Detection of homogentisic acid by electrospray ionization mass spectrometry.

OBJECTIVE: Homogentisic acid (HGA) is excreted in excessive amounts in the urine of patients with alkaptonuria, which is a hereditary metabolic disorder of phenylalanine and tyrosine. Therefore, the detection of HGA in urine is useful for the diagnosis of alkaptonuria. To evaluate the detection of HGA, we confirmed the color shift of HGA solutions and analyzed them by electrospray ionization mass spectrometry (ESI-MS). METHODS: We observed the color change of the HGA solutions under different pH conditions (pH 6.0, 7.0, and 8.0) and examined the influences of adding potassium hydroxide (KOH) and ascorbic acid (AA) to the HGA solutions. Then, we analyzed the chemical reaction in HGA solutions using ESI-MS. RESULTS: The HGA solution at pH 8.0 became brown after incubation at room temperature for 24 h and became darker brown with the addition of KOH; however, HGA solutions at pH 6.0 and 7.0 showed no color changes. The brown color change of the HGA solution at pH 8.0 was also inhibited by AA. Moreover, all HGA sample solutions showed the deprotonated molecular ion peak at m/z 167.035 in the negative ion mode after incubation at room temperature for 24 h and with the addition of KOH and AA. CONCLUSION: We identified the molecular ion of HGA in all sample solutions by ESI-MS, regardless of different pH conditions, color changes, or the presence of AA. These results suggest that spectral analysis by ESI-MS is suitable for the detection of HGA and the diagnosis of alkaptonuria.

12,20-Epoxypregnane Glycosides from the Roots of Oxypetalum caeruleum.

The MeOH extract from dried roots of Oxypetalum caeruleum (Apocynaceae, formerly known as Asclepiadaceae) plants yielded twenty new pregnane glycosides, some of which had a new 12,20-epoxy type aglycone. The structures of these compounds were established using NMR, MS spectroscopic analysis and chemical evidence.

Synthesis and rotational dynamics of diazamacrocycles having bridged 1,4-naphthylene as framed molecular rotors.

The rotation of a part of a molecule has attracted attention because of its possible role in the development of an artificial molecular rotor. In this study, 1,4-naphthylene bridged diazamacrocycles, in which the 1,4-diaminonaphthalene moiety is bridged by two long alkyl chains, were designed as novel framed molecular rotors, and the dependence of the rotation on the frame size were investigated. Framed rotors CnNp (n = 12, 14, 16, 18), where the number in the compound name indicates the length of a side chain, were synthesized by direct cyclization of 1,4-naphthalenediamine with α,ω-dihaloalkane in the presence of Na2CO3 as the base. The rotation of the naphthylene rotor in C12Np, C14Np, and C16Np was nearly suppressed in solution, whereas the rotor in C18Np showed rotation, as confirmed by the temperature-dependent coalescence of the NMR signals of the α-CH2.

C21 Steroidal Glycosides from the Roots of Oxypetalum caeruleum.

The MeOH extract from dried roots of Oxypetalum caeruleum (Apocynaceae) plants yielded seventeen new pregnane glycosides, some of which had the acylated-ramanone or -isoramanone type aglycone. The structures of these compounds were established using NMR, MS spectroscopic analysis and chemical evidence.

Preparation of cage-shaped hexakis(spiroborate)s.

