Formylation Reaction of Amines Using N-Formylcarbazole.

Formamides are useful starting materials for pharmaceutical syntheses. Although various synthetic methods have been documented in this regard, the use of N-formylcarbazole as a formylation reagent for amines has not yet been reported. We report here the first examples of the use of N-formylcarbazole for the formylation of amines. The characteristic reactivity of N-formylcarbazole enables the selective formylation of sterically less hindered aliphatic primary and secondary amines. In contrast, sterically bulkier amines and weakly nucleophilic amines such as anilines are less reactive under the reaction conditions.

Phenylpropanoids and neolignans isolated from Myristica fragrans enhance glucose uptake in myotubes.

Nutmeg is the seed of Myristica fragrans or its powder and is used as a spice and a traditional medicine. The antidiabetic effect of nutmeg is not fully understood yet. In this study, we examine the isolation and identification of the active compounds of Myristica fragrans with regards to glucose uptake and elucidate their mechanism in L6 myotubes. Myrisiticin, licarin B, erythro-2-(4-allyl-2,6-dimethoxy-phenoxy)-1-(3,4-dimethoxyphenyl)-propan-1-ol (ADDP) and (7S,8R)-2-(4-allyl-2,6-dimethoxyphenoxy)-1-(3,4,5-trimethoxyphenyl)-propan-1-ol (ADTP) were isolated and identified as the active compounds. Myristicin or a mixture of ADDP and ADTP promoted the translocation of glucose transporter 4 (GLUT4) through phosphorylation of AMP-activated protein kinase in L6 myotubes 15 min after treatment, while licarin B promoted it 240 min after treatment. Oral administration of the fraction from Myristica fragrans containing these active compounds to ICR mice suppressed post-prandial hyperglycemia. Thus, Myristica fragrans is a promising functional food to prevent post-prandial hyperglycemia and type 2 diabetes mellitus by promoting glucose uptake in muscle.

Anti-allergic property of 4,8-sphingadienine stereoisomers in vivo and in vitro model.

4,8-Sphingadienines (SD), metabolites of glucosylceramides (GlcCer), are sometimes determined as key mediators of the biological activity of dietary GlcCer, and cis/trans geometries of 4,8-SD have been reported to affect its activity. Since regulating excessive activation of mast cells seems an important way to ameliorate allergic diseases, this study was focused on cis/trans stereoisomeric-dependent inhibitory effects of 4,8-SD on mast cell activation. Degranulation of RBL-2H3 was inhibited by treatment of 4-cis-8-trans- and 4-cis-8-cis-SD, and their intradermal administrations ameliorated ear edema in passive cutaneous anaphylaxis reaction, but 4-trans-8-trans- and 4-trans-8-cis-SD did not. Although the activation of mast cells depends on the bound IgE contents, those stereoisomers did not affect IgE contents on RBL-2H3 cells after the sensitization of anti-TNP IgE. These results indicated that 4-cis-8-trans- and 4-cis-8-cis-SD directly inhibit the activation of mast cells. In conclusion, it was assumed that 4,8-SD stereoisomers with cis double bond at C4-position shows anti-allergic activity by inhibiting downstream pathway after activation by the binding of IgE to mast cells.

Expression of recombinant apopholasin using a baculovirus-silkworm multigene expression system and activation via dehydrocoelenterazine.

Pholasin is a photoprotein derived from the glowing bivalve mollusk, Pholas dactylus. Even though the chemical structure of the prosthetic group (chromophore) responsible for the light emission character of the mollusk remains unknown, research has shown that the presence of dehydrocoelenterazine (DCL) increased light emission and that the dithiothreitol adduct of DCL was isolated from Pholasin®. To date, our research has been focused on activating apopholasin, the naturally occurring apoprotein of Pholasin®, using DCL. In the current study, the expression of recombinant apopholasin via a baculovirus-silkworm multigene expression system is reported. Additionally, the purification of apopholasin using a Flag®-affinity column, the activation of apopholasin using DCL, and the initiation of its luminescent character through the addition of a peroxidase-hydrogen peroxide mixture are reported. The peroxidase-H2O2-dependent luminescence was observed from the recombinant apopholasin activated with DCL.

Concise synthesis of heliolactone, a non-canonical strigolactone isolated from sunflower.

Heliolactone is one of the earliest identified non-canonical strigolactones. Its concise synthesis was achieved by employing Knoevenagel-type condensation and semi-reduction of a malonate intermediate as the key steps. This synthesis was performed in a non-stereoselective manner, and thus a racemic and diastereomeric mixture of heliolactone was obtained. The developed synthetic route is fairly concise and straightforward.

First synthesis and absolute configuration of phorbasin H, a diterpene carboxylic acid isolated from the sponge Phorbas gukulensis.

The first synthesis of phorbasin H, a diterpene carboxylic acid isolated from the sponge Phorbas gukulensis, was achieved using 1,4-trans-cyclohexanedimethanol and (S)-citronellal as starting materials. The data collected in this synthesis indicated the absolute configuration of the naturally occurring phorbasin H to be S.

