16729-01-4

Despite its distance from the active site the flexible amino-terminal segment (NTS) in the β-domain of the plant sesquiterpene cyclase δ-cadinene synthase (DCS) is essential for active site closure and desolvation events during catalysis.

Recombinant (+)-δ-cadinene synthase (DCS) from Gossypium arboreum catalyzes the metal-dependent cyclization of (E,E)-farnesyl diphosphate (FDP) to the cadinane sesquiterpene δ-cadinene, the parent hydrocarbon of cotton phytoalexins such as gossypol. In contrast to some other sesquiterpene cyclases, DCS carries out this transformation with >98% fidelity but, as a consequence, leaves no mechanistic traces of its mode of action. The formation of (+)-δ-cadinene has been shown to occur via the enzyme-bound intermediate (3R)-nerolidyl diphosphate (NDP), which in turn has been postulated to be converted to cis-germacradienyl cation after a 1,10-cyclization. A subsequent 1,3-hydride shift would then relocate the carbocation within the transient macrocycle to expedite a second cyclization that yields the cadinenyl cation with the correct cis stereochemistry found in (+)-δ-cadinene. An elegant 1,10-mechanistic pathway that avoids the formation of (3R)-NDP has also been suggested. In this alternative scenario, the final cadinenyl cation is proposed to be formed through the intermediacy of trans, trans-germacradienyl cation and germacrene D. In addition, an alternative 1,6-ring closure mechanism via the bisabolyl cation has previously been envisioned. We report here a detailed investigation of the catalytic mechanism of DCS using a variety of mechanistic probes including, among others, deuterated and fluorinated FDPs. Farnesyl diphosphate analogues with fluorine at C2 and C10 acted as inhibitors of DCS, but intriguingly, after prolonged overnight incubations, they yielded 2F-germacrene(s) and a 10F-humulene, respectively. The observed 1,10-, and to a lesser extent, 1,11-cyclization activity of DCS with these fluorinated substrates is consistent with the postulated macrocyclization mechanism(s) en route to (+)-δ-cadinene. On the other hand, mechanistic results from incubations of DCS with 6F-FPP, (2Z,6E)-FDP, neryl diphosphate, 6,7-dihydro-FDP, and NDP seem to be in better agreement with the potential involvement of the alternative biosynthetic 1,6-ring closure pathway. In particular, the strong inhibition of DCS by 6F-FDP, coupled to the exclusive bisabolyl- and terpinyl-derived product profiles observed for the DCS-catalyzed turnover of (2Z,6E)-farnesyl and neryl diphosphates, suggested the intermediacy of α-bisabolyl cation. DCS incubations with enantiomerically pure [1- 2H1](1R)-FDP revealed that the putative bisabolyl-derived 1,6-pathway proceeds through (3R)-nerolidyl diphosphate (NDP), is consistent with previous deuterium-labeling studies, and accounts for the cis stereochemistry characteristic of cadinenyl-derived sesquiterpenes. While the results reported here do not unambiguously rule in favor of 1,6- or 1,10-cyclization, they demonstrate the mechanistic versatility inherent to DCS and highlight the possible existence of multiple mechanistic pathways.

Sesquiterpene synthases catalyze with different catalytic fidelity the cyclization of farnesyl pyrophosphate (FPP) into hundreds of known compounds with diverse structures and stereochemistries. Two sesquiterpene synthases, Cop4 and Cop6, were previously isolated from Coprinus cinereus as part of a fungal genome survey. This study investigates the reaction mechanism and catalytic fidelity of the two enzymes. Cyclization of all-trans-FPP ((E,E)-FPP) was compared to the cyclization of the cis-trans isomer of FPP ((Z,E)-FPP) as a surrogate for the secondary cisoid neryl cation intermediate generated by sesquiterpene synthases, which are capable of isomerizing the C2-C3 π bond of all-trans-FPP. Cop6 is a "high-fidelity" α-cuprenene synthase that retains its fidelity under various conditions tested. Cop4 is a catalytically promiscuous enzyme that cyclizes (E,E)-FPP into multiple products, including (-)-germacrene D and cubebol. Changing the pH of the reaction drastically alters the fidelity of Cop4 and makes it a highly selective enzyme. Cyclization of (Z,E)-FPP by Cop4 and Cop6 yields products that are very different from those obtained with (E,E)-FPP. Conversion of (E,E)-FPP proceeds via a (6R)-β-bisabolyl carbocation in the case of Cop6 and an (E,E)-germacradienyl carbocation in the case of Cop4. However, (Z,E)-FPP is cyclized via a (6S)-β-bisabolene carbocation by both enzymes. Structural modeling suggests that differences in the active site and the loop that covers the active site of the two enzymes might explain their different catalytic fidelities.

