Young J. Hong

Consequences of conformational preorganization in sesquiterpene biosynthesis: theoretical studies on the formation of the bisabolene, curcumene, acoradiene, zizaene, cedrene, duprezianene, and sesquithuriferol sesquiterpenes.

Quantum chemical calculations on cyclization mechanisms for several sesquiterpene families proposed to be closely related to each other in a biogenic sense (the bisabolene, curcumene, acoradiene, zizaene (zizaene, isozizaene, epi-zizaene, and epi-isozizaene), cedrene (alpha/beta-cedrenes and 7-epi-alpha/beta-cedrenes), duprezianene, and sesquithuriferol families) are described. On the basis of the results of these calculations, we suggest that the conformation of the bisabolyl cation attainable in an enzyme active site is a primary determinant of the structure and relative stereochemistry of the sesquiterpenes formed. We also suggest that substantial conformational changes of initially formed conformers of the bisabolyl cation are necessary in order to form zizaene and epi-cedrene. Given that the productive conformation of the bisabolyl cation does not necessarily reflect the original orientation of farnesyl diphosphate bound in the corresponding enzyme active site, we conclude that folding of farnesyl diphosphate alone does not always dictate the structure and relative stereochemistry of cyclization products. In addition, the potential roles of dynamic matching in determining product distributions and enzyme-promoted formation of secondary carbocations are discussed.

Biosynthetic consequences of multiple sequential post-transition-state bifurcations

Selectivity in chemical reactions that form complex molecular architectures from simpler precursors is usually rationalized by comparing competing transition-state structures that lead to different possible products. Herein we describe a system for which a single transition-state structure leads to the formation of many isomeric products via pathways that feature multiple sequential bifurcations. The reaction network described connects the pimar-15-en-8-yl cation to miltiradiene, a tricyclic diterpene natural product, and isomers via cyclizations and/or rearrangements. The results suggest that the selectivity of the reaction is controlled by (post-transition-state) dynamic effects, that is, how the carbocation structure changes in response to the distribution of energy in its vibrational modes. The inherent dynamical effects revealed herein (characterized through quasiclassical direct dynamics calculations using density functional theory) have implications not only for the general principles of selectivity prediction in systems with complex potential energy surfaces, but also for the mechanisms of terpene synthase enzymes and their evolution. These findings redefine the challenges faced by nature in controlling the biosynthesis of complex natural products.

A potential energy surface bifurcation in terpene biosynthesis

Terpenes comprise a class of natural products that includes molecules with thousands of distinct structurally and stereochemically complex molecular architectures. The core hydrocarbon frameworks of these molecules are constructed via carbocation rearrangements promoted by terpene synthase (cyclase) enzymes. Although many mechanistic details for such reactions have been uncovered, the factors that control which carbocation intermediates and transition-state structures form are not well understood. Here we show that rearrangement pathways that pass through particular transition-state structures can bifurcate after the transition state. The resulting pathways lead to terpenes with distinctly different skeletons from each other. Although these types of bifurcating pathways have been described previously for some small molecules, the possibility that they may have an important role in the production of complex molecules in nature has, to our knowledge, not previously been considered.

Formation of the Unusual Semivolatile Diterpene Rhizathalene by the Arabidopsis Class I Terpene Synthase TPS08 in the Root Stele Is Involved in Defense against Belowground Herbivory

This work reports that Arabidopsis roots produce at least four related semivolatile diterpenes named rhizathalenes that have not been previously identified in this or any other plant species. It shows that rhizathalenes act as antifeedants against root-feeding insects. Secondary metabolites are major constituents of plant defense against herbivore attack. Relatively little is known about the cell type–specific formation and antiherbivore activities of secondary compounds in roots despite the substantial impact of root herbivory on plant performance and fitness. Here, we describe the constitutive formation of semivolatile diterpenes called rhizathalenes by the class I terpene synthase (TPS) 08 in roots of Arabidopsis thaliana. The primary enzymatic product of TPS08, rhizathalene A, which is produced from the substrate all-trans geranylgeranyl diphosphate, represents a so far unidentified class of tricyclic diterpene carbon skeletons with an unusual tricyclic spiro-hydrindane structure. Protein targeting and administration of stable isotope precursors indicate that rhizathalenes are biosynthesized in root leucoplasts. TPS08 expression is largely localized to the root stele, suggesting a centric and gradual release of its diterpene products into the peripheral root cell layers. We demonstrate that roots of Arabidopsis tps08 mutant plants, grown aeroponically and in potting substrate, are more susceptible to herbivory by the opportunistic root herbivore fungus gnat (Bradysia spp) and suffer substantial removal of peripheral tissue at larval feeding sites. Our work provides evidence for the in vivo role of semivolatile diterpene metabolites as local antifeedants in belowground direct defense against root-feeding insects.