Enhanced elevations of hypo-osmotic shock-induced cytosolic and nucleic calcium concentrations in tobacco cells by pretreatment with dimethyl sulfoxide.

Dimethyl sulfoxide (DMSO) is a dipolar aprotic solvent widely used in biological assays. Here, we observed that DMSO enhanced the hypo-osmotically induced increases in the concentration of Ca2+ in cytosolic and nucleic compartments in the transgenic cell-lines of tobacco (BY-2) expressing aequorin.

First synthesis of (S)-(+)-hymenoic acid, a DNA polymerase λ inhibitor isolated from Hymenochaetaceae sp.

Hymenoic acid, isolated from cultures of the fungus, Hymenochaetaceae sp., is a specific inhibitor of DNA polymerase λ. The first synthesis of (S)-(+)-hymenoic acid was achieved by starting from trans-1,4-cyclohexanedimethanol and methyl (R)-(-)-3-hydroxyisobutyrate, and Julia-Kocienski olefination was employed as the key step.

Identification of (Z)-3:(E)-2-Hexenal Isomerases Essential to the Production of the Leaf Aldehyde in Plants.

The green odor of plants is characterized by green leaf volatiles (GLVs) composed of C6 compounds. GLVs are biosynthesized from polyunsaturated fatty acids in thylakoid membranes by a series of enzymes. A representative member of GLVs (E)-2-hexenal, known as the leaf aldehyde, has been assumed to be produced by isomerization from (Z)-3-hexenal in the biosynthesis pathway; however, the enzyme has not yet been identified. In this study, we purified the (Z)-3:(E)-2-hexenal isomerase (HI) from paprika fruits and showed that various plant species have homologous HIs. Purified HI is a homotrimeric protein of 110 kDa composed of 35-kDa subunits and shows high activity at acidic and neutral pH values. Phylogenetic analysis showed that HIs belong to the cupin superfamily, and at least three catalytic amino acids (His, Lys, Tyr) are conserved in HIs of various plant species. Enzymatic isomerization of (Z)-3-hexenal in the presence of deuterium oxide resulted in the introduction of deuterium at the C4 position of (E)-2-hexenal, and a suicide substrate 3-hexyn-1-al inhibited HI irreversibly, suggesting that the catalytic mode of HI is a keto-enol tautomerism reaction mode mediated by a catalytic His residue. The gene expression of HIs in Solanaceae plants was enhanced in specific developmental stages and by wounding treatment. Transgenic tomato plants overexpressing paprika HI accumulated (E)-2-hexenal in contrast to wild-type tomato plants mainly accumulating (Z)-3-hexenal, suggesting that HI plays a key role in the production of (E)-2-hexenal in planta.

A concise synthesis of optically active solanacol, the germination stimulant for seeds of root parasitic weeds.

Solanacol, isolated from tobacco (Nicotiana tabacum L.), is a germination stimulant for seeds of root parasitic weeds. A concise synthesis of optically active solanacol has been achieved by employing enzymatic resolution as a key step.

Chromophores in photoproteins of a glowing squid and mollusk.

Bioluminescence is a chemical reaction process for light emission in vivo. An organic substance is normally oxidized in the protein to obtain the energy required for the light emission. Determination of the structure of the substance is one of the most important parts of bioluminescent research. Photoproteins of a flying squid and a mollusk contain chromophores that are formed by connecting an apo-protein and dehydrocoelenterazine. The chromophore has a chemical structure that can emit light in a photoprotein. The structural analysis of the chromophores in the photoproteins is described.

Synthesis of 7-oxo-5-deoxystrigol, a 7-oxygenated strigolactone analog.

A straightforward synthesis of 7-oxo-5-deoxystrigol, a 7-oxygenated strigolactone analog, was achieved by starting from 2,2-dimethylcyclohexane-1,3-dione.

Dynamic chirality determines critical roles for bioluminescence in symplectin-dehydrocoelenterazine system.

Symplectin is a photoprotein containing the dehydrocoelenterazine (DCL) chromophore, which links to a cysteine residue through a covalent bond with the emission of blue light. This study focuses on the stereochemical process of the emerging stereogenic centers. Two isomeric fluorinated DCL analogs (2,4-diF- and 2,6-diF-DCL) were employed owing to their different bioluminescence activities, these being 200% and 20% compared to natural DCL, respectively. Each of these diF-DCLs was found to exchange with the natural DCL in symplectin at pH 6.0. The emerging stereogenic carbons were racemic at the binding sites. Changing the pH of this storage form to the protein's optimum solubility pH (pH 7.8) resulted in 2,4-diF-DCL-bound symplectin luminescence, and the spent solutions were then analyzed and coelenteramide-390-CGLK-peptide and coelenteramine were detected after a peptidase digestion. The same analysis of the 2,6-diF-DCL-bound symplectin, on the other hand, afforded coelenteramine only but no coelenteramide. When the racemic storage diF-DCLs moved to the active site at pH 7.8, a change in the chirality with the 390-Cys residue resulted. Model experiments using L-cysteine-containing CGLK-peptide supported two diastereoisomers from each diF-DCL. The significant difference in the luminescence from these two chromophores is attributed to a plausible mechanism including the dynamically variable stereogenic center emerging at the storage and then the active site on the symplectin. It is concluded that such dynamic chirality plays a significant role in the symplectoteuthis bioluminescence.

