On the Mechanism of Au-Catalyzed Enynamide-yne Dehydro-Diels-Alder Reactions: An Experimental and Computational Study.

Gold-catalyzed dehydro-Diels-Alder reactions of ynamide derivatives allow efficient access to a variety of N-containing aromatic heterocycles. A dual gold catalysis mechanism was postulated for transformations involving the formation of C-C bonds by reaction between a terminal alkyne and an enynamide fragment. In this article, complete experimental and computational investigations into the mechanism of such a transformation are reported. Support for a dual gold catalysis was found and it was shown that the concerted or stepwise nature of the cyclization event depends on the substitution of the ynamide moiety. The reaction was found to proceed in three stages: 1) formation of a σ,π-digold complex from the terminal alkyne, 2) cyclization to produce a gem-diaurated aryl complex, and 3) catalyst transfer to free the product and regenerate the σ,π-digold complex.

Diterpene Synthase-Catalyzed Biosynthesis of Distinct Clerodane Stereoisomers.

The diterpene synthase clerodienyl diphosphate synthase 1 (PvCPS1) from the crop plant switchgrass (Panicum virgatum) stereoselectively converts (E,E,E)-geranylgeranyl diphosphate (GGPP) into the clerodane natural product, cis-trans-clerodienyl diphosphate (CLPP, 1). Structure-guided point mutations of PvCPS1 redirected product stereoselectivity toward the formation of a rare cis-clerodane diastereomer, cis-cis-CLPP (2). Additionally, an alternative cis-clerodane diastereomer, (5S,8S,9R,10R)-13Z-CLPP (3), was produced when treating PvCPS1 and select variants thereof with the cis-prenyl substrate (Z,Z,Z)-nerylneryl diphosphate (NNPP). These results support the hypothesis that substrate configuration and minor active-site alterations impact precatalysis substrate folding in the stereoselective biosynthesis of clerodane diterpenoid scaffolds, and can be employed to provide enzymatic access to a broader range of bioactive clerodane natural products.

A Maze of Dyotropic Rearrangements and Triple Shifts: Carbocation Rearrangements Connecting Stemarene, Stemodene, Betaerdene, Aphidicolene, and Scopadulanol.

Results of quantum chemical investigations shed new light on the mechanisms of formation of the stemarene, stemodene, betaerdene, aphidicolene, and scopadulanol diterpenes from syn-copalyl diphosphate ( syn-CPP). These terpenes are shown to be connected by a complex network of reaction pathways involving concerted but asynchronous dyotropic rearrangements and triple shift rearrangements. The interconnection of these pathways leads to multiple routes for formation of each diterpene, which could lead to different origins for some carbon atoms in a given diterpenes under different conditions.

Premutilin Synthase: Ring Rearrangement by a Class II Diterpene Cyclase.

Biosynthesis of the complex diterpenoid antibiotic pleuromutilin relies on a bifunctional (di)terpene synthase, and here site-directed mutagenesis was used to knockout either of the two active sites. This enabled characterization of the novel ring contracted intermediate produced by the initiating class II diterpene cyclase active site. Quantum chemical calculations further indicate the importance of reactant configuration for this intriguing ring rearrangement.

Diverged Plant Terpene Synthases Reroute the Carbocation Cyclization Path towards the Formation of Unprecedented 6/11/5 and 6/6/7/5 Sesterterpene Scaffolds.

Sesterterpenoids are a relatively rare class of plant terpenes. Sesterterpene synthase (STS)-mediated cyclization of the linear C25 isoprenoid precursor geranylfarnesyl diphosphate (GFPP) defines sesterterpene scaffolds. So far only a very limited number of STSs have been characterized. The discovery of three new plant STSs is reported that produce a suite of sesterterpenes with unprecedented 6/11/5 and 6/6/7/5 fused ring systems when transiently co-expressed with a GFPP synthase in Nicotiana benthamiana. Structural elucidation, feeding experiments, and quantum chemical calculations suggest that these STSs catalyze an unusual cyclization path involving reprotonation, intramolecular 1,6 proton transfer, and concerted but asynchronous bicyclization events. The cyclization is diverted from those catalyzed by the characterized plant STSs by forming unified 15/5 bicyclic sesterterpene intermediates. Mutagenesis further revealed a conserved amino acid residue implicated in reprotonation.

Synthesis and Structure Revision of Dichrocephones†A and B.

Herein, we report the first enantioselective synthesis of dichrocephones A and B, which are cytotoxic triquinane sesquiterpenes with a dense array of stereogenic centers within a strained polycyclic environment. Key features include the application of a catalytic asymmetric Wittig reaction, followed by stereoselective functionalization of the propellane core into a pentacyclic intermediate. Double reductive ring cleavage yielded the proposed structure of dichrocephone A. Mismatched spectroscopic data for our synthetic material compared to the natural isolate led us to revise the previously proposed configuration based on biosynthetic considerations and NMR calculations. Implementation of these findings culminated in the synthesis of dichrocephones A and B.

