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Juvenile hormone (JH) plays a pivotal role in almost every aspect of insect development and reproduction. The chemical structure of the JH in heteropteran species has long remained elusive until methyl (2R,3S,10R)-2,3;10,11-bisepoxyfarnesoate, commonly named as juvenile hormone III skipped bisepoxide (JHSB3), was isolated from Plautia stali (Hemiptera: Heteroptera: Pentatomidae). Recently, several groups reported the presence of JHSB3 in other heteropteran species. However, most of the studies paid no attention to the determination of the relative and absolute structure of the JH. In this study, we investigated the JH of the cabbage bug Eurydema rugosa (Hemiptera: Heteroptera: Pentatomidae), known as a pest for wild and cultivated crucifers. JHSB3 was detected in the hexane extract from the corpus allatum (CA) product using a chiral ultraperformance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) which can inform the absolute stereochemistry of the JH. Its stereoisomers were not detected. Topical application of the synthetic JHSB3 to the last instar nymphs inhibited their metamorphosis and induced nymphal-type colouration of the dorsal abdomen in a dose-dependent manner. Additionally, the topical application of JHSB3 effectively terminated summer and winter diapauses in females. These results indicate that the JH of E. rugosa is JHSB3. Although individuals in summer and winter diapauses are physiologically distinct in E. rugosa, the results suggest that the physiological differences between these diapauses are based, not on the responsiveness to JH, but on the processes governing activation of the CA or on its upstream cascades.

Natural products containing an aziridine ring, such as mitomycin C and azinomycin B, exhibit antitumor activities by alkylating DNA via their aziridine rings; however, the biosynthetic mechanisms underlying the formation of these rings have not yet been elucidated. We herein investigated the biosynthesis of vazabitide A, the structure of which is similar to that of azinomycin B, and demonstrated that Vzb10/11, with no similarities to known enzymes, catalyzed the formation of the aziridine ring via sulfate elimination. To elucidate the detailed reaction mechanism, crystallization of Vzb10/11 and the homologous enzyme, AziU3/U2, in the biosynthesis of azinomycin B was attempted, and the structure of AziU3/U2, which had a new protein fold overall, was successfully determined. The structural analysis revealed that these enzymes adjusted the dihedral angle between the amino group and the adjacent sulfate group of the substrate to almost 180° and enhanced the nucleophilicity of the C6-amino group temporarily, facilitating the S

The C-H alkenylation of N-acetylcarbazoles proceeds smoothly at the C1-position in the presence of a cationic Cp*Rh(III) catalyst to produce 1-alkenylcarbazoles. The use of a cationic CpERh(III) catalyst enables further alkenylation to give 1,8-dialkenylcarbazoles. The direct alkenylation procedure in combination with the ready removal of the acetyl directing group provides a straightforward synthetic pathway to 1- and/or 8-alkenyl-N-H-carbazole derivatives. One of 1-alkenyl-N-H-carbazoles obtained by the present C-H alkenylation/deacetylation exhibits solvatochromism.

The mevalonate pathway is an upstream terpenoid biosynthetic route of terpenoids for providing the two five-carbon units, dimethylallyl diphosphate, and isopentenyl diphosphate. Recently, trans-anhydromevalonate-5-phosphate (tAHMP) was isolated as a new biosynthetic intermediate of the archaeal mevalonate pathway. In this study, we would like to report the first synthesis of tAHMP and its enzymatic transformation using one of the key enzymes, mevalonate-5-phosphate dehydratase from a hyperthermophilic archaeon, Aeropyrum pernix. Starting from methyl tetrolate, a Cu-catalyzed allylation provided an E-trisubstituted olefin in a stereoselective manner. The resulting E-olefin was transformed to tAHMP by cleavage of the olefin and phosphorylation. The structure of the synthetic tAHMP was unambiguously determined by NOESY analysis.

The rhodium(III)-catalyzed annulative coupling of 9-benzoylcarbazoles with internal alkynes proceeds efficiently through ortho C–H and C–N bond cleavages. This reaction provides direct access to va...

