RESUMO
Arboreous fruit of Schisandra chinensis Baillon, Schisandra Fruit (SF), is a crude drug used in Japanese traditional Kampo medicine. The marker compounds of SF for quality control are lignans, such as schizandrin (Sz) and gomisin A (GmA). Kampo formulation containing SF is usually prepared as decoctions in the dosage form of whole crude drug (W), as its size is small enough to measure using a spoon. However, in some traditional books, it has been described that SF must be used in the dosage form of crushed or cut pieces (Cr). In this study, we evaluated the transferring ratio of lignans from SF to the decoction, and the stability and taste of the decoctions of shoseiryuto (SST) and ninjin'yoeito (NYT) using each dosage form, i.e., Cr and W, of SF. The transferring ratio of Sz and GmA was significantly higher in the decoction prepared with the Cr form than that prepared using the W form in both SST and NYT. The concentration of Sz and GmA in the decoctions was stable when maintained at 4 °C for 35 days. The taste of SST decoction prepared using the Cr form was more acidic, harsher, and bitterer than SST decoction prepared using the W form, and the taste of NYT decoction prepared using the Cr form was harsher than NYT decoction prepared using the W form. In conclusion, when SF is used in Kampo prescription, crushing the fruits and seeds can increase its effectiveness.
Assuntos
Ciclo-Octanos/análise , Dioxóis/análise , Medicamentos de Ervas Chinesas/química , Lignanas/análise , Compostos Policíclicos/análise , Schisandra/química , Adulto , Feminino , Frutas/química , Humanos , Masculino , Medicina Kampo , Paladar , Adulto JovemRESUMO
The glycosphingolipid neurosporaside (α-D-Glcp-(1 â 2)-ß-D-Galp-(1 â 6)-ß-D-Galp-(1 â 6)-ß-D-Galp-(1 â)-Cer) occurs in Neurospora crassa. We attempted to synthesize neurosporaside by block synthesis (route A) and linear synthesis (route B). Oligosaccharide derivatives were synthesized using trimethylsilyltrifluoromethanesulfonate and N-iodosuccinimide/trifluoromethane sulfonic acid as promoters. The target tetrasaccharide could not be attained via route A, but route B showed potential: glycosidic bonds (ß-D-Galp-(1 â 6)-ß-D-Galp-(1 â 6)-ß-D-Galp) were formed stereoselectively, leading to the synthesis of glycosphingolipid 2.
Assuntos
Glicoesfingolipídeos/química , Glicoesfingolipídeos/síntese química , Neurospora crassa/química , Configuração de Carboidratos , Mesilatos/química , Estereoisomerismo , Succinimidas/química , Compostos de Trimetilsilil/químicaRESUMO
Aconite tuber is a representative crude drug for warming the body internally in Japanese Kampo medicine and Chinese traditional medicine. The crude drug is used in major prescriptions for the aged. Varieties of Aconitum plants are distributed throughout the Japanese Islands, especially Hokkaido. With the aim of identifying the medicinal potential of Aconitum plants from Hokkaido, 107 specimens were collected from 36 sites in the summer of 2011 and 2012. Their nuclear DNA region, internal transcribed spacer (ITS), and aconitine alkaloid contents were analyzed. Phylogenic analysis of ITS by maximum parsimony analysis showed that the majority of the specimens were grouped into one cluster (cluster I), separated from the other cluster (cluster II) consisting of alpine specimens. The aconitine alkaloid content of the tuberous roots of 76 specimens showed 2 aspects-specimens from the same collection site showed similar aconitine alkaloid profiles, and cluster I specimens from different habitats showed various alkaloid profiles. Environmental pressure of each habitat is presumed to have caused the morphology and aconitine alkaloid profile of these genetically similar specimens to diversify.
Assuntos
Aconitina/análise , Aconitum/química , Aconitum/classificação , Aconitum/genética , Japão , Tubérculos/químicaRESUMO
Photoaffinity labeling technology is a highly efficient method for cloning carbohydrate-binding proteins. When the carbohydrate probes are synthesized according to conventional methods, however, the reducing terminus of the sugar is opened to provide an acyclic structure. Our continued efforts to solve this problem led to the development of new molecular tools with an oligosaccharide structure that contains a phenyldiazirine group for the elucidation of carbohydrate-protein interactions. We investigated whether carbohydrate-lectin interactions are affected by differences in the glycosidic formation and synthesized three types of molecular tools containing Galp-GlcpNAc disaccharide ligands and a photoreactive group (1, 2, 3). Photoaffinity labeling validated the recognition of the new ligand by different glycosidic bonds. Photoaffinity labeling also demonstrated that both the reducing end sugar and non-reducing end sugar recognized the Erythrina cristagalli agglutinin.
