ABSTRACT
We report the first total synthesis of silybin A (1). Key synthetic steps include the construction of the 1,4-benzodioxane neolignan skeleton, a modified Julia-Kocienski olefination reaction between m-nitrophenyltetrazole sulfone (m-NPT sulfone) 10 and aldehyde 21, the formation of the flavanol lignan skeleton 28 via a quinomethide intermediate under acidic conditions, and stepwise oxidation of the benzylic position of flavanol 29.
Subject(s)
Silybin , Silybin/chemical synthesis , Silybin/chemistry , Stereoisomerism , Molecular Structure , Silymarin/chemical synthesis , Silymarin/chemistry , Oxidation-ReductionABSTRACT
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.
Subject(s)
Harmful Algal Bloom , Receptors, Ionotropic Glutamate , Kainic AcidABSTRACT
The applicability of a method for simultaneous analysis of Acromelic acids A, B, and Clitidine, which are venomous constituents of Paralepistopsis acromelalga, was assessed for three simulations: tempura, chikuzenni, and soy sauce soup. All components were detectable for all cooking methods. No interfering peak affecting the analysis was observed. The findings indicate that samples of leftover cooked products can be used to ascertain causes of food poisoning by Paralepistopsis acromelalga. Additionally, results showed that most of the toxic components were eluted into the soup broth. This property is useful for rapid screening for Paralepistopsis acromelalga in edible mushrooms.
Subject(s)
Agaricales , Foodborne Diseases , Mushroom Poisoning , Soy Foods , Mushroom Poisoning/diagnosis , Food, Processed , CookingABSTRACT
An isolation method for Acromelic acids A, B and Clitidine, which are venomous constituents of Paralepistopsis acromelalga was developed. Highly purified products were obtained from the mushroom extract using silica gel, ODS, ion-exchange column chromatography and preparative TLC. Using those results, we optimized the LC-MS/MS conditions. Finally we developed a method for simultaneous analysis. In recovery tests, the average recovery was 80.8-112.4%, repeatability was 1.4-3.8RSD%. The limits of quantification of the respective compounds were estimated as 0.25 µg/g. Based on the results, this method can reveal causes of food poisoning by Paralepistopsis acromelalga.
Subject(s)
Tandem Mass Spectrometry , Venoms , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methodsABSTRACT
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).
Subject(s)
Aminoimidazole Carboxamide/analogs & derivatives , Oryza/metabolism , S-Adenosylhomocysteine/analogs & derivatives , Saccharomyces cerevisiae/metabolism , Aminoimidazole Carboxamide/chemistry , Aminoimidazole Carboxamide/metabolism , Molecular StructureABSTRACT
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.
Subject(s)
Agaricales/metabolism , Aminoimidazole Carboxamide/analogs & derivatives , Hypoxanthines/metabolism , Aminoimidazole Carboxamide/chemistry , Aminoimidazole Carboxamide/isolation & purification , Aminoimidazole Carboxamide/metabolism , Carbon-13 Magnetic Resonance Spectroscopy , Chromatography, Liquid , Fruiting Bodies, Fungal/chemistry , Hypoxanthines/chemistry , Hypoxanthines/isolation & purification , Tandem Mass SpectrometryABSTRACT
Staphylococcal enterotoxin A (SEA) functions both as superantigens that stimulate non-specific T cell proliferation as well as potent gastrointestinal toxins. We previously reported that (-)-epigallocatechin gallate (EGCG) binds to SEA. Therefore, the ability of EGCG to inhibit SEA toxin activity was examined. As a result, EGCG significantly decreased SEA-induced expression and production of interferon gamma (IFN-γ). In addition, EGCG inhibited SEA-induced spleen cell proliferation. To investigate the role of the galloyl group in EGCG on SEA cytotoxicity in more detail, the effect of the binding of a hydroxyl group at position 3 of the galloyl group in EGCG to SEA on SEA cytotoxicity was examined using two methylated EGCG. SEA cytotoxicity was significantly controlled in both (-)-3''-Me-EGCG and (-)-4''-Me-EGCG. These results suggest that EGCG inhibits toxic activity via direct interaction with SEA or without any interaction with SEA. The binding affinity between SEA and EGCG under in vivo conditions was examined using a model solution. Although after treatment under acidic and alkaline conditions, the presence of protein and the digestive tract model solution, EGCG still interacted with SEA. Our studies are the first to demonstrate the effect of the binding of EGCG to SEA on toxin activity.