In previous research studies, various types of prismatic cage-shaped molecular containers have been prepared and evaluated in terms of their guest inclusion properties. Basically, most of these molecular cages have a cationic or electron-deficient nature, and exhibit strong affinity mainly toward electron-rich aromatic guests. On the other hand, there is no report concerning anionic prismatic cages that are expected to recognize cationic polyaromatic guests with various structures and functions. In this manuscript, we present the preparation of hexakis(spiroborate)-type molecular cages, which was achieved by the reaction of phenylene- or biphenylenebis(dihydroxynaphthalene), hexahydroxytriphenylene, and boric acid in N,N-dimethylformamide. Their triangular prismatic hollow structures were confirmed by X-ray crystallographic analysis, and it was found that both phenylene- and biphenylene-bridged spiroborate cages have internal cavities of the corresponding size. It was also revealed that tetra(n-butylammonium) cations located inside the cavity and between the two adjacent spiroborate cages resulted in the formation of a one-dimensional columnar array. The molecular recognition behavior of the spiroborate cages was evaluated using tris(pyridinium)triazines as tricationic aromatic guests. 1H NMR measurement implied that a discrete 1 : 1 host-guest complex was formed when 1 equiv. of guest was added to the cage, whereas infinite one-dimensional aromatic stacks were constructed by the addition of 2 equiv. of guest.

Hollow and Solid Spheres Assembled from Functionalized Macrocycles Containing Adamantane.

An adamantane-based macrocycle possessing eight hydroxyl groups (1) was synthesized, in which the macrocyclic framework comprises two disubstituted adamantane molecules bearing phenyl derivatives connected to two biphenylene spacers by oxygen atoms. Furthermore, functionalized macrocycles containing methyl (2) and methoxycarbonylmethyl (3) groups were prepared. From the X-ray crystallographic analysis, the backbone of the macrocycles in all crystals had a nearly hexagonal shape with a cavity and these macrocycles could be arranged into different tubular structures dependent on the substituents. In acetone, macrocycle (1) formed stable hollow spherical aggregates with multilayer membranes. In contrast, macrocycle (3) exhibited no production of self-assembled materials in chloroform. The addition of hexane into the solution caused the generation of solid spheres and their fused network aggregates, which were finally transformed into crystals owing to the solvent effects.

Formation of a Ruthenium(V)-Imido Complex and the Reactivity in Substrate Oxidation in Water through the Nitrogen Non-Rebound Mechanism.

A RuII-NH3 complex, 2, was oxidized through a proton-coupled electron transfer (PCET) mechanism with a CeIV complex in water at pH 2.5 to generate a RuV═NH complex, 5. Complex 5 was characterized with various spectroscopies, and the spin state was determined by the Evans method to be S = 1/2. The reactivity of 5 in substrate C-H oxidation was scrutinized in acidic water, using water-soluble organic substrates such as sodium ethylbenzene-sulfonate (EBS), which gave the corresponding 1-phenylethanol derivative as the product. In the substrate oxidation, complex 5 was converted to the corresponding RuIII-NH3 complex, 3. The formation of 1-phenylethanol derivative from EBS and that of 3 indicate that complex 5 as the oxidant does not perform nitrogen-atom transfer, in sharp contrast to other high-valent metal-imido complexes reported so far. Oxidation of cyclobutanol by 5 afforded only cyclobutanone as the product, indicating that the substrate oxidation by 5 proceeds through a hydride-transfer mechanism. In the kinetic analysis on the C-H oxidation, we observed kinetic isotope effects (KIEs) on the C-H oxidation with use of deuterated substrates and remarkably large solvent KIE (sKIE) in D2O. These positive KIEs indicate that the rate-determining step involves not only cleavage of the C-H bond of the substrate but also proton transfer from water molecules to 5. The unique hydride-transfer mechanism in the substrate oxidation by 5 is probably derived from the fact that the RuIV-NH2 complex (4) formed from 5 by 1e-/1H+ reduction is unstable and quickly disproportionates into 3 and 5.

Combined analysis of 1,3-benzodioxoles by crystalline sponge X-ray crystallography and laser desorption ionization mass spectrometry.

