Role of hypoxanthine-guanine phosphoribosyltransferase in the metabolism of fairy chemicals in rice.

Fairy chemicals (FCs), 2-azahypoxanthine (AHX), imidazole-4-carboxamide (ICA), and 2-aza-8-oxohypoxanthine (AOH), are molecules with many diverse functions in plants. The defined biosynthetic pathway for FCs is a novel purine metabolism in which they are biosynthesized from 5-aminoimidazole-4-carboxamide. Here, we show that one of the purine salvage enzymes, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), recognizes AHX and AOH as substrates. Two novel compounds, AOH ribonucleotide and its ribonucleoside which are the derivatives of AOH, were enzymatically synthesized. The structures were determined by mass spectrometry, 1D and 2D NMR spectroscopy, and X-ray single-crystal diffraction analysis. This report demonstrates the function of HGPRT and the existence of novel purine metabolism associated with the biosynthesis of FCs in rice.

The role of xanthine dioxygenase in the biosynthetic pathway of 2-aza-8-oxohypoxanthine of Lepista sordida.

2-Azahypoxanthine (AHX) and 2-aza-8-oxohypoxanthine (AOH), discovered as causal substances of fairy rings are known to be endogenous in the fairy ring-forming Lepista sordida. In this study, we showed that xanthine dioxygenase, an a-ketoglutarate-dependent dioxygenase, might catalyze the conversion of AHX to AOH in the fungus. Furthermore, this enzyme is the first reported molybdopterin-independent protein of hypoxanthine metabolism.

Identification of Biosynthetic and Metabolic Genes of 2-Azahypoxanthine in Lepista sordida Based on Transcriptomic Analysis.

2-Azahypoxanthine was isolated from the fairy ring-forming fungus Lepista sordida as a fairy ring-inducing compound. 2-Azahypoxanthine has an unprecedented 1,2,3-triazine moiety, and its biosynthetic pathway is unknown. The biosynthetic genes for 2-azahypoxanthine formation in L. sordida were predicted by a differential gene expression analysis using MiSeq. The results revealed that several genes in the purine and histidine metabolic pathways and the arginine biosynthetic pathway are involved in the biosynthesis of 2-azahypoxanthine. Furthermore, nitric oxide (NO) was produced by recombinant NO synthase 5 (rNOS5), suggesting that NOS5 can be the enzyme involved in the formation of 1,2,3-triazine. The gene encoding hypoxanthine-guanine phosphoribosyltransferase (HGPRT), one of the major phosphoribosyltransferases of purine metabolism, increased when 2-azahypoxanthine content was the highest. Therefore, we hypothesized that HGPRT might catalyze a reversible reaction between 2-azahypoxanthine and 2-azahypoxanthine-ribonucleotide. We proved the endogenous existence of 2-azahypoxanthine-ribonucleotide in L. sordida mycelia by LC-MS/MS for the first time. Furthermore, it was shown that recombinant HGPRT catalyzed reversible interconversion between 2-azahypoxanthine and 2-azahypoxanthine-ribonucleotide. These findings demonstrate that HGPRT can be involved in the biosynthesis of 2-azahypoxanthine via 2-azahypoxanthine-ribonucleotide generated by NOS5.

Total synthesis of (-)-domoic acid, a potent ionotropic glutamate receptor agonist and the key compound in oceanic harmful algal blooms.

The stereo-controlled total synthesis of (-)-domoic acid is described. The critical construction of the C1'-C2' Z-configuration was accomplished by taking advantage of an unsaturated lactam structure. The side chain fragment was introduced in the final stages of synthesis through a modified Julia-Kocienski reaction, aiming for its efficient derivatization.

1,2,3-Triazine formation mechanism of the fairy chemical 2-azahypoxanthine in the fairy ring-forming fungus Lepista sordida.