Assuntos
Carboidratos/química , Lectinas/metabolismo , Marcadores de Fotoafinidade/química , Aglutininas/química , Aglutininas/metabolismo , Carboidratos/síntese química , Dissacarídeos/química , Dissacarídeos/metabolismo , Erythrina/metabolismo , Lectinas/química , Ligantes , Ligação Proteica , Raios UltravioletaRESUMO
In vitro display technologies such as ribosome display and messenger RNA (mRNA)/complementary DNA (cDNA) display are powerful methods that can generate library diversities on the order of 10(10-14). However, in mRNA and cDNA display methods, the end use diversity is two orders of magnitude lower than initial diversity and is dependent on the downstream processes that act as limiting factors. We found that in our previous cDNA display protocol, the purification of protein fusions by the use of streptavidin matrices from cell-free translation mixtures had poor efficiency (â¼10-15%) that seriously affected the diversity of the purified library. Here, we have investigated and optimized the protocols that provided remarkable purification efficiencies. The stalled ribosome in the mRNA-ribosome complex was found to impede this purification efficiency. Among the various conditions tested, destabilization of ribosomes by appropriate concentration of metal chelating agents in combination with an optimal temperature of 30°C were found to be crucial and effective for nearly complete isolation of protein fusions from the cell-free translation system. Thus, this protocol provided 8- to 10-fold increased efficiency of purification over the previous method and results in retaining the diversity of the library by approximately an order of magnitude-important for directed evolution. We also discuss the possible effects in the fabrication of protein chips.
Assuntos
DNA Complementar/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Sequência de Bases , DNA Complementar/química , Biblioteca Gênica , Conformação de Ácido Nucleico , Puromicina/química , Puromicina/metabolismo , RNA Mensageiro/química , Ribossomos/químicaRESUMO
Glycosphingolids and glycoproteins in cell membrane are thought to play important roles in variety biological phenomena. Our interests focused on elucidation of their functions at the molecular level. We are especially interested in organic chemistry and physical chemistry of carbohydrates. Physical part-Surface of Langmuir-Blodgett (LB) films which comprised of various gangliosides, sphingolipids and cholesterol are observed by atomic force microscopy (AFM). We studied distribution of model compounds related to an antigenic epitope from Bupleurum falcatum L. in G(M3) (GM3)-DPPC monolayers. We also investigated relation between disease and glycolids in cell membrane. Chemical part-New molecular tools for the elucidation of carbohydrate roles using photoaffinity labeling were developed. We showed that the structural difference between closed ring- and open ring-type carbohydrates affected binding affinity to proteins. This study confirmed that carbohydrate structure, in both non-reducing end and reducing end sugars, is deeply related to the recognition event between carbohydrates and proteins. As a result, they will be used in future studies to elucidate carbohydrate functions on cell surfaces.
Assuntos
Carboidratos/fisiologia , Membrana Celular , Glicolipídeos/fisiologia , Imagem Molecular/métodos , Técnicas Biossensoriais/métodos , Ligantes , Glicoproteínas de Membrana , Microscopia de Força Atômica , Marcadores de FotoafinidadeRESUMO
Oligomerization, conformational changes, and the consequent neurodegeneration of Alzheimer's ß-amyloid protein (AßP) play crucial roles in the pathogenesis of Alzheimer's disease (AD). Mounting evidence suggests that oligomeric AßPs cause the disruption of calcium homeostasis, eventually leading to neuronal death. We have demonstrated that oligomeric AßPs directly incorporate into neuronal membranes, form cation-sensitive ion channels ("amyloid channels"), and cause the disruption of calcium homeostasis via the amyloid channels. Other disease-related amyloidogenic proteins, such as prion protein in prion diseases or α-synuclein in dementia with Lewy bodies, exhibit similarities in the incorporation into membranes and the formation of calcium-permeable channels. Here, based on our experimental results and those of numerous other studies, we review the current understanding of the direct binding of AßP into membrane surfaces and the formation of calcium-permeable channels. The implication of composition of membrane lipids and the possible development of new drugs by influencing membrane properties and attenuating amyloid channels for the treatment and prevention of AD is also discussed.
RESUMO
Because carbohydrates and proteins bind with such low affinity, the nature of their interactions is not clear. Photoaffinity labeling with diazirin groups is useful for elucidating the roles of carbohydrates in these binding processes. However, when carbohydrate probes are synthesized according to this conventional method, the reducing terminus of the sugar is opened to provide an acyclic structure. Because greater elucidation of carbohydrate-protein interactions requires a closed-ring carbohydrate in addition to the photoreactive group, we synthesized new molecular tools. The carbohydrate ligands were synthesized in three steps (glycosylation with allyl alcohol, deprotection, and ozonolysis). Specific binding proteins for carbohydrate ligands were obtained by photoaffinity labeling. Closed ring-type carbohydrate ligands, in which the reducing sugar is closed, bound to lectins more strongly than open ring-type sugars. Carbohydrate to protein binding was observed using AFM.
