RESUMO
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.
Assuntos
Silibina , Silibina/síntese química , Silibina/química , Estereoisomerismo , Estrutura Molecular , Silimarina/síntese química , Silimarina/química , OxirreduçãoRESUMO
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.
Assuntos
Proliferação Nociva de Algas , Receptores Ionotrópicos de Glutamato , Ácido CaínicoRESUMO
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.
Assuntos
Agaricales , Triazinas , Agaricales/metabolismo , Hipoxantinas , Marasmius , Nitrogênio , Triazinas/metabolismoRESUMO
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.
Assuntos
Agaricales/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Hipoxantinas/metabolismo , Aminoimidazol Carboxamida/química , Aminoimidazol Carboxamida/isolamento & purificação , Aminoimidazol Carboxamida/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Cromatografia Líquida , Carpóforos/química , Hipoxantinas/química , Hipoxantinas/isolamento & purificação , Espectrometria de Massas em TandemRESUMO
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.
RESUMO
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.
Assuntos
Eletroforese em Gel de Poliacrilamida Nativa/métodos , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/isolamento & purificação , Cristalografia por Raios X/métodos , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
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.
Assuntos
Benzopiranos/química , Fenóis/química , Fenóis/síntese química , Catálise , Técnicas de Química Sintética , Paládio/química , EstereoisomerismoRESUMO
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.
Assuntos
Ácido Caínico/análogos & derivados , Agaricales/química , Ácido Caínico/síntese química , Ácido Caínico/química , Ácido Caínico/isolamento & purificação , Estrutura MolecularRESUMO
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.
Assuntos
Espermina/análogos & derivados , Espermina/síntese química , Estrutura Molecular , Espermina/químicaRESUMO
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.
Assuntos
Biotina/química , Química Orgânica/métodos , Hipoxantinas/síntese química , Reguladores de Crescimento de Plantas/síntese química , Coloração e Rotulagem , Alcinos/química , Azidas/química , Hipoxantinas/química , Reguladores de Crescimento de Plantas/químicaRESUMO
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.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Flavonas/farmacologia , Agregados Proteicos/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Flavonoides/farmacologia , Flavonóis , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/ultraestrutura , Cultura Primária de Células , Conformação Proteica/efeitos dos fármacos , RatosRESUMO
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.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Ácidos Graxos/farmacologia , Perna (Organismo)/química , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/isolamento & purificação , Artrite Experimental/tratamento farmacológico , Ácidos Graxos/química , Ácidos Graxos/isolamento & purificação , Feminino , Furanos/química , Furanos/isolamento & purificação , Furanos/farmacologia , Humanos , Lipídeos/química , Masculino , Estrutura Molecular , Oncorhynchus keta/metabolismo , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Testículo/químicaRESUMO
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.
Assuntos
Hipoxantinas/metabolismo , Oryza/metabolismo , Purinas/metabolismo , Cristalografia por Raios X , Hipoxantinas/síntese química , Hipoxantinas/química , Conformação Molecular , Purinas/química , Xantina Oxidase/metabolismoRESUMO
Many overseas healthcare researchers have advocated the importance of inter-professional education (IPE). However, to what extent laypersons, who reside in Japanese rural areas, understand IPE is not well known. To clarify this issue, we interviewed laypersons regarding their perception of IPE. We selected one rural area in Japan, where over 40% of the residents were more than 65 years old. Participants, who had an interest in their community healthcare system, were nominated. Semi-structured interviews of approximately 60 minutes were performed and topics focused on were: 1. laypersons' perception of IPE, 2. laypersons' views on educating future medical professions, and 3. what laypersons could contribute to the educating of future medical professionals. Nine participants (58 to 88 years, 1 female and 8 males, mean 76 years) took part. We found three common themes among the interviewees. These were: 1. little knowledge about IPE, 2. positive attitudes towards educating future medical professionals, 3. original ideas concerning how to educate medical professionals. We also found that laypersons in this rural region did not fully understand IPE. As one potential educational method, we should promote IPE involving laypersons. For future study, we plan to explore medical professionals' opinions, and compare them with those of laypersons'.
Assuntos
Educação Profissionalizante/métodos , Conhecimentos, Atitudes e Prática em Saúde , Ocupações em Saúde/educação , Relações Interprofissionais , População Rural , Idoso , Idoso de 80 Anos ou mais , Serviços de Saúde Comunitária , Feminino , Humanos , Entrevistas como Assunto/métodos , Japão , Masculino , Pessoa de Meia-IdadeRESUMO
In Japan in 2004, 59 people who had consumed angel-wing mushroom, Pleurocybella porrigens, experienced acute encephalopathy, and of these 17 died. We purified a lethal protein to mice, pleurocybelline (PC), from P. porrigens. Although PC caused no damage to the brain, PC formed a complex with a lectin (PPL) and showed exo-protease activity, degrading substrates from both N- and C-termini. In addition, the presence of an unstable toxic compound, pleurocybellaziridine (PA), in the mushroom was demonstrated. We hypothesized that the complex and PA are involved in disease development and verified that apoptotic cells in the hippocampus were significantly increased by injection of the mixture of PC, PPL, and PA, indicating that these substances might be involved in acute encephalopathy.
