土肥 博史

<jats:p>Along with the increasing resistance of Candida spp. to some antibiotics, it is necessary to find new antifungal drugs, one of which is from the medicinal plant Red Betel (Piper crocatum). The purpose of this research is to isolate antifungal constituents from P. crocatum and evaluate their activities as ergosterol biosynthesis inhibitors via an in silico study of ADMET and drug-likeness analysis. Two new active compounds 1 and 2 and a known compound 3 were isolated, and their structures were determined using spectroscopic methods, while their bioactivities were evaluated via in vitro and in silico studies, respectively. Antifungal compound 3 was the most active compared to 1 and 2 with zone inhibition values of 14.5, 11.9, and 13.0 mm, respectively, at a concentration of 10% w/v, together with MIC/MFC at 0.31/1.2% w/v. Further in silico study demonstrated that compound 3 had a stronger ΔG than the positive control and compounds 1 and 2 with −11.14, −12.78, −12.00, and −6.89 Kcal/mol against ERG1, ERG2, ERG11, and ERG24, respectively, and also that 3 had the best Ki with 6.8 × 10−3, 4 × 10−4, 1.6 × 10−3, and 8.88 μM. On the other hand, an ADMET analysis of 1–3 met five parameters, while 1 had one violation of Ro5. Based on the research data, the promising antifungal constituents of P. crocatum allow P. crocatum to be proposed as a new antifungal candidate to treat and cure infections due to C. albicans.</jats:p>

<jats:p>Glycerol is a symmetrical, small biomolecule with high flexibility in molecular conformations. Using a 1H-NMR spectroscopic Karplus analysis in our way, we analyzed a rotational isomerism in the glycero backbone which generates three kinds of staggered conformers, namely gt (gauche-trans), gg (gauche-gauche), and tg (trans-gauche), at each of sn-1,2 and sn-2,3 positions. The Karplus analysis has disclosed that the three rotamers are consistently equilibrated in water keeping the relation of ‘gt:gg:tg = 50:30:20 (%)’ at a wide range of concentrations (5 mM~540 mM). The observed relation means that glycerol in water favors those symmetric conformers placing 1,2,3-triol groups in a gauche/gauche geometry. We have found also that the rotational isomerism is remarkably changed when the solvent is replaced with DMSO-d6 or dimethylformamide (DMF-d7). In these solvents, glycerol gives a relation of ‘gt:gg:tg = 40:30:30 (%)’, which means that a remarkable shift occurs in the equilibrium between gt and tg conformers. By this shift, glycerol turns to also take non-symmetric conformers orienting one of the two vicinal diols in an antiperiplanar geometry.</jats:p>

Osteoporosis is associated with increase in fat tissue in bone marrow in humans. Mesenchymal stem cells in bone marrow are induced to differentiate into osteoblasts rather than adipocytes by the stimulation of peroxisome proliferator-activated receptor (PPAR) γ antagonists. PPARγ antagonists are expected to be useful to prevent osteoporosis by regulating the lineages of mesenchymal stem cells in bone marrow, as well as the prevention of obesity. In this study, we explored natural components suppressing PPARγ transcriptional activity in rosemary. Separation of active fraction of rosemary extract by repeated high performance liquid chromatograph and PPARγ luciferase reporter assay identified erucic acid, one of the monounsaturated fatty acids, as an active component. Twenty-five-micrometer erucic acid significantly decreased PPARγ luciferase activity and enhanced the differentiation of mouse-delivered C3H10T1/2 cells into osteoblasts rather than adipocytes. Furthermore, 25-μM erucic acid significantly decreased the expression of adipocyte marker genes, while accelerating osteoblast marker genes. In conclusion, erucic acid is a novel natural component derived from rosemary regulating mesenchymal stem cell differentiation via suppression of PPARγ transcriptional activity.