Assuntos
Carboidratos/química , Lectinas/química , Marcadores de Fotoafinidade/química , Azirinas/química , Di-Hidropiridinas/química , Ligantes , Microscopia de Força Atômica , Marcadores de Fotoafinidade/síntese química , Marcadores de Fotoafinidade/farmacologia , Ligação ProteicaRESUMO
Mass spectrometry, especially tandem mass spectrometry, has been widely used in the field of analytical sciences for handling biological and chemical samples. The technique resolves molecular and fragment ions based on the mass to charge ratio. Energy-resolved mass spectrometry (ERMS) further provides an activation energy-related factor in the dissociation reaction. Therefore, it is a very powerful technique that can discriminate isomeric compounds. Despite the power of ERMS, useful information cannot be obtained when an analyte contains structural isomers. Carbohydrates carry multiple chiral centers, thus oligomers of monosaccharides can form a vast number of structural isomers. We decided to use such species in our endeavors to establish a method of identifying the 'purity' of an analyte solely based on mass spectrometry. In the present paper, we describe a stage-discriminated spectral correlation of ERMS, which not only enables identification of the presence of contaminants in an analyte, but also provides information regarding the 'purity' of fragment ions.
Assuntos
Espectrometria de Massas/métodos , Oligossacarídeos/química , Cromatografia Líquida de Alta Pressão , Isomerismo , Estrutura Molecular , Oligossacarídeos/classificaçãoRESUMO
Glycans, a family of compounds often attached to proteins and ceramides, are diverse molecules involved in a wide range of biological functions. Their structural analysis is necessary and is often carried out at the microscale level. Methods based on mass spectrometry are therefore used, although they do not provide information regarding isomeric structures often found in glycan structures. If one finds "factors" characteristic of a certain isomer, this information can be used to elucidate an unknown oligosaccharide sequence. One potential technique is to use energy-resolved mass spectrometry (ERMS) that has been used to distinguish a pair of isomeric compounds. Thus, compounds in a combinatorial library might be effectively used for this purpose. We analyzed a set of 16 isomeric disaccharides, the structures of which consisted of all possible combinations of anomeric configurations and interglycosidic linkage positions. All of the compounds were distinguished based on ERMS where normal collision-induced dissociation could distinguish only seven compounds. Furthermore, it was shown that alpha-glycosidic linkages of fucose were more reactive than the beta-isomers and the secondary glycosides were more reactive than the primary glycosides.
Assuntos
Algoritmos , Galactosídeos/química , Espectrometria de Massas/métodos , Análise Discriminante , Isomerismo , Conformação MolecularRESUMO
6-N-[2-(Tetradecyl)hexadecanamido]hexyl beta-D-glucopyranosyluronic acid-(1-->6)-beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside (1) and its clustering compound (2) carrying a tetravalent sugar unit, which are new model compounds related to a major antigenic epitope from antiulcer pectic polysaccharide of Bupleurum falcatum L., were synthesized and the distributions of 1 and 2 in mixed ganglioside (GM1, GD1a or GT1b)/phospholipid (DPPC) monolayers were observed using atomic force microscopy (AFM). AFM images showed that 1 was distributed in the GM1, GD1a and GT1b region of the mixed monolayers, in which 1 was miscible with GD1a. Specific distribution of 1 was observed in the mixed GM1/DPPC monolayer. Compound 2 was miscible with GM1, while 2 formed associations with GD1a and GT1b in the mixed monolayers. The distribution mode of 1 and 2 was different among the mixed ganglioside/DPPC monolayers.
Assuntos
Bupleurum/química , Epitopos/química , Gangliosídeos/química , Membranas Artificiais , Fosfolipídeos/síntese química , Configuração de Carboidratos , Sequência de Carboidratos , Epitopos/imunologia , Microscopia de Força Atômica , Dados de Sequência Molecular , Tamanho da Partícula , Pectinas/química , Fosfolipídeos/química , Sensibilidade e EspecificidadeRESUMO
A library composed of a complete set of fucopyranosyl-galactopyranosides was synthesized. A perbenzylated phenylthio fucopyranoside and a series of tri-O-benzyl-galactopyranosyl fluorides having single hydroxyl groups at the 2-, 3-, 4-, and 6-positions were used as the glycosyl donor and glycosyl acceptors, respectively. The chosen set of functionalities at the anomeric centers enabled rapid access to the oligosaccharides based on chemoselective activation. The first coupling reaction was achieved by the action of dimethyl(methylthio)sulfonium trifluoromethanesulfonate (DMTST). The resulting disaccharide fluoride was readily activated by hafnocene bistrifluoromethanesulfonate [Cp2Hf(OTf)2] and glycosidated with n-octanol.
