Metal free regio - and stereoselective semireduction of CF3-substituted N-allenamides.

We developed a chemoselective metal-free access for the 1,2- and 2,3-semireduction of CF3-N-allenamides. The enamide functionality of CF3-substituted N-allenamides could be efficiently reduced by Et3SiH/BF3·OEt2 in total regioselectivity and good stereoselectivity, whereas DBU promoted the isomerization of the resulting allyl amide leading exclusively to the E-configurated enamide.

Evolutionary metabolomics of specialized metabolism diversification in the genus Nicotiana highlights N-acylnornicotine innovations.

Specialized metabolite (SM) diversification is a core process to plants' adaptation to diverse ecological niches. Here, we implemented a computational mass spectrometry-based metabolomics approach to exploring SM diversification in tissues of 20 species covering Nicotiana phylogenetics sections. To markedly increase metabolite annotation, we created a large in silico fragmentation database, comprising >1 million structures, and scripts for connecting class prediction to consensus substructures. Together, the approach provides an unprecedented cartography of SM diversity and section-specific innovations in this genus. As a case study and in combination with nuclear magnetic resonance and mass spectrometry imaging, we explored the distribution of N-acylnornicotines, alkaloids predicted to be specific to Repandae allopolyploids, and revealed their prevalence in the genus, albeit at much lower magnitude, as well as a greater structural diversity than previously thought. Together, the data integration approaches provided here should act as a resource for future research in plant SM evolution.

Silver-Catalyzed Domino Reaction of CF3-Substituted N-Allenamides with Primary Amines for the Construction of 2-Amido-5-fluoropyrroles.

We report herein a domino reaction to construct 2-amido-5-fluoropyrroles from CF3-substituted N-allenamides. The in situ generated gem-difluorinated ene-ynamides derived from CF3-substituted N-allenamides, when subjected to silver catalysis with a primary amine, undergo simultaneous hydroamination of the ynamide moiety followed by a 5-endo-trig addition/ß-fluoride elimination sequence, enabling the construction of 2-amido-5-fluoropyrroles. This transformation features excellent functional group compatibility. By employing 2-aminophenols, functionalized benzo-oxazoles were produced.

Construction of C-N and C-O bonds based on N-allenamide functionalization.

The two-faced reactivity of N-allenamides allows regio- and stereo-controlled functionalization at the α-, ß- and γ-positions of the nitrogen atom. The contingency to obtain either proximal or distal adducts makes these substrates essential for the construction of complex heterocyclic scaffolds. This review covers the recent advances made in the development of new methods involving amination and alkoxylation reactions on N-allenamides leading to a broad array of N-heterocycles. The syntheses reported herein are classified based on their reaction type. In addition, mechanistic perceptions are provided for the majority of the discussed transformations.

Noncanonical Strigolactone Analogues Highlight Selectivity for Stimulating Germination in Two Phelipanche ramosa Populations.

Strigolactones (SLs) are plant hormones exuded in the rhizosphere with a signaling role for the development of arbuscular mycorrhizal (AM) fungi and as stimulants of seed germination of the parasitic weeds Orobanche, Phelipanche, and Striga, the most threatening weeds of major crops worldwide. Phelipanche ramosa is present mainly on rape, hemp, and tobacco in France. P. ramosa 2a preferentially attacks hemp, while P. ramosa 1 attacks rapeseed. The recently isolated cannalactone (14) from hemp root exudates has been characterized as a noncanonical SL that selectively stimulates the germination of P. ramosa 2a seeds in comparison with P. ramosa 1. In the present work, (-)-solanacol (5), a canonical orobanchol-type SL exuded by tobacco and tomato, was established to possess a remarkable selective germination stimulant activity for P. ramosa 2a seeds. Two cannalactone analogues, named (±)-SdL19 and (±)-SdL118, have been synthesized. They have an unsaturated acyclic carbon chain with a tertiary hydroxy group and a methyl or a cyclopropyl group instead of a cyclohexane A-ring, respectively. (±)-SdL analogues are able to selectively stimulate P. ramosa 2a, revealing that these minimal structural elements are key for this selective bioactivity. In addition, (±)-SdL19 is able to inhibit shoot branching in Pisum sativum and Arabidopsis thaliana and induces hyphal branching in the AM fungus Rhizophagus irregularis, like SLs.

Synthesis of Non-natural Aza-Iridoids via Ynamides and Molecular Networking-Based Tracing of Their In Planta Bioconversion.