Mycoplasma pneumoniae expresses β-glycolipids (β-GGLs) in cytoplasmic membranes, which possess a unique β(1 → 6)-linked disaccharide epitope, which has high potential in biochemical and medicinal applications. In the present study, a series of β-GGLs homologues with different acyl chains (C12, C14, C16, and C18) were prepared from a common precursor. An ELISA assay using an anti-(β-GGLs) monoclonal antibody indicated that the synthetic homologues with long acyl chains had greater diagnostic potential in the order C18 &gt C16 &gt C14 &gt C12. Toward a simultaneous detection of natural glycolipids by mass spectrometry (MS), a deuterium-labeled C16 homologue (β-GGL-C16-d3) was prepared and applied as an internal standard for a high-resolution electrospray ionization MS (ESI-MS) analysis. The ESI-MS analysis was used to identify and quantify acyl homologues (C16/C16, C16/C18, and C18/C18) of β-GGL-C16 in cultured M. pneumoniae. A β-GGLs homologue with a 1,2-diacetyl group (C2) was also prepared as a “water soluble” glycolipid homologue and characterized by 1H NMR spectroscopy. We envisage that each of these chemosynthetic homologues will provide promising approaches to solve medical and biological problems associated with mycoplasma infectious diseases (MIDs).

A facile methodology is proposed for regioselective conjugation between glycosyl donors and acceptors towards the development of regioselective 1,2-cis-glycosylation method. ortho-Formylphenyl 1-thio-β-d-galactopyranoside was regioselectively tethered to methyl α-d-glucopyranoside under acidic condition to furnish an 4,6-O-arylidene acetal-linked conjugate. This conjugate can be readily converted to an ether-linked 4-O- or 6-O-derivative by regioselective cleavage of the acetal ring. In the glycosylation reaction, the ether-linked 4-OH conjugate was found to show excellent 1,2-cis selectivity via an intramolecular 1,9-transfer.

Cell-membrane glycerophospholipids and glycolipids have a common asymmetric skeleton of 1,2-diacyl-sn-glycerols. The 1,2-diacyl moiety in solutions permits a rapid equilibrium among three helical conformers, namely gt(+), gg(-), and tg, to display diverse conformational properties. The conformational property changes variably not only by head groups at the sn-3 position, but also by the solvent conditions applied. Recently, we came across an empirical rule in the conformational diversity in the solution state when we assumed the term of 'helical disparity' for the equilibrium between gt(+) and gg(-) conformers with reversed helical signs for each other. The sign and magnitude of the helical disparity (%) governs the (+)- or (-)-chirality around the lipid tail and corresponds to the magnitude of the exciton couplet CD (circular dichroism) bands. The empirical rule expresses that the disparity (%) changes linearly by the function of gt(+) population (%). Herein, the rule was verified by H-1 NMR spectroscopy using different types of 1,2-diacyl-sn-glycerols as model compounds. The present paper describes that the rule is formulated with a general equation (Eq-1): 'helical disparity (%)' = [gt(+)-gg(-)] (%) = A[gt(+)-B], in which A and B are constants taking values around 1.3 and 38, respectively. This rule is maintained regardless of the 1,2-diacyl and sn-3 substituent groups as far as examined here, while affording several exceptions. With Eq-1 (A = 1.3, B = 38), a conformational diagram can be obtained. This allows us to overview the diverse helical conformational properties of the asymmetric 1,2-diacyl-sn-glycerols in the solutions state. (C) 2017 Elsevier Ltd. All rights reserved.

Cell-membrane glycerolipids exhibit a common structural backbone of asymmetric 1,2-diacyl-sn-glycerol bearing polar head groups in the sn-3 position. In this study, the possible effects of sn-3 head groups on the helical conformational property around the 1,2-diacyl moiety in the solution state were examined. 1H NMR Karplus relation studies were carried out using a series of 1,2dipalmitoyl-sn-glycerols bearing different sn-3 substituents (namely palmitoyl, benzyl, hydrogen, and phosphates). The 1H NMR analysis indicated that the helical property around the 1,2-diacyl moiety is considerably affected by these sn-3 substituents. The sn-3 hydroxy group induced a unique helical property, which was considerably dependent on the solvents used. In CDCl3 solution, three staggered conformers, namely gt(+), gg(-) and tg, were randomized, while in more polar solvents, the gt(+) conformer with (+)-helicity was amplified at the expense of gg(-) and tg conformers. The sn-3 phosphocholine in phosphatidylcholine exhibited a greater effect on the gt(+) conformer, which was independent of the solvents used. From the 1H NMR analysis, the helical conformational properties around the 1,2-diacyl moiety conformed to a simple empirical rule, which permitted the proposal of a conformational diagram for 1,2-dipalmitoyl-sn-glycerols in the solution states.