Assuntos
Dissacarídeos/síntese química , Configuração de Carboidratos , Dissacarídeos/química , Fucose/análogos & derivados , Galactosídeos/síntese química , Estereoisomerismo , Tioglicosídeos/químicaRESUMO
A new, efficient synthesis of oligosaccharides, which involves solid-phase reactions without mixing in combination with an orthogonal-glycosylation strategy, is described. Despite a great deal of biological interest, the combinatorial chemistry of oligosaccharides is an extremely difficult subject. The problems include 1) lengthy synthetic protocols required for the synthesis and 2) the variety of glycosylation conditions necessary for individual reactions. These issues were addressed and solved by using the orthogonal-coupling protocol and the application of a temperature gradient to provide appropriate conditions for individual reactions. Furthermore, we succeeded in carrying out solid-phase reactions with neither mechanical mixing nor flow. In this report, the synthesis of a series of trisaccharides, namely, alpha/beta-L-Fuc-(1-->6)-alpha/beta-D-Gal-(1-->2/3/4/6)-alpha/beta-D-Glc-octyl, is reported to demonstrate the eligibility of the synthetic method in combinatorial chemistry.
Assuntos
Técnicas de Química Combinatória/métodos , Oligossacarídeos/síntese química , Configuração de Carboidratos , Glicosilação , Espectroscopia de Ressonância Magnética , Oligossacarídeos/química , Trissacarídeos/síntese química , Trissacarídeos/químicaRESUMO
The penetration of bovine serum albumin (BSA) into dipalmitoylphosphatidylglycerol (DPPG) monolayers was observed using atomic force microscopy (AFM) and surface pressure measurements. The effects of surface pressure, amount of BSA and the addition of ganglioside GM1 (GM1) were investigated. The surface pressure of the DPPG monolayer was increased by the penetration of BSA, and the increase in surface pressure was greater in the liquid-expanded film than that in the liquid-condensed film. The AFM images indicated that BSA penetrated into the DPPG monolayer. The amount of BSA that penetrated into the DPPG monolayer increased with time and with the amount of BSA added. On the contrary, the AFM image showed that BSA penetration into the mixed DPPG/GM1 (9 : 1) monolayer scarcely occurred. GM1 inhibited the penetration of BSA into the DPPG monolayer.
Assuntos
Membranas Artificiais , Microscopia de Força Atômica/métodos , Fosfatidilgliceróis/metabolismo , Soroalbumina Bovina/metabolismo , Animais , Bovinos , Permeabilidade/efeitos dos fármacos , Soroalbumina Bovina/análiseRESUMO
Glycosphingolipids isolated from larvae of the green-bottle fly, Lucilia caesar, have quite unique structures containing GlcNAcbeta-(1 --> 3)-Man and GalNAcbeta-(1 --> 4)-GlcNAcbeta-(1 --> 3)-Man. We have synthesized two glycosphingolipids, beta-D-GlcNAcp-(1 --> 3)-beta-D-Manp-(1 --> 4)-beta-D-Glcp-(1 --> 1)-Cer and beta-D-GalNAcp-(1 --> 4)-beta-D-GlcNAcp-(1 --> 3)-beta-D-Manp-(1 --> 4)-beta-D-Glcp-(1 --> 1)-Cer. A key reaction in the synthetic sequence is the application of the intramolecular aglycon delivery (IAD) approach for the synthesis of the beta-mannopyranosidic linkages.
Assuntos
Ascaris suum/química , Glicoesfingolipídeos/síntese química , Animais , Fucose , Invertebrados/química , Espectroscopia de Ressonância Magnética , Manose , Monossacarídeos/síntese química , Oligossacarídeos/síntese químicaRESUMO
A novel amphoteric glycosphingolipid, cholinephosphoryl-(-->6)-beta-D-GlcpNAc-(1-->3)-beta-D-Manp-(1-->4)-beta-D-Glcp-(1-->)-Cer, isolated from the porcine parasitic nematode, Ascaris suum, may be expected to be involved in host-parasite interactions. This glycosphingolipid analogue containing octyl residue in place of ceramide was synthesized as follows: The key reaction of this synthetic procedure is the formation of a intramolecular aglycon delivery (IAD) approach for beta-selective mannosylation. Then, a coupling of phosphocholine group at the position C-6'' of 16 was attempted using 2-chloro-2-oxo-1,3,2-dioxaphospholane, followed by reaction of the resulting cyclic phosphate intermediate with anhydrous trimethylamine to give 17. Subsequent debenzylation and debenzylidenation afforded target compound (2).