Insect juvenile hormone (JH) mimics (JHMs) are known to have ovicidal effects if applied to adult females or eggs. Here, we examined the effects of exogenous JHMs on embryonic development of the bean bug, Riptortus pedestris. The expression profiles of JH early response genes and JH biosynthetic enzymes indicated that JH titer was low for the first 3 days of the egg stage and increased thereafter. Application of JH III skipped bisepoxide (JHSB3) or JHM on Day 0 eggs when JH titer was low caused reduced hatchability, and the embryos mainly arrested in mid- or late embryonic stage. Application of JHMs on Day 5 eggs also resulted in an arrest, but this was less effective compared with Day 0 treatment. Interestingly, ovicidal activity of synthetic JHMs was much lower than that of JHSB3. This study will contribute to developing novel insecticides that are selective among insect species.

Natural products containing an o-dialkylbenzene moiety exhibit a wide variety of bioactivities, including antibacterial, antifungal, antitumor, and antiangiogenic activities. However, the biosynthetic scheme of the o-dialkylbenzene moiety remains unclear. In this study, we identified the biosynthetic gene cluster (BGC) of compounds 1 and 2 in Streptomyces sp. SANK 60404, which contains a rare o-dialkylbenzene moiety, and successfully reconstituted the biosynthesis of 1 using 22 recombinant enzymes in vitro. Our study established a biosynthetic route for the o-tolyl group within the o-dialkylbenzene moiety, where the triene intermediate 3 loaded onto a unique acyl carrier protein (ACP) is elongated by a specific ketosynthase-chain length factor pair of a type II polyketide synthase system with the aid of a putative isomerase to be termed "electrocyclase" and a thioesterase-like enzyme in the BGC. The C2-elongated all-trans diketo-triene intermediate is subsequently isomerized to the 6Z configuration by the electrocyclase to allow intramolecular 6π-electrocyclization, followed by coenzyme FAD/FMN-dependent dehydrogenation. Bioinformatics analysis showed that the key genes are all conserved in BGCs of natural products containing an o-dialkylbenzene moiety, suggesting that the proposed biosynthetic scheme is a common strategy to form o-dialkylbenzenes in nature.

A facile method for deutrium incorporation into aldehydes by mild reduction of NaBD4 of aldehydes and MnO2 oxidation (98% D) is disclosed.

Fourteen aromatic metabolites (6-19) were isolated from an aqueous extract of the solitary tunicate Cnemidocarpa irene collected in Hokkaido, Japan. The structures of the metabolites were determined based on the spectroscopic interpretations, including one- and two-dimensional NMR, mass spectra, UV, and circular dichroism data. The biopterin analogue 10 modulated the behavior of mice after intracerebroventricular injection and showed a weak affinity to ionotropic glutamate receptor subtypes. Analyses of fluorescent coelomic fluid of the tunicate revealed that pterin 12 was responsible for the fluorescence of the blood cells, while β-carbolines 1 and 3 were fluorescent compounds in the serum. The metabolic profiles in adults, juveniles, larvae, and eggs of the animal differed substantially, suggesting that the metabolism of the animal, especially biosynthesis of aromatic secondary metabolites, changes over different life stages.

N-acylation reaction offers an opportunity to develop an efficient synthesis of amide group-containing molecules. We found that N-acyl carbazoles showed remarkable selectivity in transamidation. St...

The stereocontrolled total synthesis of the allene and carbonyl conjugated apocarotenoids, paracentrone and 19-hexanoyloxyparacentrone 3-acetate, was achieved by sequential cross-coupling reactions using boronic acid ester and iodine- or tin-substituted C5 dienes, which were the building blocks for the elongation of the conjugated polyene systems at both terminals.

The juvenile hormone (JH) is a key hormone of insects. In this study, the first structure determination of JH of Rhodnius prolixus (Heteroptera; Reduviidae) known historically as the insect for whi...

