 hydride shift to generate the branch-point eudesmyl carbocation 3b (Scheme 1). [16] Subsequent methyl or methylene migration followed by proton transfers give rise to epiaristolochene (4) and premnaspirodiene (5), respectively. The isolation of germacrene A from incubations of the catalytically impaired mutant form of TEAS, Y520F, [3] as well as the incorporation of a single deuterium at the 1β position of 4 and the retention of the label in 5 from incubations in deuterated media, [16] support the mechanism shown.…”

“…[16] Subsequent methyl or methylene migration followed by proton transfers give rise to epiaristolochene (4) and premnaspirodiene (5), respectively. The isolation of germacrene A from incubations of the catalytically impaired mutant form of TEAS, Y520F, [3] as well as the incorporation of a single deuterium at the 1β position of 4 and the retention of the label in 5 from incubations in deuterated media, [16] support the mechanism shown. In this paper, we report the efficient TEAS-catalyzed conversion of (2E,6Z)-6-fluorofarnesyl PP to (-)-1-fluorogermacrene A and the physical characterization of the resultant sesquiterpene derivative.…”