Demonstration of Germacrene A as an Intermediate in 5-Epi-aristolochene Synthase Catalysis

Abstract

Sesquiterpene synthases are a family of enzymes that catalyze farnesyl pyrophosphate (FPP) cyclization via alternative pathways to produce a variety of cyclic sesquiterpene products. Catalysis by several of these enzymes, including tobacco 5-epi-aristolochene synthase (TEAS), has been proposed to include the formation of germacrene A as a stable intermediate. Neither germacrene A nor any other intermediate is released from sesquiterpene synthase active sites during normal catalysis. Evidence to support the intermediacy of germacrene A has been derived from investigations of aristolochene synthases from Aspergillus terreus and Penicillium roquefortii (Cane, D. E. Chem. Rev. 1990, 90, 1089−1103 and references therein. Cane, D. E.; Bryant, C. J. Am. Chem. Soc. 1994, 116, 12063−12064. Cane, D. E.; Tsantrizos, Y. S. J. Am. Chem. Soc. 1996, 118, 10037−10040). However, until the present investigations of TEAS, formation of this postulated intermediate has never been directly demonstrated. TEAS catalyzes the cyclization of FPP to 5-epi-aristolochene, a precursor of a tobacco phytoalexin, capsidiol. Based upon the three-dimensional structure of TEAS, a detailed mechanism has been proposed for TEAS catalysis that includes the prediction that proton donation by Y520 is responsible for the activation of germacrene A to a eudesmane cation (Starks, C. M.; Back, K.; Chappell, J.; Noel, J. P. Science 1997, 277, 1815−1820). In the present investigation, a Y520F point mutation is introduced into TEAS (TEAS-Y520F) by site-directed mutagenesis. In the presence of 3H−FPP, TEAS-Y520F produces hexanes−extractable 3H with a catalytic efficiency approximately 3% that of nonmutated, recombinant TEAS. The hexanes−extractable 3H is identified as germacrene A, m/z 204, through direct GC-MS comparison to an authentic sample. This observation confirms the intermediacy of germacrene A in TEAS catalysis, supports the postulated production of germacrene A by a variety of other sesquiterpene synthases, and also confirms the proposed role of Y520 in TEAS catalysis.

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

“…[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.…”

“…Thus, the suprafacial pericyclic transition state with partial bonding between C2/C3 and C10/ C5 resembling a strain-free trans-decalin would be readily accessible from 13a. In contrast, a concerted, suprafacial [3,3]-rearrangementof the UD conformer 13b would necessarily produce the C5 epimer of 14 through a highly strained boat/twist-boat conformation of the alternative cisdecalin-like transition state. The lower energy of chair-like transition states in [3,3]sigmatropic rearrangements, compared to the boat-like or twisted alternatives, is well established.…”

“…The similarity of the conditions required for the thermal [3,3]sigmatropic rearrangements of 1-fluorogermacrene A and germacrene A (3 h vs. 2 h at 110 °C), the formation of single isomers in both cases, and the similarity of the 1 H NMR spectra of the two products were considered good reasons to assign the structure as the 1-fluoro derivative (14)of β-elemene, a sesquiterpene of well-established structure and stereochemistry. [33] The 5,10-trans diequatorial and 5,7-cis diequatorial orientations of the vinyl and the two isopropenyl substituents predicted for the stable chair conformer of 14 (Scheme 5) were confirmed by the appearance of a doubletof doublets (2.49 ppm, J=13 and 4 Hz) in the proton NMR spectrum attributable to an axial H5 proton coupled to axial and equatorial protons on the adjacent C6 methylene group.…”

“…The stereospecificity of this [3,3]sigmatropic process can be rationalized by the structural similarity of the UU conformer 13a (Scheme 4) to the stable chair conformation of 14 (Scheme 5). Thus, the suprafacial pericyclic transition state with partial bonding between C2/C3 and C10/ C5 resembling a strain-free trans-decalin would be readily accessible from 13a.…”

“…Examples of cryptic deprotonated intermediates include the tertiary diphosphates (PPs), (R)-and (S)-linalyl and (R)-and (S)-nerolidyl PPs, which are converted to cyclic monoterpene and sesquiterpene products, respectively, faster than they are produced by the initial isomerase activity of the cyclases. [2] Neutral intermediates include the macrocyclic sesquiterpenes (R)-and (S)-germacrene A, humulene, and bicyclogermacrene, which all remain as tightly bound intermediates during the respective cyclizations catalyzed by epiaristolochene, [3] aristolochene, [4] pentalenene, [5] and germacradienol-germacrene D [6] synthases.…”