Terpene synthases (TS) are classified into two broad types, Class I and II, based on the chemical strategy for initial carbocation formation and motif sequences of the catalytic site. We have recently identified a new class of enzymes, Class IB, showing the acceptability of long (C₂₀–C₃₅) prenyl-diphosphates as substrates and no amino acid sequence homology with known TS. Conversion of long prenyl-diphosphates such as heptaprenyl-diphosphate (C₃₅) is unusual and has never been reported for Class I and II enzymes. Therefore, the characterization of Class IB enzymes is crucial to understand the reaction mechanism of the extensive terpene synthesis. Here, we report the crystal structure bound with a substrate surrogate and biochemical analysis of a Class IB TS, using the enzyme from Bacillus alcalophilus (BalTS). The structure analysis revealed that the diphosphate part of the substrate is located around the two characteristic Asp-rich motifs, and the hydrophobic tail is accommodated in a unique hydrophobic long tunnel, where the C₃₅ prenyl-diphosphate, the longest substrate of BalTS, can be accepted. Biochemical analyses of BalTS showed that the enzymatic property, such as Mg^2⁺ dependency, is similar to those of Class I enzymes. In addition, a new cyclic terpene was identified from BalTS reaction products. Mutational analysis revealed that five of the six Asp residues in the Asp-rich motifs and two His residues are essential for the formation of the cyclic skeleton. These results provided a clue to consider the application of the unusual large terpene synthesis by Class IB enzymes.

The products released by the corpus allatum (CA), an endocrine gland producing juvenile hormone (JH), were analyzed in three species of stink bugs. Liquid chromatography–mass spectrometry (LC–MS) analysis of the CA products from female adults of Halyomorpha halys (Stal), Nezara viridula (Linnaeus), and Nezara antennata Scott (Hemiptera: Pentatomidae) revealed that the CA biosynthesized JH III skipped bisepoxyside (JHSB3) in all three species. In H. halys, in addition to JHSB3, the CA also produced its stereoisomer, 10S-JHSB3. A bioassay focusing on the number of antennal segments was adopted to examine biological activity of these products. When last instar nymphs were treated with either of the two JHs, they emerged as adults bearing nymphal type antennae with four segments in a dose-dependent fashion, indicating that both JHSB3 and 10S-JHSB3 had the JH activity in H. halys. Therefore, for the first time, 10S-JHSB3 in H. halys was found to be a naturally occurring JH molecule with 10S-configuration.

Terpene synthase (TS) from Bacillus alcalophilus (BalTS) is the only Class IB TS for which a 3D structure has been elucidated. Recently, geranyl-β-phellandrene, a novel cyclic diterpene, was identified as a product of BalTS in addition to the acyclic β-springene. In the present study, we have provided insight into the mechanism of geranyl-β-phellandrene formation. Deuterium labeling experiments revealed that the compound is produced via a 1,3-hydride shift. In addition, nonenzymatic reactions using divalent metal ions were performed. The enzyme is essential for the geranyl-β-phellandrene formation. Furthermore, BalTS variants targeting tyrosine residues enhanced the yield of geranyl-β-phellandrene and the proportion of the compound of the total products. It was suggested that the expansion of the active site space may allow the conformation of the intermediates necessary for cyclization. The present study describes the first Class IB TSs to successfully alter product profiles while retaining high enzyme activity.

Some enzymes annotated as squalene synthase catalyze the prenylation of carbazole-3,4-quinone-containing substrates in bacterial secondary metabolism. Their reaction mechanisms remain unclear because of their low sequence similarity to well-characterized aromatic substrate prenyltransferases (PTs). We determined the crystal structures of the carbazole PTs, and these revealed that the overall structure is well superposed on those of squalene synthases. In contrast, the stacking interaction between the prenyl donor and acceptor substrates resembles those observed in aromatic substrate PTs. Structural and mutational analyses suggest that the Ile and Asp residues are essential for the hydrophobic and hydrophilic interactions with the carbazole-3,4-quinone moiety of the prenyl acceptor, respectively, and a deprotonation mechanism of an intermediary σ-complex involving a catalytic triad is proposed. Our results provide a structural basis for a new subclass of aromatic substrate PTs.

The mevalonate pathway is an upstream terpenoid biosynthetic route of terpenoids for providing the two five-carbon units, dimethylallyl diphosphate, and isopentenyl diphosphate. Recently

The nucleophilic γ-phenylation and γ-alkylation of α,β-unsaturated amides have been developed. This umpolung reaction allows the regioselective introduction of phenyl and alkyl groups to a vinylketene N,O-acetal, which is generated in situ from an α,β-unsaturated N-alkoxyamide, followed by N–O bond cleavage in a two-step, one-pot process.

All-trans polyprenols consisting of one hundred carbons (C100) have been synthesized for the first time via the sulfone coupling of C45 and C55 fragments. The resulting coupling product with four b...