A synthesis of new-to-nature aza-iridoids via ynamides is presented. ZrCl4 proved to be the best acid to perform this transformation. Various ynamides were accommodated, and seco-iridoids could be obtained as well. Aza-iridoids were infiltrated into leaves of Scrophularia Nodosa, an iridoid-producing plant species. High-resolution mass spectrometry coupled to computational metabolomic approaches was employed for the detection of aza-iridoid bioconversion products.

Regio- and Stereoselective Addition to gem-Difluorinated Ene-Ynamides: Access to Stereodefined Fluorinated Dienes.

The first synthesis of gem-difluorinated ene-ynamides is presented via deprotonation of trifluoromethylated N-allenamides and δ extrusion of fluorine. These highly reactive building blocks, owing to their dual functional groups, offer a unique entry to difluorinated dienes and to stereodefined, monofluoro-substituted dienes. Stereoselective addition to the ynamide moiety led to difluorinated dienes. A stereocontrolled domino δ elimination reaction followed by an addition/elimination sequence from trifluoromethylated N-allenamides provided exclusively stereodefined monofluorinated ene-ynamides.

One-Pot anti-Michael Regio- and Stereoselective Hydroamination of Activated N-Allenamides.

N-Allenamides, substituted by an ester at the γ-position, were obtained through addition of terminal ynamides with ethyl diazoacetate under copper catalysis for the first time. Regio- and stereoselective hydroamination of those activated N-allenamides provided exclusively E-configured captodative enamimes through a one-pot anti-Michael addition. Numerous ynamides as well as various secondary amines were adapted in this process.

Copper-Catalyzed Synthesis of Terminal vs. Fluorine-Substituted N-Allenamides via Addition of Diazo Compounds to Terminal Ynamides.

A copper-mediated coupling reaction between ynamides and diazo-compounds to produce N-allenamides is reported for the first time. This method enables facile and rapid access to terminal N-allenamides by using commercially available TMS-diazomethane with wide functional group compatibility on the nitrogen. Furthermore, the ubiquity of molecules containing a fluorine moiety in medicine, in agricultural, and material science requires the continuous search of new building blocks, including this unique surrogate. The CuI/diazo protocol was successfully applied to the synthesis of fluorine-substituted N-allenamides. DFT calculations provided insights in the mechanism involved.

Direct Synthesis of CF2H-Substituted 2-Amidofurans via Copper-Catalyzed Addition of Difluorinated Diazoacetone to Ynamides.

The significance of molecules containing difluoromethyl groups is driven by their potential applications in pharmaceutical and agrochemical science. Methods for the incorporation of lightly fluorinated groups such as CF2H have been less well developed. Here we report the use of difluorinated diazoacetone as a practical reagent for the direct synthesis of CF2H-substituted 2-amidofurans through addition to ynamides. These newly designed difluorinated amidofurans were elaborated to create new nitrogen-containing frameworks that would be challenging to obtain otherwise.

Innate promiscuity of the CYP706 family of P450 enzymes provides a suitable context for the evolution of dinitroaniline resistance in weed.

Increased metabolism is one of the main causes for evolution of herbicide resistance in weeds, a major challenge for sustainable food production. The molecular drivers of this evolution are poorly understood. We tested here the hypothesis that a suitable context for the emergence of herbicide resistance could be provided by plant enzymes with high innate promiscuity with regard to their natural substrates. A selection of yeast-expressed plant cytochrome P450 enzymes with well documented narrow to broad promiscuity when metabolizing natural substrates was tested for herbicide metabolism competence. The positive candidate was assayed for capacity to confer herbicide tolerance in Arabidopsis thaliana. Our data demonstrate that Arabidopsis thaliana CYP706A3, with the most promiscuous activity on monoterpenes and sesquiterpenes for flower defence, can also oxidize plant microtubule assembly inhibitors, dinitroanilines. Ectopic overexpression of CYP706A3 confers dinitroaniline resistance. We show, in addition, that the capacity to metabolize dinitroanilines is shared by other members of the CYP706 family from plants as diverse as eucalyptus and cedar. Supported by three-dimensional (3D) modelling of CYP706A3, the properties of enzyme active site and substrate access channel are discussed together with the shared physicochemical properties of the natural and exogenous substrates to explain herbicide metabolism.

Tertiary Enamide-Promoted Diastereoselective Domino: N-Acyliminium Ion Trapping and Nazarov Cyclization.