The titled 1,2-diacyl-sn-glycerol constitutes a common asymmetric backbone of cell-membrane phospholipids and glycolipids. The acyclic sn-glycerol skeleton allows flexible conformational changes by free rotation along each of the glycerol sn-1,2 and sn-2,3 C-C single bonds in solution. It is generally believed, however, that the sn-1,2 tail position adopts two gauche conformers [gt-(+) and gg-(-)] near exclusively to display an ordered conformation. In the present study, our interest was focused on the fact that the two gauche conformers possess helical signs which are the reverse of each other. We examined (+)- and (-)-chirality arising from the disparity between the two helical conformers using a series of 1,2-dibenzoyl-sn-glycerols 2-8 as model compounds, which possess 1,2-dibenzoate chromophore for circular dichroism analysis, while having variable substituent groups at the sn-3 position. First, the analysis was conducted with the circular dichroism 'dibenzoate exciton coupling methodology' of Nakanishi and Harada followed by H-1 NMR analysis to calculate the time-averaged populations (%) of the three staggered conformers, that is, gt-(+), gg-(-) and tg. The results are discussed in terms of the 'helicity index' which covers information on the sign and intensity, of circular dichroism bands in circular dichroism analysis as well as on the helical disparity (%) and the total volume (%) of the two helical conformers in H-1 NMR analysis. The two independent spectroscopic approaches were well matched in the estimation of the helical index, which in turn allowed us to conclude that the sn-3 substituting groups have diverse effects on the helical properties of 1,2-diacyl-sn-glycerols in solution. (C) 2015 Elsevier Ltd. All rights reserved.

Glycosyl-[60]fullerenes were first used as decontaminants against ricin, a lactose recognition proteotoxin in the <italic>Ricinus communis</italic> family. A fullerene glycoconjugate carrying two lactose units was synthesized by a [3 + 2] cycloaddition reaction between C₆₀ and the azide group in 6-azidohexyl β-lactoside per-<italic>O</italic>-acetate. A colloidal aqueous solution with brown color was prepared from deprotected bis(lactosyl)-C₆₀ and was found stable for more than 6 months keeping its red color. Upon mixing with an aqueous solution of <italic>Ricinus communis</italic> agglutinin (RCA₁₂₀), the colloidal solution soon caused precipitations, while becoming colorless and transparent. In contrast, a solution of concanavalin A (Con A) caused no apparent change, indicating that the precipitation was caused specifically by carbohydrate–protein interactions. This notable phenomenon was quantified by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the results were discussed in terms of detection and decontamination of the deadly biological toxin in the <italic>Ricinus communis</italic> family.

We investigated in this study the effect of modified arabinoxylan from rice bran (MGN-3) and its fractions on <small>D</small>-galactosamine (<small>D</small>-GalN)-induced IL-18 expression and hepatitis in rats. Male Wistar rats were pretreated with MGN-3 or fractions of the MGN-3 hydrolysate, or with saline 1 h before administering <small>D</small>-GalN (400 mg/kg B.W.). The serum transaminase activities, IL-18 mRNA expression level in the liver and IL-18 concentration in the serum were determined 24 h after injecting <small>D</small>-GalN. Both the oral and intraperitoneal administration of MGN-3 (20 mg/kg B.W.) alleviated <small>D</small>-GalN-induced hepatic injury under these experimental conditions. The low-molecular-weight fraction (LMW) of MGN-3 showed the strongest protective effect on <small>D</small>-GalN-induced liver injury, its main sugar component being glucose. Moreover, the <small>D</small>-GalN-induced IL-18 expression was significantly reduced by treating with MGN-3 and LMW. The results suggest that MGN-3 and LMW could provide significant protection against <small>D</small>-GalN liver injury, and that IL-18 might be involved in their protective influence.

