Variations of Indole Metabolites and NRPS-PKS Loci in Two Different Virulent Strains of Xenorhabdus hominickii.

Xenorhabdus hominickii ANU1 is known to be an entomopathogenic bacterium symbiotic to nematode Steinernema monticolum. Another bacterial strain X. hominickii DY1 was isolated from a local population of S. monticolum. This bacterial strain X. hominickii DY1 was found to exhibit high insecticidal activities against lepidopteran and coleopteran species after hemocoelic injection. However, these two X. hominickii strains exhibited significant variations in insecticidal activities, with ANU1 strain being more potent than DY1 strain. To clarify their virulence difference, bacterial culture broths of these two strains were compared for secondary metabolite compositions. GC-MS analysis revealed that these two strains had different compositions, including pyrrolopyrazines, piperazines, cyclopeptides, and indoles. Some of these compounds exhibited inhibitory activities against phospholipase A2 to block eicosanoid biosynthesis and induce significant immunosuppression. They also exhibited significant insecticidal activities after oral feeding, with indole derivatives being the most potent. More kinds of indole derivatives were detected in the culture broth of ANU1 strain. To investigate variations in regulation of secondary metabolite production, expression level of leucine-responsive regulatory protein (Lrp), a global transcription factor, was compared. ANU1 strain exhibited significantly lower Lrp expression level than DY1 strain. To assess genetic variations associated with secondary metabolite synthesis, bacterial loci encoding non-ribosomal protein synthase and polyketide synthase (NRPS-PKS) were compared. Three NRPS and four PKS loci were predicted from the genome of X. hominickii. The two bacterial strains exhibited genetic variations (0.12∼0.67%) in amino acid sequences of these NRPS-PKS. Most NRPS-PKS genes exhibited high expression peaks at stationary phase of bacterial growth. However, their expression levels were significantly different between the two strains. These results suggest that differential virulence of the two bacterial strains is caused by the difference in Lrp expression level, leading to difference in the production of indole compounds and other NRPS-PKS-associated secondary metabolites.

Phenylethylamides derived from bacterial secondary metabolites specifically inhibit an insect serotonin receptor.

Serotonin (5-hydroxytryptamine: 5-HT) is a biogenic monoamine that mediates immune responses and modulates nerve signal in insects. Se-5HTR, a specific receptor of serotonin, has been identified in the beet armyworm, Spodoptera exigua. It is classified into subtype 7 among known 5HTRs. Se-5HTR was expressed in all developmental stages of S. exigua. It was expressed in all tested tissues of larval stage. Its expression was up-regulated in hemocytes and fat body in response to immune challenge. RNA interference (RNAi) of Se-5HTR exhibited significant immunosuppression by preventing cellular immune responses such as phagocytosis and nodulation. Treatment with an inhibitor (SB-269970) specific to 5HTR subtype 7 resulted in significant immunosuppression. Furthermore, knockout mutant of Se-5HTR by CRISPR-Cas9 led to significant reduction of phagocytotic activity of S. exigua hemocytes. Such immunosuppression was also induced by bacterial secondary metabolites derived from Xenorhabdus and Photorhabdus. To determine specific bacterial metabolites inhibiting Se-5HTR, this study screened 37 bacterial secondary metabolites with respect to cellular immune responses associated with Se-5HTR and selected 10 potent inhibitors. These 10 selected compounds competitively inhibited cellular immune responses against 5-HT and shared phenylethylamide (PEA) chemical skeleton. Subsequently, 46 PEA derivatives were screened and resulting potent chemicals were used to design a compound to be highly inhibitory against Se-5HTR. The designed compound was chemically synthesized. It showed high immunosuppressive activities along with specific and competitive inhibition activity for Se-5HTR. This study reports the first 5HT receptor from S. exigua and provides its specific inhibitor designed from bacterial metabolites and their derivatives.

Catalytic access to α-oxo gold carbenes by N-O bond oxidants.

Metal carbenes are highly versatile species that mediate various transformations. Recent advances in gold catalysis have allowed catalytic access to α-oxo metal carbenes from the alkyne functionality. Compared with traditional methods that rely upon metal-catalyzed decomposition of diazo precursors, the generation of this synthon occurs in an environmentally more appealing fashion by gold-catalyzed alkyne oxygenation. Hydroxylamine derivatives are typically prepared from hydroxylamine salts that are cheap and can be handled without special precaution. In reactions with an alkyne activated by gold, relatively stable nitrones and related reagents undergo efficient O-atom transfer to form putative α-oxo gold carbenes. The highly reactive nature of these species could be utilized in a variety of cascade transformations. Herein, recent synthetic methods based on this reactivity as well as the currently available mechanistic and structural studies through computational and experimental methods have been discussed. A variety of tandem reactions performed by our laboratory and others have demonstrated the synthetic utility of catalytically generated α-oxo gold carbenes and enabled access to various heterocycles. For example, a reaction between nitrones and alkynes led to azomethine ylides for the [3 + 2] dipolar cycloaddition. Alternatively, α-oxo gold carbenes can be transformed into enolate equivalents through a 1,2-pinacol shift. The addition of hydroxylamine derivatives across triple bonds led to oxoamination, providing α-aminocarbonyl compounds or regioselective Fisher indole-type synthesis. N-O bond cleaving redox chemistry paved the way for intermolecular redox processes, most notably by use of pyridine-N-oxide derivatives with expanding synthetic applications. In closing, other metal-based oxygenations using N-O bond oxidants will be highlighted. One particularly interesting aspect is the process leading to metal vinylidene complexes. Trapping of this intermediate resulted in opposite regioselectivity from gold catalysis in alkyne oxygenation and led to ketene intermediates for use in subsequent cascade transformations.