N-Acyliminium ions generated from enamidyl vinyl ketones provided cyclopentenoid-fused diazepines diastereoselectively using BF3·Et2O in one pot through a domino N-acyliminium ion trapping/Nazarov reaction, simultaneously generating three new stereogenic centers. The particular structural design of the cross-conjugated dienone dictates the torquoselectivity observed in this polarized Nazarov reaction. Various N-bridgehead polycyclic scaffolds of putative pharmacological interest were obtained. Cyclic voltammetry was used to support the preferred reaction sequence within this domino reaction.

A Promiscuous CYP706A3 Reduces Terpene Volatile Emission from Arabidopsis Flowers, Affecting Florivores and the Floral Microbiome.

Flowers are essential but vulnerable plant organs, exposed to pollinators and florivores; however, flower chemical defenses are rarely investigated. We show here that two clustered terpene synthase and cytochrome P450 encoding genes (TPS11 and CYP706A3) on chromosome 5 of Arabidopsis (Arabidopsis thaliana) are tightly coexpressed in floral tissues, upon anthesis and during floral bud development. TPS11 was previously reported to generate a blend of sesquiterpenes. By heterologous coexpression of TPS11 and CYP706A3 in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana, we demonstrate that CYP706A3 is active on TPS11 products and also further oxidizes its own primary oxidation products. Analysis of headspace and soluble metabolites in cyp706a3 and 35S:CYP706A3 mutants indicate that CYP706A3-mediated metabolism largely suppresses sesquiterpene and most monoterpene emissions from opening flowers, and generates terpene oxides that are retained in floral tissues. In flower buds, the combined expression of TPS11 and CYP706A3 also suppresses volatile emissions and generates soluble sesquiterpene oxides. Florivory assays with the Brassicaceae specialist Plutella xylostella demonstrate that insect larvae avoid feeding on buds expressing CYP706A3 and accumulating terpene oxides. Composition of the floral microbiome appears also to be modulated by CYP706A3 expression. TPS11 and CYP706A3 simultaneously evolved within Brassicaceae and form the most versatile functional gene cluster described in higher plants so far.plantcell;31/12/2947/FX1F1fx1.

Characterization of Jasmonoyl-Isoleucine (JA-Ile) Hormonal Catabolic Pathways in Rice upon Wounding and Salt Stress.

BACKGROUND: Jasmonate (JA) signaling and functions have been established in rice development and response to a range of biotic or abiotic stress conditions. However, information on the molecular actors and mechanisms underlying turnover of the bioactive jasmonoyl-isoleucine (JA-Ile) is very limited in this plant species. RESULTS: Here we explored two gene families in rice in which some members were described previously in Arabidopsis to encode enzymes metabolizing JA-Ile hormone, namely cytochrome P450 of the CYP94 subfamily (CYP94, 20 members) and amidohydrolases (AH, 9 members). The CYP94D subclade, of unknown function, was most represented in the rice genome with about 10 genes. We used phylogeny and gene expression analysis to narrow the study to candidate members that could mediate JA-Ile catabolism upon leaf wounding used as mimic of insect chewing or seedling exposure to salt, two stresses triggering jasmonate metabolism and signaling. Both treatments induced specific transcriptional changes, along with accumulation of JA-Ile and a complex array of oxidized jasmonate catabolites, with some of these responses being abolished in the JASMONATE RESISTANT 1 (jar1) mutant. However, upon response to salt, a lower dependence on JAR1 was evidenced. Dynamics of CYP94B5, CYP94C2, CYP94C4 and AH7 transcripts matched best the accumulation of JA-Ile catabolites. To gain direct insight into JA-Ile metabolizing activities, recombinant expression of some selected genes was undertaken in yeast and bacteria. CYP94B5 was demonstrated to catalyze C12-hydroxylation of JA-Ile, whereas similarly to its Arabidopsis bi-functional homolog IAR3, AH8 performed cleavage of JA-Ile and auxin-alanine conjugates. CONCLUSIONS: Our data shed light on two rice gene families encoding enzymes related to hormone homeostasis. Expression data along with JA profiling and functional analysis identifies likely actors of JA-Ile catabolism in rice seedlings. This knowledge will now enable to better understand the metabolic fate of JA-Ile and engineer optimized JA signaling under stress conditions.

Metal-free synthesis of activated ynesulfonamides and tertiary enesulfonamides.

An operationally simple synthesis of activated ynesulfonamides and enesulfonamides is described. Ynesulfonamides can be obtained through reaction of sulfonylamides with activated bromoalkynes and Triton B in a short time at room temperature. Likewise, terminal alkynes react with sulfonylamides to provide enesulfonamides. Z/E enesulfonamides can be transformed exclusively into E enesulfonamides.