<italic>Mycoplasma fermentans</italic> possesses unique α-glycolipid antigens (GGPL-I and GGPL-III) at the cytoplasm membrane, which carry a phosphocholine group at the sugar primary (6-OH) position. This paper describes a practical synthetic pathway to a GGPL-I homologue (C_16:0) and its diastereomer, in which our one-pot α-glycosylation method was effectively applied. The synthetic GGPL-I isomers were characterized with ¹H NMR spectroscopy to determine the equilibrium among the three conformers (<italic>gg</italic>, <italic>gt</italic>, <italic>tg</italic>) at the acyclic glycerol moiety. The natural GGPL-I isomer was found to prefer <italic>gt</italic> (54%) and <italic>gg</italic> (39%) conformers around the lipid tail<italic>,</italic> while adopting all of the three conformers with equal probability around the sugar position. This property was very close to what we have observed with respect to the conformation of phosphatidylcholine (DPPC), suggesting that the <italic>Mycoplasma</italic> glycolipids GGPLs may constitute the cytoplasm fluid membrane together with ubiquitous phospholipids, without inducing stereochemical stress.

Galacto-trehalose (GT) is a novel class of 1,1'-linked nonreducing disaccharide having an alpha-galactoside epitope. In this study, a pair of alpha,alpha- and alpha,beta-GT isomers were prepared in one pot with our alpha-glycosylation method, converted into vinyl monomers and then subjected to radical copolymerization with a second sugar (4-acrylamidophemyl beta-Glc or beta-GlcNAc) in the presence of acrylamide. The derived glycopolymers were assayed with alpha-galactoside-specific proteins (BSI-B-4 lectin and Shiga toxin-1) to show the results that both alpha,alpha- and alpha,beta-isomers are recognized by these carbohydrate-binding proteins more strongly in forms of the GT polymers. Moreover, the glycopolymer carrying both alpha,alpha-GT and beta-GlcNAc along the polymer chain showed an integrated detoxifying activity to the E. coli toxin as the result of a "module effect" of the second sugar.

We have purified and characterized an oligoxylosyl transfer enzyme (OxtA) from Bacillus sp. strain KT12. In the present study, a N-terminally His-tagged recombinant form of the enzyme, OxtA(H)(E), was overproduced in Escherichia coli and applied to the reaction with xylan and hydroquinone to produce 4-hydroxyphenyl beta-D-oligoxylosides, beta-(Xyl)(n)-HQ (n = 1-4), by one step reaction. The obtained beta-(Xyl)(n)-HQ inhibited mushroom tyrosinase, which catalyzes the oxidation of L-DOPA to L-DOPA quinine, and the IC50 values of beta-Xyl-HQ, beta-(Xyl)(2)-HQ, beta-(Xyl)(3)-HQ, and beta-(Xyl)(4)-HQ were 3.0, 0.74, 0.48, and 0.18 mM respectively. beta-(Xyl)(4)-HQ showed 35-fold more potent inhibitory activity than P-arbutin (4-hydroxyphenyl beta-D-glucopyranoside), of which the IC50 value was measured to be 6.3 mM. Kinetic analysis revealed that beta-(Xyl)(2)-HQ, beta-(Xyl)(3)-HQ, and beta-(Xyl)(4)-HQ competitively inhibited the enzyme, and the corresponding K-i values were calculated to be 0.20, 0.29, and 0.057 mM respectively.

just recently, a pair of beta-glycolipids was isolated from the cell membrane of Mycoplasma pneumoniae as a mixture of the two compounds. They are the major immunodeterminants of this pathogenic Mycoplasma and indicate high medicinal potential. They have a beta-(1 -&gt; 6)-linked disaccharide structure close to each other: one has beta-D-galactopyranoside (beta-Gal-type 1) at the non-reducing terminal, and another has beta-D-glucopyranoside (beta-Glc-type 2). In the present Study, the first stereoselective synthesis was conducted for each of the two beta-glycolipids 1 and 2. H-1 NMR and TLC-immunostaining studies of the synthetic compounds enable LIS to establish the absolute structures having the beta-(1 -&gt; 6)-linked disaccharides at the glycerol sn-3 position. (C) 2008 Elsevier Ltd. All rights reserved.