Subject(s)
Catechin/analogs & derivatives , Enterotoxins/toxicity , Animals , Catechin/chemistry , Catechin/pharmacology , Cell Proliferation/drug effects , Cytokines/biosynthesis , Cytokines/genetics , Drug Interactions , Enterotoxins/pharmacology , Female , Gene Expression Regulation/drug effects , Hydrogen-Ion Concentration , Mice , Molecular Structure , Pancreatin , Pepsin A/pharmacology , Protein BindingABSTRACT
The first total synthesis of (7â³R,8â³R)-, (7â³S,8â³S)-isomers of princepin (1) and (7â³R,8â³R)-, (7â³S,8â³S)-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.
ABSTRACT
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.
Subject(s)
Native Polyacrylamide Gel Electrophoresis/methods , Receptor, Adenosine A2A/chemistry , Receptor, Adenosine A2A/isolation & purification , Crystallography, X-Ray/methods , Humans , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purificationABSTRACT
Staphylococcal enterotoxin A (SEA) is a toxin protein, and is the most common cause of staphylococcal food poisoning. Polyphenols, such as catechins, are known to interact with proteins. In this study, we investigated the binding of catechins to SEA using SPR (Biacore), Fourier transform infrared spectroscopy (FT-IR), isothermal titration calorimetry (ITC), and protein-ligand docking. We found that (−)-epigallocatechin gallate (EGCG) could strongly bind to SEA. According to thermodynamic parameters, a negative ΔG indicated that the interaction between EGCG and SEA was spontaneous, and the electrostatic force accompanied by hydrophobic binding forces may play a major role in the binding. Data from Western blot analysis and docking simulation suggest that the hydroxyl group at position 3 of the galloyl group in the catechin structure was responsible for binding affinity with the Y91 of the A-6 region of SEA active sites. Our results provide further understanding of the binding interactions between catechins and SEA, and the inhibition of toxin activities by catechins.
Subject(s)
Catechin/metabolism , Enterotoxins/metabolism , Calorimetry , Catalytic Domain , Catechin/chemistry , Enterotoxins/chemistry , Molecular Docking Simulation , Protein Binding , Spectroscopy, Fourier Transform Infrared , Surface Plasmon Resonance , ThermodynamicsABSTRACT
We accomplished divergent synthesis of potent kinase inhibitor BAY 61-3606 (1) and 27 derivatives via conjugation of imidazo[1,2-c]pyrimidine and indole ring compounds with aromatic (including pyridine) derivatives by means of palladium-catalyzed cross-coupling reaction. Spleen tyrosine kinase (Syk) and germinal center kinase (Gck, MAP4K2) inhibition assays showed that some of the synthesized compounds were selective Gck inhibitors.
Subject(s)
Imidazoles/chemistry , Protein Kinase Inhibitors/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrimidines/chemistry , Catalysis , Drug Evaluation, Preclinical , Germinal Center Kinases , Humans , Imidazoles/chemical synthesis , Imidazoles/metabolism , Indoles/chemistry , Inhibitory Concentration 50 , Niacinamide/analogs & derivatives , Niacinamide/chemistry , Niacinamide/metabolism , Palladium/chemistry , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Protein Serine-Threonine Kinases/metabolism , Pyrimidines/chemical synthesis , Pyrimidines/metabolism , Structure-Activity Relationship , Syk Kinase/antagonists & inhibitors , Syk Kinase/metabolismABSTRACT
Total syntheses of teadenols A and B, isolated from fermented tea, were accomplished in a highly stereocontrolled manner. Key steps were an organocatalytic asymmetric α-aminoxylation reaction of an aldehyde and a palladium-catalyzed intramolecular allylic substitution with phenol. In the latter reaction, we utilized the different conformational preferences of cyclic and acyclic carbonate precursors to obtain cis- and trans-fused benzopyran rings, respectively, via intramolecular etherification.