2-Azahypoxanthine (AHX) was first isolated from the culture broth of the fungus Lepista sordida as a fairy ring-inducing compound. It has since been found that a large number of plants and mushrooms produce AHX endogenously and that AHX has beneficial effects on plant growth. The AHX molecule has an unusual, nitrogen-rich 1,2,3-triazine moiety of unknown biosynthetic origin. Here, we establish the biosynthetic pathway for AHX formation in L. sordida. Our results reveal that the key nitrogen sources that are responsible for the 1,2,3-triazine formation are reactive nitrogen species (RNS), which are derived from nitric oxide (NO) produced by NO synthase (NOS). Furthermore, RNS are also involved in the biochemical conversion of 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranosyl 5'-monophosphate (AICAR) to AHX-ribotide (AHXR), suggesting that a novel biosynthetic route that produces AHX exists in the fungus. These findings demonstrate a physiological role for NOS in AHX biosynthesis as well as in biosynthesis of other natural products containing a nitrogen-nitrogen bond.

S-Adenosylhomocysteine Analogue of a Fairy Chemical, Imidazole-4-carboxamide, as its Metabolite in Rice and Yeast and Synthetic Investigations of Related Compounds.

During the course of our investigations of fairy chemicals (FCs), we found S-ICAr-H (8a), as a metabolite of imidazole-4-carboxamide (ICA) in rice and yeast (Saccharomyces cerevisiae). In order to determine its absolute configuration, an efficient synthetic method of 8a was developed. This synthetic strategy was applicable to the preparation of analogues of 8a that might be biologically very important, such as S-ICAr-M (9), S-AICAr-H (10), and S-AICAr-M (11).

Biosynthesis of the Fairy Chemicals, 2-Azahypoxanthine and Imidazole-4-carboxamide, in the Fairy Ring-Forming Fungus Lepista sordida.

Fairy rings resulting from a fungus-plant interaction appear worldwide. 2-Azahypoxanthine (AHX) and imidazole-4-carboxamide (ICA) were first isolated from the culture broth of one of the fairy ring-forming fungi, Lepista sordida. Afterward, a common metabolite of AHX in plants, 2-aza-8-oxohypoxanthine (AOH), was found in AHX-treated rice. The biosynthetic pathway of the three compounds that are named as fairy chemicals (FCs) in plants has been partially elucidated; however, that in mushrooms remains unknown. In this study, it was revealed that the carbon skeletons of AHX and ICA were constructed from Gly in L. sordida mycelia and the fungus metabolized 5-aminoimidazole-4-carboxamide (AICA) to both of the compounds. These results indicated that FCs were biosynthesized by a diversion of the purine metabolic pathway in L. sordida mycelia, similar to that in plants. Furthermore, we showed that recombinant adenine phosphoribosyltransferase (APRT) catalyzed reversible interconversion not only between 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranosyl 5'-monophosphate (AICAR) and AICA but also between ICA-ribotide (ICAR) and ICA. Furthermore, the presence of ICAR in L. sordida mycelia was proven for the first time by LC-MS/MS detection, and this study provided the first report that there was a novel metabolic pathway of ICA in which its ribotide was an intermediate in the fungus.

Total Syntheses and Cytotoxic Evaluations of Cryptolactones A1, A2, B1, B2, and Their Derivatives.

The cryptolactones A1, A2, B1, and B2 isolated from a Cryptomyzus sp. aphid were synthesized via the Mukaiyama aldol reaction and olefin metathesis. Their antipodes and derivatives were also synthesized by the same strategy to investigate structure-activity relationships. These compounds exhibited cytotoxic activity against human promyelocytic leukemia HL-60 cells with IC50 values of 2.1-42 µM.

Synthetic Studies on Pactamycin: A Synthesis of Johnson's Intermediate.

A formal total synthesis of pactamycin (1) has been accomplished by face-selective and regioselective nitroso Diels-Alder (NDA) reaction of acyl nitroso compound 14, which contains a camphorsultam chiral auxiliary, and chiral cyclopentadiene 12. Construction of the chiral secondary alcohol of 12 was performed by (S,S)-Ts-DENEB catalyst-mediated reduction, and the NDA adduct 15a was readily converted to Johnson's intermediate 21.

Total Synthesis of Sophoraflavanone H and Confirmation of Its Absolute Configuration.