Terpene synthases are responsible for a large diversity of terpene carbon skeletons found in nature. The multiproduct sesquiterpene synthase MtTPS5 isolated from Medicago truncatula produces 27 products from farnesyl diphosphate (1, FDP). In this paper, we report the reaction steps involved in the formation of these products using incubation experiments with deuterium-containing substrates; we determined the absolute configuration of individual products to establish the stereochemical course of the reaction cascade and the initial conformation of the cycling substrate. Additional labeling experiments conducted with deuterium oxide showed that cadalane sesquiterpenes are mainly produced via the protonation of the neutral intermediate germacrene D (5). These findings provide an alternative route to the general accepted pathway via nerolidyl diphosphate (2, NDP) en route to sesquiterpenes with a cadalane skeleton. Mutational analysis of the enzyme demonstrated that a tyrosine residue is important for the protonation process.

[3H](+)-δ-Cadinene and its 8-hydroxy derivative, prepared from (1RS)-[1-3H]FPP by the action of one and two recombinant enzymes, respectively, were infiltrated into cotyledons of bacterial blight-resistant cotton plants as they biosynthesized sesquiterpene phytoalexins in response to infection by Xanthomonas campestris pv. malvacearum. Following both treatments, tritium appeared in the HPLC fraction that contained hemigossypol. Hemigossypol was isolated from the cotyledons that had been treated with [3H](+)-8-hydroxy-δ-cadinene and was trimethylsilylated and purified. In two experiments, specific radioactivity of the hemigossypol derivative indicated that 5% and 10%, respectively, of the [3H](+)-8-hydroxy-δ-cadinene had been converted to hemigossypol.

Germacrene D is considered as a precursor of many sesquiterpene hydrocarbons. We have investigated the acid catalyzed as well as the photochemically and thermally induced rearrangement processes of germacrene D isolated from several Solidago species, which contain both enantiomers of germacrene D. Enantiomeric mixtures of sesquiterpenes of the cadinane, eudesmane (selinane), oppositane, axane, isodaucane, and bourbonane group as well as isogermacrene D were identified as main products and made available as reference compounds for structure investigations and stereochemical assignments of plant constituents. δ-Amorphene, one of the rearrangement products, was identified as a natural product for the first time. The absolute configuration of γ-amorphene was revised by correlation with the absolute configuration of germacrene D. The mechanisms of the rearrangement reactions are discussed. (C) 2000 Elsevier Science Ltd.

Conversion of the previously reported, carvacrol-based 4(S*)-isopropyl-7(R*)-chlorobicyclo[3.1.1]heptan-6-one and its bromo equivalent into (±)-α- and (±)-β-copaene it described. Model 5-nor-β-copaene was synthesized in the following manner, (a) γ-(trimet

Tricyclic compounds were obtained from α-murolene and α-copaene in superacidic media, and this agrees with the scheme for the biogenetic transformations of these substances.It was found that the temperature at which the stable cations are generated from α-murolene has an effect on the ratio of the paths leading to the formation of the bi- and tricyclic ions.The prediction of the most probable paths for the transformation of α-copaene agrees with the actually observed transformation of the stable ions generated from this substance.The preferred direction in the ske letal rearrangements of the investigated compounds depends on the nature of the acid medium.

This paper reports on a total synthesis of racemic δ-cadinene (2), which had been obtained previously in optically active form by acid catalyzed cyclization of (-)-germacene D.The Robinson annelation using cyclohexenone enamine (12) proceeded stereoselectively to form δ-cadinenone (3), whose oxygen was removed by the thioketal-Raney Ni Method to produce δ-cadinene.

Epicubebol has been isolated from the methanol extracts of the brown alga Dictyopteris divaricata as the major constituent.Cadinane-type sesquiterpenes, cubebenes, δ-cadinene, cubenol, and epicubenol, as well as two sesquiterpene methyl ethers as minor constituents have also been obtained from the extracts.

E,E- and 2Z,6E-hedycaryol phenyl sulfides were converted by sigmatropy of their sulfoxides to the same allyl alcohol which in turn afforded cadinane derivatives stereospecifically by the action of acids.

From the oleoresin of the Yeddo spruce we have isolated a new sesquiterpene alcohol for which, on the basis of spectral and chemical chracteristics, the structure of 5S,8S-germacra-1E,6E-dien-5-ol (I) is suggested.The photocyclization of the alcohol isolated has given bourbonol, and its reaction with formic acid has yielded γ- and δ-cadinenes, the formates of T-muurolol and of T- and α-cadinols, and free T-muurolol and T- and α-cadinols