Heptosides are found in important bacterial glycolipids such as lipopolysaccliaride (LPS). the biosynthesis of which is targeted for the develoopment of novel antibacterial agents. This work describes the synthesis of a fluorinated analogue of ADP-L-glycero-beta-D-manno-heptopyranose, the donor substrate of the heptosyl transferase WaaC, which catalyzes the incorporation of this carbohydrate into LPS. Synthetically, the key step for the preparation of ADP-2F-heptose is the Simultaneous and stercoselective installation of both the fluorine atom at C-2 and the phosphoryl group at C-1 through a selectfluor-mediated (selectfluor= 1-clilol-omettivi-4-fluorodiazonia-bicy[2.2.2]octane bis(triflate)) electrophific addition/nucleophilic substitution involving a heptosylplycal. Therefore, we detail in this article 1) the stereoselective preparation of the key intermediates heptosylglycals, 2) the development of a new fluorophosphorylation procedure allowing an excellent beta-gluco stereoselectivity with "all-equatorial" glycals, 3) the synthesis of the target ADP-2F-heptose, and 4) some comments on the contacts observed between the fluorine atom of the final molecule and the protein in the crystallographic structure of heptosyltransferase WaaC.

Single-wall carbon nanotubes (SWNTs) can be well-dispersed in water by wrapping with short segments of natural DNA from salmon sperm. We report here the isolated DNA-wrapped SWNT hybrids. Measurements were carried out using UV-vis-NIR, near-infrared photoluminescence (PL) spectrum and atomic force microscopy (AFM). A possible charge transport between SWNTs and salmon-DNA is discussed in terms of observed spectral shifts in the photoluminescence spectra.

Lipopolysaccharides constitute the outer leaflet of the outer membrane of Gram-negative bacteria and are therefore essential for cell growth and viability. The heptosyltransferase WaaC is a glycosyltransferase (GT) involved in the synthesis of the inner core region of LPS. It catalyzes the addition of the first L-glycero-D-manno-heptose (heptose) molecule to one 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue of the Kdo(2)-lipid A molecule. Heptose is an essential component of the LPS core domain; its absence results in a truncated lipopolysaccharide associated with the deep-rough phenotype causing a greater susceptibility to antibiotic and an attenuated virulence for pathogenic Gram-negative bacteria. Thus, WaaC represents a promising target in antibacterial drug design. Here, we report the structure of WaaC from the Escherichia coli pathogenic strain RS218 alone at 1.9 angstrom resolution, and in complex with either ADP or the non-cleavable analog ADP-2-deoxy-2-fluoro-heptose of the sugar donor at 2.4 angstrom resolution. WaaC adopts the GT-B fold in two domains, characteristic of one glycosyltransferase structural superfamily. The comparison of the three different structures shows that WaaC does not undergo a domain rotation, characteristic of the GT-B family, upon substrate binding, but allows the substrate analog and the reaction product to adopt remarkably distinct conformations inside the active site. In addition, both binary complexes offer a close view of the donor subsite and, together with results from site-directed mutagenesis studies, provide evidence for a model of the catalytic mechanism. (c) 2006 Published by Elsevier Ltd.

A novel multivalent carbohydrate-CNT hybrid has been synthesized from SWNT and glycoconjugate polymer, carrying carbohydrate as pendant groups at side chain. Dispersion of SWNT in aqueous solution by using glycoconjugate polymer is achieved by sonication and ultracentrifugation. The multivalent carbohydrate-CNT hybrid is revealed to be photoluminescence active by near-infrared fluorescence spectroscopy. AFM measurements of the hybrid support not only the wrapping of SWNT by glycoconjugate polymer but also successful carbohydrate attachment of individual SWNT. (c) 2006 Elsevier B.V. All rights reserved.

A new hybrid material C10H2@SWNTs, single-wall carbon nanotubes (SWNTs) encapsulating C10H2 polyyne molecules, has been synthesized and confirmed by Raman spectroscopy and X-ray diffraction measurement. The C10H2 polyyne molecules composed of linearly bonded sp-carbon atoms are aligned inside the SWNTs. This hybrid material exhibits a characteristic Raman signal at 2066 cm(-1) attributable to the stretching vibration of the C10H2 molecules interacting with SWNTs. Being trapped inside the SWNTs, the otherwise unstable C10H2 molecules are shown to be stable well above 300 degrees C under dry-air conditions. (c) 2006 Elsevier B.V. All rights reserved.