The crystalline sponge (CS) method, which employs single-crystal X-ray diffraction to determine the structure of an analyte present as a liquid or an oil and having a low melting point, was used in combination with laser desorption ionization mass spectrometry (LDI-MS). 1,3-Benzodioxole derivatives were encapsulated in CS and their structures were determined by combining X-ray crystallography and MS. After the X-ray analysis, the CS was subjected to imaging mass spectrometry (IMS) with an LDI spiral-time-of-flight mass spectrometer (TOF-MS). The ion detection area matched the microscopic image of the encapsulated CS. In addition, the accumulated 1D mass spectra showed that fragmentation of the guest molecule (hereafter, guest) can be easily visualized without any interference from the fragment ions of CS except for two strong ion peaks derived from the tridentate ligand TPT (2,4,6-tris(4-pyridyl)-1,3,5-triazine) of the CS and its fragment. X-ray analysis clearly showed the presence of the guest as well as the π-π, CH-halogen, and CH-O interactions between the guest and the CS framework. However, some guests remained randomly diffused in the nanopores of CS. In addition, the detection limit was less than sub-pmol order based on the weight and density of CS determined by X-ray analysis. Spectroscopic data, such as UV-vis and NMR, also supported the encapsulation of the guest through the interaction between the guest and CS components. The results denote that the CS-LDI-MS method, which combines CS, X-ray analysis and LDI-MS, is effective for structure determination.

Identification of significant amino acids in multiple transmembrane domains of human transient receptor potential ankyrin 1 (TRPA1) for activation by eudesmol, an oxygenized sesquiterpene in hop essential oil.

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel that is activated by various noxious or irritant substances in nature, including spicy compounds. Many TRPA1 chemical activators have been reported; however, only limited information is available regarding the amino acid residues that contribute to the activation by non-electrophilic activators, whereas activation mechanisms by electrophilic ligands have been well characterized. We used intracellular Ca(2+) measurements and whole-cell patch clamp recordings to show that eudesmol, an oxygenated sesquiterpene present at high concentrations in the essential oil of hop cultivar Hallertau Hersbrucker, could activate human TRPA1. Gradual activation of inward currents with outward rectification by eudesmol was observed in human embryonic kidney-derived 293 cells expressing human TRPA1. This activation was completely blocked by a TRPA1-specific inhibitor, HC03-0031. We identified three critical amino acid residues in human TRPA1 in putative transmembrane domains 3, 4, and 5, namely threonine at 813, tyrosine at 840, and serine at 873, for activation by ß-eudesmol in a systematic mutational study. Our results revealed a new TRPA1 activator in hop essential oil and provide a novel insight into mechanisms of human TRPA1 activation by non-electrophilic chemicals.

Homogeneous Photocatalytic Water Oxidation with a Dinuclear Co(III)-Pyridylmethylamine Complex.

A bis-hydroxo-bridged dinuclear Co(III)-pyridylmethylamine complex (1) was synthesized and the crystal structure was determined by X-ray crystallography. Complex 1 acts as a homogeneous catalyst for visible-light-driven water oxidation by persulfate (S2O8(2-)) as an oxidant with [Ru(II)(bpy)3](2+) (bpy = 2,2'-bipyridine) as a photosensitizer affording a high quantum yield (44%) with a large turnover number (TON = 742) for O2 formation without forming catalytically active Co-oxide (CoO(x)) nanoparticles. In the water-oxidation process, complex 1 undergoes proton-coupled electron-transfer (PCET) oxidation as a rate-determining step to form a putative dinuclear bis-µ-oxyl Co(III) complex (2), which has been suggested by DFT calculations. Catalytic water oxidation by 1 using [Ru(III)(bpy)3](3+) as an oxidant in a H2(16)O and H2(18)O mixture was examined to reveal an intramolecular O-O bond formation in the two-electron-oxidized bis-µ-oxyl intermediate, prior to the O2 evolution.

Matured hop extract reduces body fat in healthy overweight humans: a randomized, double-blind, placebo-controlled parallel group study.