Subject(s)
Benzopyrans/chemistry , Phenols/chemistry , Phenols/chemical synthesis , Catalysis , Chemistry Techniques, Synthetic , Palladium/chemistry , StereoisomerismABSTRACT
Practical total syntheses of acromelic acids A (1) and B (2), which were scarce natural products isolated from toxic mushroom by Shirahama and Matsumoto, were accomplished in 13 (36% total yield) and 17 steps (6.9% total yield), respectively, from 2,6-dichloropyridine (8). Beginning with regioselective transformation of symmetric 8 by either ortho-lithiation or bromination, nitroalkenes 15 and 16 were provided. Stereoselective construction of the vicinal stereocenters at the C-3, 4 positions of 1 and 2 was performed by a Ni-catalyzed asymmetric conjugate addition of α-ketoesters to the nitroalkenes. Construction of the pyrrolidine ring was accomplished in a single operation via a sequence consisting of reduction of the nitro group, intramolecular condensation with the ketone, and reduction of the resulting ketimine.
Subject(s)
Kainic Acid/analogs & derivatives , Agaricales/chemistry , Kainic Acid/chemical synthesis , Kainic Acid/chemistry , Kainic Acid/isolation & purification , Molecular StructureABSTRACT
Polyamines, such as spermine (1), thermospermine (2) and norspermine (3), are widely distributed in nature, and have multiple biological activities. In addition, many of their conjugates have potential for pharmacological use. Here, we present a solid-phase synthesis using our nitrobenzenesulfonyl (Ns) strategy, which can provide 1, 2 and 3 on a gram scale. This approach should be suitable for facile construction of a diverse library of polyamines.
Subject(s)
Spermine/analogs & derivatives , Spermine/chemical synthesis , Molecular Structure , Spermine/chemistryABSTRACT
We describe a practical, large-scale synthesis of the "fairy-ring" plant-growth regulator 2-azahypoxanthine (AHX), and its biologically active hydroxyl metabolite (AOH) and riboside derivative (AHXr). AHXr, a biosynthetic intermediate, was synthesized from inosine via a biomimetic route. Biotinylated derivatives of AHX and AHXr were also synthesized as probes for mechanistic studies.
Subject(s)
Biotin/chemistry , Chemistry, Organic/methods , Hypoxanthines/chemical synthesis , Plant Growth Regulators/chemical synthesis , Staining and Labeling , Alkynes/chemistry , Azides/chemistry , Hypoxanthines/chemistry , Plant Growth Regulators/chemistryABSTRACT
Amyloid ß protein (Aß) self-assembles into insoluble fibrils, and forms the senile plaques associated with Alzheimer's disease. 3,3',4',5'-Tetrahydroxyflavone, a synthetic analogue of the natural flavonoid fisetin, has been found to potently inhibit Aß fibril formation. In the present study, we investigated how inhibition of Aß fibril formation by this flavonoid affects Aß conformation and neurotoxicity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of Aß1-42 (20 µM) incubated with or without 3,3',4',5'-tetrahydroxyflavone demonstrated that 3,3',4',5'-tetrahydroxyflavone (100 µM) rapidly caused formation of atypical Aß conformers, which appeared as a very broad, smear-like band in the high molecular weight region and were distinguishable from soluble Aß oligomers or mature Aß fibrils. Transmission electron microscopy (TEM) revealed that large spherical Aß aggregates were preferentially formed in the presence of 3,3',4',5'-tetrahydroxyflavone. The SDS-resistant, smear-like band on SDS-PAGE and the large spherical aggregates in TEM both disappeared after heat treatment (100°C, 10 min). Furthermore, a neurotoxicity assay with cultured rat hippocampal neurons demonstrated that Aß incubated with 3,3',4',5'-tetrahydroxyflavone was significantly less toxic than Aß incubated without the flavonoid. These results suggest that the newly synthesized fisetin analogue 3,3',4',5'-tetrahydroxyflavone directly produces atypical, large Aß aggregates and reduces Aß toxicity.