Assuntos
Agaricales , Encefalopatias , Intoxicação Alimentar por Cogumelos , Animais , Camundongos , Encéfalo , Encefalopatias/induzido quimicamente , Lectinas , Intoxicação Alimentar por Cogumelos/complicaçõesRESUMO
Angiogenesis, a process of construction of new blood capillaries, is crucial for tumor progression and metastasis. Our previous studies demonstrated that a component of green tea, epigallocatechin-3-gallate (EGCG), suppressed angiogenesis and subsequent tumor growth. In this study, to elucidate the detailed mechanism of the anti-angiogenic effect of EGCG and to enhance the antiangiogenic activity of EGCG, we designed and synthesized EGCG derivatives and examined their biological effect and intracellular localization in human umbilical vein endothelial cells (HUVECs). EGCG derivatives aminopentyl dideoxyEGCG and aminopentyl dideoxygallocatechin-3-gallate (cis-APDOEGCG and trans-APDOEGCG) had an enhanced inhibitory effect on the proliferation when used at more than 30 µM. To elucidate antiangiogenic effect of EGCG, we used a 1 µM concentration for subsequent experiments where no effect on proliferation was observed. These EGCG derivatives showed a stronger inhibitory effect on migration, invasion, and tube formation by HUVECs than the non-derivatized EGCG. Furthermore, the derivatives induced a change in the distribution of F-actin and subsequent morphology of the HUVECs. Next, we synthesized fluorescent TokyoGreen-conjugated EGCG derivative (EGCG-TG) and observed the distribution in HUVECs under a confocal laser scanning microscope. Abundant fluorescence was observed in the cells after a 3-h incubation, and was localized in mitochondria as well as in cytoplasm. These results suggest that EGCG was incorporated into the HUVECs, that a portion of it entered into their mitochondria.
Assuntos
Inibidores da Angiogênese/farmacologia , Camellia sinensis/química , Catequina/análogos & derivados , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Mitocôndrias/metabolismo , Extratos Vegetais/farmacologia , Actinas/metabolismo , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/uso terapêutico , Transporte Biológico , Catequina/síntese química , Catequina/farmacologia , Catequina/uso terapêutico , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citoplasma/metabolismo , Células Endoteliais/fisiologia , Células Endoteliais/ultraestrutura , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Humanos , Neovascularização Patológica/prevenção & controleRESUMO
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.
RESUMO
We have explored the utility, strength, and limitation of through-space exciton-coupled circular dichroism in determination of the secondary structure of optically active chromophoric nanoarrays using the example of end-capped porphyrin- and metalloporphyrin-oligodeoxynucleotide conjugates. We put special emphasis on the explanation of the origin and significance of the distinctive multiple bands in the CD spectra (trisignate and tetrasignate CD bands). Such CD profiles are often observed in chiral aggregates or multichromophoric arrays but have never before been studied in detail. We found that variation of temperature and ionic strength has a profound effect on the geometry of the porphyrin-DNA conjugates and thus the nature of electronic interactions. At lower temperatures and in the absence of NaCl all three 5'-DNA-porphyrin conjugates display negative bisignate CD exciton couplets of variable intensity in the Soret region resulting from through-space interaction between the electric transition dipole moments of the two end-capped porphyrins. As the temperature is raised these exciton couplets are transformed into single positive bands originating from the porphyrin-single-strand DNA interactions. At higher ionic strengths and low temperatures, multisignate CD bands are observed in the porphyrin Soret region. These CD signature bands originate from a combination of intermolecular, end-to-end porphyrin-porphyrin stacking between duplexes and porphyrin-DNA interactions. The intermolecular aggregation was confirmed by fluorescence and absorption spectroscopy and resonance light scattering. DeVoe theoretical CD calculations, in conjunction with molecular dynamics simulations and Monte Carlo conformational searches, were used to mimic the observed bisignate exciton-coupled CD spectra as well as multiple CD bands. Calculations correctly predicted the sign and shape of the experimentally observed CD spectra. These studies reveal that the exciton-coupled circular dichroism is a very useful technique for the determination of the structure of optically active arrays.
Assuntos
DNA/química , Porfirinas/química , Absorção , Sequência de Bases , Dicroísmo Circular , Cobre/química , DNA/genética , Dimerização , Luz , Metaloporfirinas/química , Simulação de Dinâmica Molecular , Método de Monte Carlo , Conformação de Ácido Nucleico , Concentração Osmolar , Espalhamento de Radiação , Cloreto de Sódio/química , Espectrometria de Fluorescência , Estereoisomerismo , Zinco/químicaRESUMO
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.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Arabidopsis/metabolismo , Redes e Vias Metabólicas/genética , Oryza/metabolismo , Purinas/metabolismo , Aminoimidazol Carboxamida/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Oryza/genética , Oryza/crescimento & desenvolvimentoRESUMO
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.