BACKGROUND: Hops are the main components of beer that provide flavor and bitterness. Iso-α-acids, the bitter components of beer, have been reported to reduce body fat in humans, but the bitterness induced by effective doses of iso-α-acids precludes their acceptance as a nutrient. The matured hop bitter acids (MHBA) of oxidized hops appear to have a more pleasant bitterness compared to the sharper bitterness of iso-α-acids. While there has been little information concerning the identity of the MHBA compounds and their physiological effects, MHBA was recently found to be primarily composed of oxides derived from α-acids, and structurally similar to iso-α-acids. Here, we investigated the effects of matured hop extract (MHE) containing MHBA on reducing abdominal body fat in healthy subjects with a body mass index (BMI) of 25 to below 30 kg/m(2), classified as "obese level 1" in Japan or as "overweight" by the WHO. TRIAL DESIGN: A randomized, double-blind, placebo-controlled parallel group study. METHODS: Two hundred subjects (male and female aged 20 to below 65 years with a BMI of 25 or more and less than 30 kg/m(2)) were randomly assigned to two groups. During a 12-week ingestion period, the subjects in each group ingested daily 350 mL of test-beverage, either containing MHE (with 35 mg MHBA), i.e. the namely active beverage, or a placebo beverage without MHE. The primary endpoint was reduction of the abdominal fat area as determined by CT scanning after continual ingestion of MHE for 12 weeks. RESULTS: Compared to the placebo group, a significant reduction was observed in the visceral fat area after 8 and 12 w, and in the total fat area after 12 w in the active group. There was also a concomitant decrease in body fat ratio in the active group compared to the placebo group. No adverse events related to the test beverages or clinically relevant abnormal changes in the circulatory, blood and urine parameters were observed in either group. CONCLUSIONS: The present study suggests that continual ingestion of MHE safely reduces body fat, particularly the abdominal visceral fat of healthy overweight subjects. TRIAL REGISTRATION: UMIN-CTR UMIN000014185.

Peptide [4]Catenane by Folding and Assembly.

A topologically complex peptide [4]catenane with the crossing number of 12 was synthesized by a folding and assembly strategy wherein the folding and metal-directed self-assembly of a short peptide fragment occur simultaneously. The latent Ω-looped conformation of the Pro-Gly-Pro sequence was found only when pyridines at the C- and N-termini coordinatively bind metal ions (Ag(I) or Au(I) ). Crystallographic studies revealed that the Ω-looped motifs formed four M3 L3 macrocycles that were intermolecularly entwined to generate an unprecedented peptide [4]catenane topology.

Capsule-Capsule Conversion by Guest Encapsulation.

Guest-induced M18 L6 -M24 L8 capsule-capsule conversion is reported. Both capsules are composed of Pd(II) ethylenediamine units (M) and 1,3,5-tris(3,5-pyrimidyl)pyrimidine (L), and form trigonal bipyramidal (M18 L6 ) and octahedral (M24 L8) closed-shell structures with huge hydrophobic inner spaces. The M18 L6 trigonal bipyramid is converted to the M24 L8 octahedron through encapsulation of large aromatic guests, with the latter capsule possessing a cavity volume three times larger than the former. Despite the dynamic properties of the capsule host, the encapsulated guests are difficult to extract and are thus isolated from the external environment.

ε-Viniferin, a resveratrol dimer, prevents diet-induced obesity in mice.

Red wines are thought to be one of the major dietary sources of trans-resveratrol. The beneficial effects of t-resveratrol against metabolic disorders have been well characterized, however, red wines also contain various resveratrol derivatives whose health benefits have not been completely elucidated. In this report, we investigated ε-viniferin, a resveratrol dimer, which is present at comparable concentrations to t-resveratrol in red wines, and has higher anti-adipogenesis activity in 3T3-L1 cells. In addition, ε-viniferin was more effective than t-resveratrol in its anti-obesity and anti-inflammatory effects in high-fat diet fed mice. These results suggested ε-viniferin may be one of the active ingredients against metabolic disorders in red wines, in addition to t-resveratrol.

Laser desorption ionization of stilbenes in crystalline sponge.