Subject(s)
Amyloid beta-Peptides/metabolism , Flavones/pharmacology , Protein Aggregates/drug effects , Animals , Cell Survival/drug effects , Flavonoids/pharmacology , Flavonols , Hippocampus/drug effects , Hippocampus/metabolism , Hippocampus/ultrastructure , Neurons/drug effects , Neurons/metabolism , Neurons/ultrastructure , Primary Cell Culture , Protein Conformation/drug effects , RatsABSTRACT
A lipid extract of Perna canaliculus (New Zealand green-lipped mussel) has reportedly displayed anti-inflammatory effects in animal models and in human controlled studies. However, the anti-inflammatory lipid components have not been investigated in detail due to the instability of the lipid extract, which has made the identification of the distinct active components a formidable task. Considering the instability of the active component, we carefully fractionated a lipid extract of Perna canaliculus (Lyprinol) and detected furan fatty acids (F-acids). These naturally but rarely detected fatty acids show potent radical-scavenging ability and are essential constituents of plants and algae. Based on these data, it has been proposed that F-acids could be potential antioxidants, which may contribute to the protective properties of fish and fish oil diets against chronic inflammatory diseases. However, to date, in vivo data to support the hypothesis have not been obtained, presumably due to the limited availability of F-acids. To confirm the in vivo anti-inflammatory effect of F-acids in comparison with that of eicosapentaenoic acid (EPA), we developed a semisynthetic preparation and examined its anti-inflammatory activity in a rat model of adjuvant-induced arthritis. Indeed, the F-acid ethyl ester exhibited more potent anti-inflammatory activity than that of the EPA ethyl ester. We report on the in vivo activity of F-acids, confirming that the lipid extract of the green-lipped mussel includes an unstable fatty acid that is more effective than EPA.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Fatty Acids/pharmacology , Perna/chemistry , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/isolation & purification , Arthritis, Experimental/drug therapy , Fatty Acids/chemistry , Fatty Acids/isolation & purification , Female , Furans/chemistry , Furans/isolation & purification , Furans/pharmacology , Humans , Lipids/chemistry , Male , Molecular Structure , Oncorhynchus keta/metabolism , Rats , Rats, Inbred Lew , Rats, Wistar , Testis/chemistryABSTRACT
We have established a novel method to evaluate the redox properties of tea polyphenols by HPLC-coulometric-array analysis. We plotted the quantity of electricity (µC) on the vertical axis and the electric potential (mV), adjusted with the associated palladium reference electrode, on the horizontal axis to provide "quantity versus potential (QP) plot". The patterns of the plots correspond to the derivative of a hydrodynamic voltammogram or a current-voltage curve, with the electric potentials of the peaks in the QP plot corresponding to the half-wave potentials in the current-voltage curve. We confirmed that catechins and theaflavins are oxidized depending on the electric potentials of their partial structures, and found that all compounds showing a peak at 0 mV in the QP plots produce hydrogen peroxide (H2O2) during the autoxidation process.
ABSTRACT
Rings or arcs of fungus-stimulated plant growth occur worldwide; these are commonly referred to as "fairy rings". In 2010, we discovered 2-azahypoxanthine (AHX), a compound responsible for the fairy-ring phenomenon caused by fungus; AHX stimulated the growth of all the plants tested. Herein, we reveal the isolation and structure determination of a common metabolite of AHX in plants, 2-aza-8-oxohypoxanthine (AOH). AHX is chemically synthesized from 5-aminoimidazole-4-carboxamide (AICA), and AHX can be converted into AOH by xanthine oxidase. AICA is one of the members of the purine metabolic pathway in animals, plants, and microorganisms. However, further metabolism of AICA remains elusive. Based on these results and facts, we hypothesized that plants themselves produce AHX and AOH through a pathway similar to the chemical synthesis. Herein, we demonstrate the existence of endogenous AHX and AOH and a novel purine pathway to produce them in plants.
Subject(s)
Hypoxanthines/metabolism , Oryza/metabolism , Purines/metabolism , Crystallography, X-Ray , Hypoxanthines/chemical synthesis , Hypoxanthines/chemistry , Molecular Conformation , Purines/chemistry , Xanthine Oxidase/metabolismABSTRACT
Staphylococcus aureus, a bacterium found on human skin, produces toxins and various virulence factors that can lead to skin infections such as atopic dermatitis. These toxins and virulence factors are carried in membrane vesicles (MVs), composed of the bacterium's own cell membranes, and are expected to reach host target cells in a concentrated form, inducing inflammation. This study investigated the effects of two polyphenols, (-)-epigallocatechin gallate (EGCG) and nobiletin (NOL), on the expression of S. aureus virulence factors and the inflammation induced by MVs. The study found that EGCG alone decreased the production of Staphylococcal Enterotoxin A (SEA), while both EGCG and NOL reduced biofilm formation and the expression of virulence factor-related genes. When S. aureus was cultured in a broth supplemented with these polyphenols, the resulting MVs showed a reduction in SEA content and several cargo proteins. These MVs also exhibited decreased levels of inflammation-related gene expression in immortalized human keratinocytes. These results suggest that EGCG and NOL are expected to inhibit inflammation in the skin by altering the properties of MVs derived from S. aureus.