Sophoraflavanone H (1) is a polyphenol with a hybrid-type structure containing 2,3-diaryl-2,3-dihydrobenzofuran and flavanone ring moieties. This compound and related analogues are promising leads for antimicrobial and antitumor drug development. Here we describe a total synthesis of 1 and its diastereomer. The dihydrobenzofuran and flavanone rings were constructed by a Rh-catalyzed asymmetric C-H insertion reaction and selective oxy-Michael reaction. The absolute configuration of 1 was established by X-ray crystallographic analysis and CD spectral investigation of synthetic derivatives.

Author Correction: Transdermal delivery of nobiletin using ionic liquids.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transdermal delivery of nobiletin using ionic liquids.

Nobiletin (NOB), a flavonoid, has extremely low water solubility and low oral bioavailability; however, despite these problems, various physiological effects have been investigated in vitro. In the present study, we investigated the transdermal delivery of NOB using choline and geranic acid (CAGE), which is a biocompatible material that has been reported to be a promising transdermal delivery approach. The feasibility was evaluated by a set of in vitro and in vivo tests. A solubility evaluation demonstrated that CAGE induced excellent solubility of NOB induced by multipoint hydrogen bonding between NOB and CAGE. In vitro transdermal tests using a Franz diffusion cell showed that CAGE was effective in enhancing transdermal absorption of NOB, compared to other penetration enhancers. Subsequent in vivo tests demonstrated that CAGE significantly improved area under the concentration-time curve of NOB in vivo and NOB/CAGE sample showed 20-times higher bioavailability than oral administration of NOB crystal. Furthermore, NOB/CAGE sample also showed significant drops of the blood glucose level in rats derived from hypoglycemic activity of NOB. Thus, transdermal administration of NOB using CAGE was shown to be feasible, which indicates that the use of CAGE may be adapted for other flavonoids that also show both low water solubility and low permeability.

Diastereodivergent and Regiodivergent Total Synthesis of Princepin and Isoprincepin in Both (7″R,8″R) and (7″S,8″S) Isomers.

The first total synthesis of (7″R,8″R)-, (7″S,8″S)-isomers of princepin (1) and (7″R,8″R)-, (7″S,8″S)-isomers of isoprincepin (2) was accomplished in a highly stereoselective manner via para quinomethide-mediated construction of the furofuran and 1,4-benzodioxane rings. Structural confirmation methods of 1 and 2 were established by CD and HPLC analysis of each diastereomers with natural products.

Ribosides and Ribotide of a Fairy Chemical, Imidazole-4-carboxamide, as Its Metabolites in Rice.

The metabolism of imidazole-4-carboxamide (ICA) in plants has been unknown. Two metabolites (1 and 2) were isolated from ICA-treated rice, and their structures were determined by spectroscopic analysis including the single-crystal X-ray diffraction technique and synthesis. The ribotide of ICA (3), whose existence was predicted, was also synthesized and detected from the treated rice by LC-MS/MS. These results indicated that rice might interconvert ICA, 1, and 3 to regulate the biological activity.

A Fairy Chemical, Imidazole-4-carboxamide, is Produced on a Novel Purine Metabolic Pathway in Rice.

Rings or arcs of fungus-regulated plant growth occurring on the floor of woodlands and grasslands are commonly called "fairy rings". Fairy chemicals, 2-azahypoxanthine (AHX), imidazole-4-carboxamide (ICA), and 2-aza-8-oxohypoxanthine (AOH), are plant growth regulators involved in the phenomenon. The endogeny and biosynthetic pathways of AHX and AOH in plants have already been proven, however, those of ICA have remained unclear. We developed a high-sensitivity detection method for FCs including ICA and the endogenous ICA was detected in some plants for the first time. The quantitative analysis of the endogenous level of ICA in rice and Arabidopsis were performed using 13C-double labeled ICA. In addition, the incorporation experiment and enzyme assay using the labeled compound into rice and partially purified fraction of rice indicated that ICA is biosynthesized from 5-aminoimidazole-4-carboxamide (AICA), a metabolite on the purine metabolic pathway. The relationship between ICA and AHX was also discussed based on quantitative analysis and gene expression analysis.

A role of uroleuconaphins, polyketide red pigments in aphid, as a chemopreventor in the host defense system against infection with entomopathogenic fungi.