Identification of a fluorescent compound in the cuticle of the train millipede Parafontaria laminata armigera.

The train millipede (Parafontaria laminata armigera) emits a blue fluorescence (λ(max)=455 nm) under black light (350 nm). The isolated fluorescent compound from the cuticle of P. laminata armigera was identified as pterin-6-carboxylic acid. The structure of this compound was identified by fluorescent, HPLC, and mass spectrometric (ESI-ion trap MS) analyses, and then compared with an authentic sample.

Prevention of copper-induced calcium influx and cell death by prion-derived peptide in suspension-cultured tobacco cells.

Impact of copper on the oxidative and calcium signal transductions leading to cell death in plant cells and the effects of the copper-binding peptide derived from the human prion protein (PrP) as a novel plant-protecting agent were assessed using a cell suspension culture of transgenic tobacco (Nicotiana tabacum L., cell line BY-2) expressing the aequorin gene. Copper induces a series of biological and chemical reactions in plant cells including the oxidative burst reflecting the production of reactive oxygen species (ROS), such as hydroxyl radicals, and stimulation of calcium channel opening, allowing a transient increase in cytosolic calcium concentrations. The former was proven by the action of specific ROS scavengers blocking the calcium responses and the latter was proven by an increase in aequorin luminescence and its inhibition by specific channel blockers. Following these early events completed within 10 min, the development of copper-induced cell death was observed during additional 1 h in a dose-dependent manner. Addition of a synthetic peptide (KTNMKHMA) corresponding to the neurotoxic sequence in human PrP, prior to the addition of copper, effectively blocked both calcium influx and cell death induced by copper. Lastly, a possible mechanism of peptide action and future applications of this peptide in the protection of plant roots from metal toxicity or in favour of phytoremediation processes are discussed.

Selective protein modification by the hydroperoxide intermediate in a photoprotein, aequorin.

During the course of protein modification program, we employed a recombinant aequorin, the apo-protein reconstituted with coelenterazine, and found out that the photolytic hyperperoxide modified three -S-SCH2CHOHCHOHCH2SH groups to -S-SCH2CHOHCH=CH-S=O)H or -S-SCH2CHOHCH=CH-S(=O)OH of terminal DTT connected to cysteine residues of the C145, C152 and C180, which turned out to locate near the chromophore.

Cysteine-390 is the binding site of luminous substance with symplectin, a photoprotein from Okinawan squid, Symplectoteuthis oualaniensis.

Symplectin is a photoprotein from a luminous squid, Symplectoteuthis oualaniensis. It has a luminous substrate, dehydrocoelenterazine (DCZ), linked through a thioether bond with a cysteine residue. We have proven the binding site of luminous substrate in symplectin by using an artificial analogue of DCZ, ortho-fluoro-DCZ (F-DCZ). F-DCZ-symplectin emitting strong blue light was reconstituted from apo-symplectin and F-DCZ. Proteolytic digestion of the reconstituted F-DCZ-symplectin afforded peptides including C(390)GLK-F-DCZ (amide), which was detected with a house assembled nano-LC-ESI-Q-TOF-MS. The chromo-peptide derived from the F-DCZ-symplectin after luminescence showed the lower molecular mass than that before the luminescence by 12 mass units, corresponding to the loss of one carbon atom upon emitting light. Thus, we have concluded that F-DCZ analogue binds to Cys390 in symplectin so as to emit light.

Pholasin luminescence is enhanced by addition of dehydrocoelenterazine.

Pholasin is a bioluminescent photoprotein of Pholas dactylus. Pholasin is a commercially available photoprotein used to measure intracellular reactive oxygen species. Although extensive efforts have been carried out to determine the chemical structure of the prosthetic (chromophore) group, it still remains unclear to date. Herein, we report the enhancement of pholasin luminescence by the addition of dehydrocoelenterazine, which is organic substance of luminous squids' photoprotein.

Determination of solvent-trapped products obtained by photolysis of aryl azides in 2,2,2-trifluoroethanol.

A series of nonfluorinated and fluorinated aryl azides with varied functionality patterns were irradiated in 2,2,2-trifluoroethanol with either a high-pressure or a low-pressure mercury lamp. Interestingly, one of the major products in these reactions was the result of the recombination of anilino and alkyl radicals to form the corresponding hemiaminal compounds. The structure of the recombination products was assigned unambiguously after proton/deuterium exchange experiments followed by MS and MS/MS analysis.