Tertiary Enamide-Triggered SEAr: Domino Allylation and Enamine-Type Addition.

Two unprecedented domino reactions are described, starting from ketospiro-enesulfonamides. By treatment with ZrCl4 and allylsilane, an intramolecular electrophilic aromatic substitution and subsequent allylation is observed. By treatment with TiCl4 and allylsilane, a double enamine-type reaction takes place, thus creating simultaneously four contiguous stereogenic centers diastereoselectively.

Trapping of N-Acyliminium Ions with Enamides: An Approach to Medium-Sized Diaza-Heterocycles.

Enamides equipped with N-acyliminium ion precursors were obtained through reduction of ynamides tethered to N-imides. Intramolecular TMSOTf-mediated trapping of N-acyliminium ions provided a variety of polyfunctionalized medium-sized diaza-heterocycles of putative pharmacological interest.

Direct Spirocyclization from Keto-sulfonamides: An Approach to Azaspiro Compounds.

Spontaneous spirocyclization of keto-sulfonamides via ynamides through a one-pot process is presented. Push-pull ynamides were obtained through Michael addition/elimination without Cu. The obtained azaspiro compounds are building blocks for indole alkaloids. Theoretical studies provide insights into the mechanism of the formal Conia-ene reaction.

Jasmonic Acid Oxidase 2 Hydroxylates Jasmonic Acid and Represses Basal Defense and Resistance Responses against Botrytis cinerea Infection.

Jasmonates (JAs) orchestrate immune responses upon wound/herbivore injury or infection by necrotrophic pathogens. Elucidation of catabolic routes has revealed new complexity in jasmonate metabolism. Two integrated pathways attenuate signaling by turning over the active hormone jasmonoyl-isoleucine (JA-Ile) through ω-oxidation or deconjugation, and define an indirect route forming the derivative 12OH-JA. Here, we provide evidence for a second 12OH-JA formation pathway by direct jasmonic acid (JA) oxidation. Three jasmonic acid oxidases (JAOs) of the 2-oxoglutarate dioxygenase family catalyze specific oxidation of JA to 12OH-JA, and their genes are induced by wounding or infection by the fungus Botrytis cinerea. JAO2 exhibits the highest basal expression, and its deficiency in jao2 mutants strongly enhanced antifungal resistance. The resistance phenotype resulted from constitutive expression of antimicrobial markers rather than from their higher induction in infected jao2 plants and could be reversed by ectopic expression of any of the three JAOs in jao2. Elevated defense in jao2 was dependent on the activity of JASMONATE RESPONSE 1 (JAR1) and CORONATINE-INSENSITIVE 1 (COI1) but was not correlated with enhanced JA-Ile accumulation. Instead, jao2 mutant lines displayed altered accumulation of several JA species in healthy and challenged plants, suggesting elevated metabolic flux through JA-Ile. Collectively, these data identify the missing enzymes hydroxylating JA and uncover an important metabolic diversion mechanism for repressing basal JA defense responses.

A grapevine cytochrome P450 generates the precursor of wine lactone, a key odorant in wine.

Monoterpenes are important constituents of the aromas of food and beverages, including wine. Among monoterpenes in wines, wine lactone has the most potent odor. It was proposed to form via acid-catalyzed cyclization of (E)-8-carboxylinalool during wine maturation. It only reaches very low concentrations in wine but its extremely low odor detection threshold makes it an important aroma compound. Using LC-MS/MS, we show here that the (E)-8-carboxylinalool content in wines correlates with their wine lactone content and estimate the kinetic constant for the very slow formation of wine lactone from (E)-8-carboxylinalool. We show that (E)-8-carboxylinalool is accumulated as a glycoside in grape (Vitis vinifera) berries and that one of the cytochrome P450 enzymes most highly expressed in maturing berries, CYP76F14, efficiently oxidizes linalool to (E)-8-carboxylinalool. Our analysis of (E)-8-carboxylinalool in Riesling × Gewurztraminer grapevine progeny established that the CYP76F14 gene co-locates with a quantitative trait locus for (E)-8-carboxylinalool content in grape berries. Our data support the role of CYP76F14 as the major (E)-8-carboxylinalool synthase in grape berries and the role of (E)-8-carboxylinalool as a precursor to wine lactone in wine, providing new insights into wine and grape aroma metabolism, and new methods for food and aroma research and production.