Catalytic asymmetric hydrogenation of dehydroamino acid esters with biscarbamate protection was examined for the first time to prepare optically active amino acids. The new method was successfully applied to the synthesis of new cystine-glutamate exchanger inhibitors.

The selective oxidation of alcohol

Natural products containing an

Most temperate multivoltine insects enter diapause, a hormonally controlled developmental suspension, in response to seasonal photoperiodic and/or thermal cues. Some insect species exhibit maternal regulation of diapause in which developmental trajectories of the offspring are determined by mothers in response to environmental cues that the mother received. Although maternally regulated diapause is common among insects, the maternal endocrinological mechanisms are largely veiled. To approach this issue, we used the jewel wasp Nasonia vitripennis, which produces non-diapause-destined offspring under long days and diapause-destined offspring under short days or low temperatures. Comparative transcriptomics of these wasps revealed possible involvement of the juvenile hormone (JH) biosynthetic cascade in maternal diapause regulation. The expression of juvenile hormone acid O-methyltransferase (jhamt) was typically downregulated in short-day wasps, and this was reflected by a reduction in haemolymph JH concentrations. RNAi targeted at jhamt reduced haemolymph JH concentration and induced wasps to produce diapause-destined offspring even under long days. In addition, topical application of JH suppressed the production of diapause-destined offspring under short days or low temperatures. These results indicate that diapause in N. vitripennis is determined by maternal jhamt expression and haemolymph JH concentration in response to day length. We therefore report a novel role for JH in insect seasonality.

Fourteen aromatic metabolites (

We report a highly primary alcohol-selective aroylation reaction using N-aroylcarbazoles (NAroCs). The aroylationproceeded smoothly in the presence of DBU, which most likely works as a general base catalyst in the reaction system. The synthetic utility was displayed in the primary alcohol-selective aroylation of complex drug molecules and natural products to their prodrugs. Stoichiometrically generated carbazole, the starting material of NAroCs could be easily recovered. We also established safer multigram and multidecagram scale preparation methods of NAroCs, which are easy-to-handle bench-stable reagents.

Class I terpene synthase (TPS) generates bioactive terpenoids with diverse backbones. Sesterterpene synthase (sester-TPS, C25), a branch of class I TPSs, was recently identified in Brassicaceae. However, the catalytic mechanisms of sester-TPSs are not fully understood. Here, we first identified three nonclustered functional sester-TPSs (AtTPS06, AtTPS22, and AtTPS29) in Arabidopsis thaliana. AtTPS06 utilizes a type-B cyclization mechanism, whereas most other sester-TPSs produce various sesterterpene backbones via a type-A cyclization mechanism. We then determined the crystal structure of the AtTPS18–FSPP complex to explore the cyclization mechanism of plant sester-TPSs. We used structural comparisons and site-directed mutagenesis to further elucidate the mechanism: (1) mainly due to the outward shift of helix G, plant sester-TPSs have a larger catalytic pocket than do mono-, sesqui-, and di-TPSs to accommodate GFPP; (2) type-A sester-TPSs have more aromatic residues (five or six) in their catalytic pocket than classic TPSs (two or three), which also determines whether the type-A or type-B cyclization mechanism is active; and (3) the other residues responsible for product fidelity are determined by interconversion of AtTPS18 and its close homologs. Altogether, this study improves our understanding of the catalytic mechanism of plant sester-TPS, which ultimately enables the rational engineering of sesterterpenoids for future applications., Sesterterpenoids, a relatively new group of terpenoids, are structurally diverse and widely distributed in bacteria, fungi, and the plant kingdom. However, the enzymatic mechanism of sesterterpene synthase (sester-TPS), the first committed biosynthesis step, remains obscure. This study reveals the critical amino acids controlling the substrate and product specificity of plant sester-TPS by determining the first crystal structure of the sester-TPS–FSPP complex.

The nucleophilic γ-phenylation and γ-alkylation of α,β-unsaturated amides have been developed. This umpolung reaction allows the regioselective introduction of phenyl and alkyl groups to a vinylketene N,O-acetal, which is generated in situ from an α,β-unsaturated N-alkoxyamide, followed by N-O bond cleavage in a two-step, one-pot process.