Intramolecular Chirality Transfer [2 + 2] Cycloadditions of Allenoates and Alkenes.

Intramolecular chirality transfer [2 + 2] cycloaddition of enantiomerically enriched allenoates and alkenes is presented. The use of a chiral catalyst was found to be critical to achieve high levels of diastereoselectivity compared to use of an achiral catalyst. The method developed leads to highly substituted cyclobutanes that would be difficult to prepare by alternative methods.

Unearthing a sesterterpene biosynthetic repertoire in the Brassicaceae through genome mining reveals convergent evolution.

Sesterterpenoids are a rare terpene class harboring untapped chemodiversity and bioactivities. Their structural diversity originates primarily from the scaffold-generating sesterterpene synthases (STSs). In fungi, all six known STSs are bifunctional, containing C-terminal trans-prenyltransferase (PT) and N-terminal terpene synthase (TPS) domains. In plants, two colocalized PT and TPS gene pairs from Arabidopsis thaliana were recently reported to synthesize sesterterpenes. However, the landscape of PT and TPS genes in plant genomes is unclear. Here, using a customized algorithm for systematically searching plant genomes, we reveal a suite of physically colocalized pairs of PT and TPS genes for the biosynthesis of a large sesterterpene repertoire in the wider Brassicaceae. Transient expression of seven TPSs from A. thaliana, Capsella rubella, and Brassica oleracea in Nicotiana benthamiana yielded fungal-type sesterterpenes with tri-, tetra-, and pentacyclic scaffolds, and notably (-)-ent-quiannulatene, an enantiomer of the fungal metabolite (+)-quiannulatene. Protein and structural modeling analysis identified an amino acid site implicated in structural diversification. Mutation of this site in one STS (AtTPS19) resulted in premature termination of carbocation intermediates and accumulation of bi-, tri-, and tetracyclic sesterterpenes, revealing the cyclization path for the pentacyclic sesterterpene (-)-retigeranin B. These structural and mechanistic insights, together with phylogenetic analysis, suggest convergent evolution of plant and fungal STSs, and also indicate that the colocalized PT-TPS gene pairs in the Brassicaceae may have originated from a common ancestral gene pair present before speciation. Our findings further provide opportunities for rapid discovery and production of sesterterpenes through metabolic and protein engineering.

Is a 1,4-Alkyl Shift Involved in the Biosynthesis of Ledol and Viridiflorol?

Results from density functional theory computations indicate that a 1,4-alkyl shift proposed as part of the carbocation cyclization/rearrangement leading to ledol, viridiflorol, and related sesquiterpenes is not energetically viable. Instead, a previously proposed mechanism that avoids such a shift is greatly preferred.

Bioinspired synthesis of pentacyclic onocerane triterpenoids.

The first chemical synthesis of pentacyclic onocerane triterpenoids has been achieved. A putative biomimetic tricyclization cascade is employed to forge a fused decalin-/oxepane ring system. The synthetic route proceeds to (+)-cupacinoxepin in seven steps and to (+)-onoceranoxide in eight steps in the longest linear sequence, when starting from geranyl chloride and (+)-sclareolide. The bioinspired epoxypolyene cyclization is supported by computational and enzymatic studies.

Product Rearrangement from Altering a Single Residue in the Rice syn-Copalyl Diphosphate Synthase.

Through site-directed mutagenesis targeted at identification of the catalytic base in the rice (Oryza sativa) syn-copalyl diphosphate synthase OsCPS4, changes to a single residue (H501) were found to induce rearrangement rather than immediate deprotonation of the initially formed bicycle, leading to production of the novel compound syn-halimadienyl diphosphate. These mutational results are combined with quantum chemical calculations to provide insight into the underlying reaction mechanism.

Blocking Deprotonation with Retention of Aromaticity in a Plant ent-Copalyl Diphosphate Synthase Leads to Product Rearrangement.

Substitution of a histidine, comprising part of the catalytic base group in the ent-copalyl diphosphate synthases found in all seed plants for gibberellin phytohormone metabolism, by a larger aromatic residue leads to rearrangements. Through a series of 1,2-hydride and methyl shifts of the initially formed bicycle predominant formation of (-)-kolavenyl diphosphate is observed. Further mutational analysis and quantum chemical calculations provide mechanistic insight into the basis for this profound effect on product outcome.

Correction: The energetic viability of an unexpected skeletal rearrangement in cyclooctatin biosynthesis.

