Alternative Synthesis of the Colorado Potato Beetle Pheromone

A concise preparation of the pheromone secreted by the male Colorado potato beetle [viz. (3<i>S</i>)-1,3-dihydroxy-3,7-dimethyl-6-octen-2-one] was accomplished in four steps starting from 2-fluoronerol or 2-fluorogeraniol. The key step in the synthesis involves a 6-endo epoxide ring-opening with ester participation that simultaneously inverts the 3<i>R</i>-configuration of the (3<i>R</i>)-2,3-epoxy-2-fluoroprenyl acetate intermediate and installs the ketone functionality of the semiochemical. Extensive NMR studies validate the proposed 6-endo mechanism of the featured rearrangement, which under anhydrous conditions resulted in the formation of two bicyclic 1,3-dioxan-5-ones via an unprecedented intramolecular Prins cyclization

Evolutionary and Mechanistic Insights from the Reconstruction of α‑Humulene Synthases from a Modern (+)-Germacrene A Synthase

Germacrene A synthase (GAS) from <i>Solidago canadensis</i> catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and <2% α-humulene, which arises from 1,11-cyclization of FDP. The origin of the 1,11-activity of GAS was investigated by amino acid sequence alignments of 1,10- and 1,11-synthases and comparisons of X-ray crystal structures with the homology model of GAS; a triad [Thr 401-Gly 402-Gly 403] that might be responsible for the predominant 1,10-cyclization activity of GAS was identified. Replacement of Gly 402 with residues of increasing size led to a progressive increase of 1,11-cyclization. The catalytic robustness of these 1,10- /1,11-GAS variants point to Gly 402 as a functional switch of evolutionary significance and suggests that enzymes with strict functionalities have evolved from less specific ancestors through a small number of substitutions. Similar results were obtained with germacrene D synthase (GDS) upon replacement of the homologous active-site residue Gly 404: GDS-G404V generated approximately 20% bicyclogermacrene, a hydrocarbon with a cyclopropane ring that underlines the dual 1,10-/1,11-cyclization activity of this mutant. This suggests that the reaction pathways to germacrenes and humulenes might be connected through a bridged 1,10,11-carbocation intermediate or transition state that resembles bicyclogermacrene. Mechanistic studies using [1-³H₁]-10-fluorofarnesyl diphosphate and deuterium-labeling experiments with [12,13-²H₆]-FDP support a germacrene–humulene rearrangement linking 1,10- and 1,11-pathways. These results support the bioinformatics proposal that modern 1,10-synthases could have evolved from promiscuous 1,11-sesquiterpene synthases

Probing the Mechanism of 1,4-Conjugate Elimination Reactions Catalyzed by Terpene Synthases

The reaction mechanisms of (<i>E</i>)-β-farnesene synthase (EBFS) and isoprene synthase (ISPS), enzymes that catalyze a formal regiospecific 1,4-conjugate elimination of hydrogen diphosphate from (<i>E</i>,<i>E</i>)-farnesyl and dimethylallyl diphosphate (FDP and DMADP) to generate the semiochemicals (<i>E</i>)-β-farnesene and isoprene, respectively, were probed with substrate analogs and kinetic measurements. The results support stepwise reaction mechanisms through analogous enzyme-bound allylic cationic intermediates. For EBFS, we demonstrate that the elimination reaction can proceed via the enzyme-bound intermediate <i>trans</i>-nerolidyl diphosphate, while for ISPS the intermediacy of 2-methylbut-3-enyl 2-diphosphate can be inferred from the product outcome when deuterated DMADPs are used as substrates. Possible implications derived from the mechanistic details of the EBFS-catalyzed reaction for the evolution of sesquiterpene synthases are discussed

Growth behaviour of <i>P. capsici</i> tdtom, after 10 days of exposure to different capsidiol concentrations.

<p>The experiment was performed 3 times.</p

Scatter plots correlating 0D600 and capsidiol concentration.

<p>The plots illustrate growth of <i>P. infestans</i> 88069td (A) and <i>P. capsici</i> tdtom (B) strains over time for a maximum of 10 days. The experiment was performed 3 times.</p

Scatter plots correlating fluorescence intensity and capsidiol concentration.

<p>The plots illustrate fluorescence intensity of <i>P. infestans</i> 88069td (A) and <i>P. capsici</i> tdtom (B) strains over time for a maximum of 10 days. The experiment was performed 3 times.</p

Capsidiol inhibits <i>P. infestans</i> growth reversibly.

<p>(A) Growth inhibition assay of <i>P. infestans</i> after 10 days of exposure of mycelial plugs to capsidiol. (B) Restoration of growth after washing treatment. Green line indicates the point after which the washing treatment was applied. The experiment was performed 3 times. Picture was taken 10 days after the washing and 20 days after initial exposure to capsidiol.</p

Provenance of <i>Phytophthora</i> samples.

<p>Provenance of <i>Phytophthora</i> samples.</p

Structural Elucidation of Cisoid and Transoid Cyclization Pathways of a Sesquiterpene Synthase Using 2-Fluorofarnesyl Diphosphates

Sesquiterpene skeletal complexity in nature originates from the enzyme-catalyzed ionization of (<i>trans</i>,<i>trans</i>)-farnesyl diphosphate (FPP) (<b>1a</b>) and subsequent cyclization along either 2,3-transoid or 2,3-cisoid farnesyl cation pathways. Tobacco 5-epi-aristolochene synthase (TEAS), a transoid synthase, produces cisoid products as a component of its minor product spectrum. To investigate the cryptic cisoid cyclization pathway in TEAS, we employed (<i>cis</i>,<i>trans</i>)-FPP (<b>1b</b>) as an alternative substrate. Strikingly, TEAS was catalytically robust in the enzymatic conversion of (<i>cis</i>,<i>trans</i>)-FPP (<b>1b</b>) to exclusively (≥99.5%) cisoid products. Further, crystallographic characterization of wild-type TEAS and a catalytically promiscuous mutant (M4 TEAS) with 2-fluoro analogues of both all-<i>trans</i> FPP (<b>1a</b>) and (<i>cis</i>,<i>trans</i>)-FPP (<b>1b</b>) revealed binding modes consistent with preorganization of the farnesyl chain. These results provide a structural glimpse into both cisoid and transoid cyclization pathways efficiently templated by a single enzyme active site, consistent with the recently elucidated stereochemistry of the cisoid products. Further, computational studies using density functional theory calculations reveal concerted, highly asynchronous cyclization pathways leading to the major cisoid cyclization products. The implications of these discoveries for expanded sesquiterpene diversity in nature are discussed