Formation of Beyerene, Kaurene, Trachylobane, and Atiserene Diterpenes by Rearrangements That Avoid Secondary Carbocations

Quantum chemical calculations on carbocation intermediates encountered during the conversion of ent-copalyl diphosphate to the diterpenes beyerene, kaurene, trachylobane, and atiserene are described. Based on the results of these computations, it is suggested that previously proposed secondary carbocation intermediates are avoided. In some cases, complex rearrangements in which up to three alkyl or hydride shifting events are coupled into concerted processes are predicted to occur instead. The potential effects of electron-rich active site groups on the inherent reactivity of key carbocations are discussed, as are complex rearrangements coupled to deprotonation events. Based on computed electrostatic potential maps, it also is proposed that ammonium ions that were previously designed as mimics of several carbocations are actually better mimics of transition state structures for carbocation deprotonation.

Quantum chemical dissection of the classic terpinyl/pinyl/bornyl/camphyl cation conundrum—the role of pyrophosphate in manipulating pathways to monoterpenes

Based on quantum chemical studies, mechanisms to form bornyl diphosphate from geranyl diphosphate are suggested. While bornyl diphosphate is usually proposed to be generated via combination of the pyrophosphate group with a secondary bornyl cation, quantum chemical computations indicate that the bornyl cation is actually not a minimum. Instead, concerted attack of the pyrophosphate group coupled with an alkyl shift could yield bornyl diphosphate from either the pinyl cation or the camphyl cation. Hints of bifurcating pathways on the energy surfaces for such reactions were also uncovered. Of particular note is the development and validation of a large model of the pyrophosphate counterion treated entirely with quantum chemistry.

A Cytochrome P450 Serves as an Unexpected Terpene Cyclase during Fungal Meroterpenoid Biosynthesis

Viridicatumtoxin (1) is a tetracycline-like fungal meroterpenoid with a unique, fused spirobicyclic ring system. Puzzlingly, no dedicated terpene cyclase is found in the gene cluster identified in Penicillium aethiopicum. Cytochrome P450 enzymes VrtE and VrtK in the vrt gene cluster were shown to catalyze C5-hydroxylation and spirobicyclic ring formation, respectively. Feeding acyclic previridicatumtoxin to Saccharomyces cerevisiae expressing VrtK confirmed that VrtK is the sole enzyme required for cyclizing the geranyl moiety. Thus, VrtK is the first example of a P450 that can catalyze terpene cyclization, most likely via initial oxidation of C17 to an allylic carbocation. Quantum chemical modeling revealed a possible new tertiary carbocation intermediate E that forms after allylic carbocation formation. Intermediate E can readily undergo concerted 1,2-alkyl shift/1,3-hydride shift, either spontaneously or further aided by VrtK, followed by C7 Friedel-Crafts alkylation to afford 1. The most likely stereochemical course of the reaction was proposed on the basis of the results of our computations.

Branching Out from the Bisabolyl Cation. Unifying Mechanistic Pathways to Barbatene, Bazzanene, Chamigrene, Chamipinene, Cumacrene, Cuprenene, Dunniene, Isobazzanene, Iso-γ-bisabolene, Isochamigrene, Laurene, Microbiotene, Sesquithujene, Sesquisabinene, Thujopsene, Trichodiene, and Widdradiene Sesquiterpenes

Quantum chemical calculations on the transformation of the bisabolyl cation into an array of sesquiterpenes (iso-γ-bisabolene, trichodiene, cuprenene, laurene, isochamigrene, chamigrene, chamipinene, sesquithujene, sesquisabinene, microbiotene, dunniene, cumacrene, isobazzanene, bazzanene, barbatene, widdradiene, and thujopsene) are described. The bisabolyl cation is the hub of a complicated web of carbocations involved in the construction of diverse and complex molecular architectures present in a large number of Natures sesquiterpenoids. The results of quantum chemical calculations on the multitude of rearrangements described herein provide reasonable answers to several persistent mechanistic questions in the world of terpene biosynthesis and also provide examples of general reactivity principles for terpene-forming (and other) carbocation rearrangements.

How cyclobutanes are assembled in nature--insights from quantum chemistry.

Biosynthetic production of cyclobutanes leads to many complex natural products. Recently, theoretical work employing quantum chemical calculations has shed light on many of the details of cyclobutane-formation, in particular, for terpene natural products. Specific insights and general principles derived from these theoretical studies are described herein.

How Many Secondary Carbocations Are Involved in the Biosynthesis of Avermitilol

Quantum chemical calculations were used to assess the viability of proposed secondary carbocations as intermediates in the biosynthesis of avermitilol. One, a cyclopropylcarbinyl cation, was found to be a true minimum, while another, a simple secondary cation, was found to exist only as part of a transition structure for water capture.