Correction for 'The energetic viability of an unexpected skeletal rearrangement in cyclooctatin biosynthesis' by Young J. Hong and Dean J. Tantillo, Org. Biomol. Chem., 2015, 13, 10273-10278.

Tension between Internal and External Modes of Stabilization in Carbocations Relevant to Terpene Biosynthesis: Modulating Minima Depth via C-H···π Interactions.

Density functional theory calculations on a carbocation rearrangement relevant to the biosynthesis of the sesquiterpenoid trefolane A are described, with a focus on the viability of altering the curvature of the potential energy surface through C-H···π interactions of the sort likely to be found at the active site of a terpene synthase enzyme. These interactions are able to remove a deep minimum from a pathway to product.

The energetic viability of an unexpected skeletal rearrangement in cyclooctatin biosynthesis.

Results of density functional theory calculations on possible mechanisms for formation of the diterpenoid cyclooctatin are described. These results are consistent with the involvement of an unexpected 1,3-alkyl shift that interconverts two cyclopropylcarbinyl carbocations and interchanges the positions of two carbon atoms in an 8-membered ring. Predictions for future experiments to provide further support of this mechanism also are described.

Generation mechanism of hydroxyl radical species and its lifetime prediction during the plasma-initiated ultraviolet (UV) photolysis.

Through this work, we have elucidated the mechanism of hydroxyl radicals (OH(•)) generation and its life time measurements in biosolution. We observed that plasma-initiated ultraviolet (UV) photolysis were responsible for the continues generation of OH(•) species, that resulted in OH(•) to be major reactive species (RS) in the solution. The density and lifetime of OH(•) species acted inversely proportional to each other with increasing depth inside the solution. The cause of increased lifetime of OH(•) inside the solution is predicted using theoretical and semiempirical calculations. Further, to predict the mechanism of conversion of hydroxide ion (OH(-)) to OH(•) or H2O2 (hydrogen peroxide) and electron, we determined the current inside the solution of different pH. Additionally, we have investigated the critical criterion for OH(•) interaction on cancer cell inducing apoptosis under effective OH(•) exposure time. These studies are innovative in the field of plasma chemistry and medicine.

Feasibility of intramolecular proton transfers in terpene biosynthesis--guiding principles.

On the basis of results from quantum chemical calculations, the feasibility of an extensive series of intramolecular proton-transfer reactions postulated to occur during terpene biosynthesis is assessed and guiding principles are proposed.

Bicyclobutonium ions in biosynthesis--interconversion of cyclopropyl-containing sterols from orchids.

Theoretical investigation of cyclopropane-to-cyclopropane rearrangements of sterols indicates a role for highly delocalized bicyclobutonium ions in biosynthesis.

5-Nitro-5'hydroxy-indirubin-3'oxime is a novel inducer of somatic cell transdifferentiation.

Patient-derived cell transplantation is an attractive therapy for regenerative medicine. However, this requires effective strategies to reliably differentiate patient cells into clinically useful cell types. Herein, we report the discovery that 5-nitro-5'hydroxy-indirubin-3'oxime (5'-HNIO) is a novel inducer of cell transdifferentiation. 5'-HNIO induced muscle transdifferentiation into adipogenic and osteogenic cells. 5'-HNIO was shown to inhibit aurora kinase A, which is a known cell fate regulator. 5'-HNIO produced a favorable level of transdifferentiation compared to other aurora kinase inhibitors and induced transdifferentiation across cell lineage boundaries. Significantly, 5'-HNIO treatment produced direct transdifferentiation without up-regulating potentially oncogenic induced pluripotent stem cell (iPSC) reprogramming factors. Thus, our results demonstrate that 5'-HNIO is an attractive molecular tool for cell transdifferentiation and cell fate research.

Biosynthesis of lycosantalonol, a cis-prenyl derived diterpenoid.

Terpenoid natural products are generally derived from isoprenyl diphosphate precursors with trans double-bond configuration, and no diterpenoid derived from the cisoid precursor (Z,Z,Z)-nerylneryl diphosphate (1) has yet been identified. Here further investigation of a terpenoid biosynthetic gene cluster from tomato is reported, which resulted in identification of a biosynthetic pathway from 1, in a pathway featuring a number of interesting transformations. Compound 1 is first cyclized to a tricyclene core ring structure analogous to that found in α-santalene, with the resulting diterpene termed here lycosantalene (2). Quantum chemical calculations indicate a role for the diphosphate anion coproduct in this cyclization reaction. Subsequently, the internal cis double bond of the neryl side chain in 2 is then further transformed to an α-hydroxy ketone moiety via an epoxide intermediate (3). Oxygen labeling studies indicate 3 undergoes oxidative conversion to lycosantalonol (4). Thus, in addition to elucidating the cisoid origins of 4, this work has further provided mechanistic insight into the interesting transformations required for its production.