Four red polyketide pigments, uroleuconaphins A1 (1) and B1 (2) and their glucosides 3 and 4, were isolated from the red goldenrod aphid Uroleucon nigrotuberculatum. Although these red pigments exist only as glucosides 3 and 4 in the intact insect body, 3 and 4 convert instantly to aglycones 1 and 2 at death. Pigments 1 and 2 inhibited the growth of Lecanicillium sp. (Ascomycota: Cordycipitaceae) and 1, 2, and 3 were active against Conidiobolus obscurus (Entomophthoromycota; Entomophthorales); these fungal species are pathogenic. We therefore regard aphid pigments 1-4 as chemopreventive agents that aid in the resistance of infection by entomopathogenic fungi at the level of the individual aphid and/or at the species level.

An efficient screening method for purifying and crystallizing membrane proteins using modified clear-native PAGE.

Membrane proteins, such as G-protein coupled receptors, control communication between cells and their environments and are indispensable for many cellular functions. Nevertheless, structural studies on membrane proteins lag behind those on water-soluble proteins, due to their low structural stability, making it difficult to obtain crystals for X-ray crystallography. Optimizing conditions to improve the stability of membrane proteins is essential for successful crystallization. However, the optimization usually requires large amounts of purified samples, and it is a time-consuming and trial-and-error process. Here, we report a rapid method for precrystallization screening of membrane proteins using Clear Native polyacrylamide gel electrophoresis (CN-PAGE) with the modified Coomassie Brilliant Blue G-250 (mCBB) stain that was reduced in sodium formate. A2A adenosine receptor (A2AAR) was selected as a target membrane protein, for which we previously obtained the crystal structure using an antibody, and was expressed as a red fluorescent protein fusion for in-gel fluorescence detection. The mCBB CN-PAGE method enabled the optimization of the solubilization, purification, and crystallization conditions of A2AAR using the solubilized membrane fraction expressing the protein without purification procedures. These data suggest the applicability of mCBB CN-PAGE technique to a wide variety of integral membrane proteins.

N-Glucosides of Fairy Chemicals, 2-Azahypoxanthine and 2-Aza-8-oxohypoxanthine, in Rice.

Plant growth stimulators, 2-azahypoxanthine (AHX) and 2-aza-8-oxohypoxanthine (AOH), were isolated from the fairy-ring-forming fungus, Lepista sordida, and AHX-treated rice, respectively. Further metabolites of AHX were detected in AHX-treated rice by HPLC, and the metabolites 1-4 were isolated from the rice. The structures of 1-4 were determined by spectroscopic analysis and synthesis. Compounds 1-4 exhibited no significant activity against rice, indicating that rice regulates the activity of AHX and AOH by converting them into their glucosides.

Preventive Effects of the Dietary Intake of Medium-chain Triacylglycerols on Immobilization-induced Muscle Atrophy in Rats.

Previous studies have shown that medium-chain triacylglycerols (MCTs) exert favorable effects on protein metabolism. This study evaluated the effects of the dietary intake of MCTs on rat skeletal muscle mass and total protein content during casting-induced hindlimb immobilization, which causes substantial protein degradation and muscle atrophy. Rats were fed a standard diet containing long-chain triacylglycerols (LCTs) or MCTs for 3 days and then a unilateral hindlimb was immobilized while they received the same diet. After immobilization for 3, 7, and 14 days, muscle mass and total protein content in immobilized soleus muscle in the LCT-fed rats had markedly decreased compared to the contralateral muscle; however, these losses were partially suppressed in MCT-fed rats. Autophagosomal membrane proteins (LC-I and -II), which are biomarkers of autophagy-lysosome activity, did not differ significantly between the LCT- and MCT-fed rats. In contrast, the immobilization-induced increase in muscle-specific E3 ubiquitin ligase MuRF-1 protein expression in immobilized soleus muscle relative to contralateral muscle was completely blocked in the MCT-fed rats and was significantly lower than that observed in the LCT-fed rats. Collectively, these results indicate that the dietary intake of MCTs at least partly alleviates immobilization-induced muscle atrophy by inhibiting the ubiquitin-proteasome pathway.