Stereoselective total synthesis of (±)-tetragocarbone A isolated from the propolis of an Australian stingless bee, Tetragonula carbonaria, has been developed by focusing on the latent symmetry of 1. The requisite 1R*, 3R*, 6S* stereogenic centers were selectively installed by hydroxy group directing reactions including the late stage desymmetrization of the 1,3-diketone. The target natural product was successfully prepared in 12 steps from phloroglucinol on 300 mg.

Thousands of terpenes have been identified to date. However, only two classes of enzymes are known to be involved in their biosynthesis, and each class has characteristic amino-acid motifs. We recently identified a novel large-terpene (C25/C30/C35) synthase, which shares no motifs with known enzymes. To elucidate the molecular mechanism of this enzyme, we determined the crystal structure of a large-β-prene synthase from B. alcalophilus (BalTS). Surprisingly, the overall structure of BalTS is similar to that of the α-domain of class I terpene synthases although their primary structures are totally different from each other. Two novel aspartate-rich motifs, DYLDNLxD and DY(F, L, W)IDxxED, are identified, and mutations of any one of the aspartates eliminate its enzymatic activity. The present work leads us to propose a new subclass of terpene synthases, class IB, which is probably responsible for large-terpene biosynthesis.

We report the three-dimensional structure of cyclolavandulyl diphosphate (CLPP) synthase (CLDS), which consecutively catalyzes the condensation of two molecules of dimethylallyl diphosphate (DMAPP) followed by cyclization to form a cyclic monoterpene, CLPP. The structures of apo-CLDS and CLDS in complex with Tris, pyrophosphate, and Mg2+ ion were refined at 2.00 A resolution and 1.73 A resolution, respectively. CLDS adopts a typical fold for cis-prenyl synthases and forms a homo-dimeric structure. An in vitro reaction using a regiospecifically 2H-substituted DMAPP substrate revealed the intramolecular proton transfer mechanism of the CLDS reaction. The CLDS structure and structure-based mutagenesis provide mechanistic insights into this unprecedented terpene synthase. The combination of structural and mechanistic insights advances the knowledge of intricate terpene synthase-catalyzed reactions.

Juvenile hormone (JH) plays a crucial role in the determination of developmental timing in insects. In Drosophila melanogaster, reports indicate that JH titers are the highest immediately following eclosion and that the mating experience increases the titers in females. However, the titers have not been successively measured for an extended period of time after eclosion. This study reveals that JH titers are increased after eclosion in virgin females when supplied with food that is occupied by eggs and larvae as well as in mated females. With the presence of eggs and larvae, food induced the virgin females to lay unfertilized eggs. When combined with previous work indicating that females are attracted to such food where they prefer to lay eggs, these results suggest that flies can prepare themselves to lay eggs by changing the titers of JH under the presence of growing larvae, ensuring that the food is an appropriate place to oviposit.

Acid-catalyzed intramolecular Friedel–Crafts cyclization of optically active α-hydroxy-α-alkenylsilanes possessing a benzene ring (>99% ee) with TMSOTf as a Lewis acid gave enantio-enriched tetrahydronaphthalenes (up to 98% ee). The silyl group attached to the chiral carbon played a crucial role in the chirality transfer.

The stereocontrolled total synthesis of the C31 apocarotenoid paracentrone was achieved by sequential cross-coupling reactions using C5 boronic acid ester and iodo-substituted dienes, which were newly developed building blocks for elongation of the conjugated polyene systems at both their terminals.

Kaitocephalin (KCP) isolated from Eupenicillium shearii PF1191 is an unusual amino acid natural product in which serine, proline, and alanine moieties are liked with carbon-carbon bonds. KCP exhibits potent and selective binding affinity for one of the ionotropic glutamate receptor subtypes, NMDA receptors (Ki=7.8nM). In this study, new structure-activity relationship studies at C9 of KCP were implemented. Eleven new KCP analogs with different substituents at C9 were prepared and employed for binding affinity tests using native ionotropic glutamate receptors. Replacement of the 3,5-dichloro-4-hydroxybenzoyl group of KCP with a 3-phenylpropionyl group resulted in significant loss of binding affinity for NMDARs (Ki=1300nM), indicating an indispensable role of the aromatic ring of KCP in the potent and selective binding to NMDARs. Other analogs showed potent binding affinity in a range of 11-270nM. These findings would directly link to develop useful chemical tools toward imaging and labeling of NMDARs.