Ynamides in ring forming transformations.

The ynamide functional group activates carbon-carbontriple bonds through an attached nitrogen atom that bears an electron-withdrawing group. As a result, the alkyne has both electrophilic and nucleophilic properties. Through the selection of the electron-withdrawing group attached to nitrogen, chemists can modulate the electronic properties and reactivity of ynamides, making these groups versatile synthetic building blocks. The reactions of ynamides also lead directly to nitrogen-containing products, which provides access to important structural motifs found in natural products and molecules of medicinal interest. Therefore, researchers have invested increasing time and research in the chemistry of ynamides in recent years. This Account surveys and assesses new organic transforma-tions involving ynamides developed in our laboratory and in others around the world. We showcase the synthetic power of ynamides for rapid assembly of complex molecular structures. Among the recent reports of ynamide transformations, ring-forming reactions provide a powerful tool for generating molecular complexity quickly. In addition to their synthetic utility, such reactions are mechanistically interesting. Therefore, we focus primarily on the cyclization chemistry of ynamides. This Account highlights ynamide reactions that are useful in the rapid synthesis of cyclic and polycyclic structural manifolds. We discuss the mechanisms active in the ring formations and describe representative examples that demonstrate the scope of these reactions and provide mechanistic insights. In this discussion, we feature examples of ynamide reactions involving radical cyclizations, ring-closing metathesis, transition metal and non-transition metal mediated cyclizations, cycloaddition reactions, and rearrangements. The transformations presented rapidly introduce structural complexity and include nitrogen within or in close proximity to a newly formed ring (or rings). Thus, ynamides have emerged as powerful synthons for nitrogen-containing heterocycles and nitrogen-substituted rings, and we hope this Account will promote continued interest in the chemistry of ynamides.

Total syntheses of cannabicyclol, clusiacyclol A and B, iso-eriobrucinol A and B, and eriobrucinol.

Total syntheses of a series of chromane natural products that contain a cyclobutane ring are described. A unified theme in the strategy employed for all these syntheses is an oxa-[3 + 3] annulation for constructing the chromane nucleus and a stepwise cationic [2 + 2] cycloaddition for the cyclobutane formation. More importantly, the two reactions could be rendered in tandem, thereby providing an expeditious approach to this family of natural products.

Entry to ß-alkoxyacrylates via gold-catalyzed intermolecular coupling of alkynoates and allylic ethers.

The first gold-catalyzed intermolecular coupling of alkynoates and allylic ethers invoking alkoxy addition and [3,3]-sigmatropic rearrangement as the key mechanism has been developed. Remarkably, the reaction showed complete chemoselectivity toward the pathway initiated by the alkoxy addition to alkynes. This unprecedented reactivity led to a new access to diversely substituted ß-alkoxyacrylates in a highly efficient manner.

Au(I)-catalyzed intramolecular oxidative cyclopropanation of 1,6-enynes derived from propiolamides with diphenyl sulfoxide.

We have developed gold(I)-catalyzed oxidative cyclopropanation of 1,6-enynes derived from propiolamides employing diphenyl sulfoxide as an oxidant. 1,6-Enynes having a terminal alkyne and a propiolamide tether efficiently transformed into cyclopropane carboxaldehyde derivatives.

Gold-catalyzed intermolecular reactions of propiolic acids with alkenes: [4 + 2] annulation and enyne cross metathesis.

A gold-catalyzed intermolecular reaction of propiolic acids with alkenes led to a [4 + 2] annulation or enyne cross metathesis. The [4 + 2] annulation proceeds with net cis-addition with respect to alkenes and provides an expedient route to α,ß-unsaturated δ-lactones, for which preliminary asymmetric reactions were also demonstrated. For 1,2-disubstituted alkenes, unprecedented enyne cross metathesis occurred to give 1,3-dienes in a completely stereospecific fashion. DFT calculations and experiments indicated that the cyclobutene derivatives are not viable intermediates and that the steric interactions during concerted σ-bond rearrangements are responsible for the observed unique stereospecificity.

Geometry-dependent divergence in the gold-catalyzed redox cascade cyclization of o-alkynylaryl ketoximes and nitrones leading to isoindoles.

We report geometry-dependent cyclizations of o-alkynylaryl ketoximes and nitrones catalyzed by gold complexes. (E)-Ketoximes undergo N-attack to give isoquinoline-N-oxides. In sharp contrast, (Z)-ketoximes undergo unprecedented O-nucleophilic attack, followed by a redox cascade leading to a novel catalytic entry to isoindoles of diverse scope. The structure of an isoindole was unambiguously supported by X-ray crystallography. We demonstrated the generality of the isoindole synthesis from either (Z)-oximes or nitrones, and presented a mechanistic model of this redox cascade based on the reaction profiles of various substrates.