The laser desorption (LD) ionization of three stilbenes in the nano porous metal-organic frameworks called "crystalline sponge" is demonstrated. The analyte position in the pore and the interaction between the analyte and the framework that functions as a matrix are discussed based on the results of single- crystal X-ray analysis. It is confirmed that the analyte/ligand ratio of 1:2 is sufficient for the analyte ionization. This method makes it possible to visualize hot spots on a target plate to be irradiated. That the sample requirement is dramatically reduced to the order of femtomoles is also an advantage. The relationship between laser interaction, analyte position in the pore, and analyte/ligand ratio is discussed as a new ionization field to elucidate the molecular structure of the analyte by LD ionization mass spectrometry.

Hollow sphere formation from a three-dimensional structure composed of an adamantane-based cage.

An adamantane-based cage with a three-dimensional (3D) framework was synthesized by the copper-mediated acetylene coupling reaction, in which two 1,3,5-triethynyladamantane units were linked by phenyldiacetylenic bridges possessing ester groups. X-ray crystallography revealed that the cage has an internal space and accommodates a solvent molecule, which afforded molecular networks through CH/O interactions between cage molecules. Furthermore, the cage containing six ester groups spontaneously self-assembled into hollow spherical aggregates with an average diameter of 230 nm in a mixed solvent.

Applications of Plant-Made Fibroblast Growth Factor for Human Pluripotent Stem Cells.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) hold great potential in regenerative medicine. These cells can be expanded indefinitely in theory and are able to differentiate into different types of cells for cell therapies, drug screening, and basic biology studies. The reliable and effective propagation of hESCs and hiPSCs is important for their downstream applications. Basic fibroblast growth factor (bFGF) is critical to hESCs and hiPSCs for maintaining their pluripotency. Plant-produced growth factors are safe to use without potential contamination of infectious viruses and are less expensive to produce. In this study, we used rice cell-made basic fibroblast growth factor (RbFGF) to propagate hESCs and hiPSCs for at least eight passages. Both hESCs and hiPSCs cultured with RbFGF not only maintained the morphology but also the specific expression (OCT4, SSEA4, SOX2, and TRA-1-60) of PSCs, similar to those cultured with the commercial Escherichia coli-produced bFGF. Furthermore, both gene chip-based PluriTest and TaqMan hPSC Scorecard pluripotency analysis demonstrated the pluripotent expression profile of the hESCs cultured with RbFGF. In vitro trilineage assays further showed that these hESCs and hiPSCs cultured on RbFGF were capable of giving rise to cell derivatives of ectoderm, mesoderm, and endoderm, further demonstrating their pluripotency. Finally, chromosome stability was also maintained in hESCs cultured with RbFGF as demonstrated by normal karyotypes. This study suggests broad applications for plant-made growth factors in stem cell culture and regenerative medicine.

Double helices of a pyridine-appended zinc chlorophyll derivative.

Self-assembled structures formed from a pyridine-appended zinc chlorophyll derivative are reported. While the zinc complex forms cyclic oligomers in chloroform solution, as indicated by (1)H NMR studies (including diffusion-ordered spectroscopy), vapor pressure osmometry, and cold-spray ionization mass spectrometry, it forms double-stranded helical coordination polymers in the solid state, as revealed by single-crystal X-ray analysis.

Quercetin-3-O-glucuronide induces ABCA1 expression by LXRα activation in murine macrophages.

Reverse cholesterol transport (RCT) removes excess cholesterol from macrophages to prevent atherosclerosis. ATP-binding cassette, subfamily A, member 1 (ABCA1) is a crucial cholesterol transporter involved in RCT to produce high density lipoprotein-cholesterol (HDLC), and is transcriptionally regulated by liver X receptor alpha (LXRα), a nuclear receptor. Quercetin is a widely distributed flavonoid in edible plants which prevented atherosclerosis in an animal model. We found that quercetin-3-O-glucuronide (Q3GA), a major quercetin metabolite after absorption from the digestive tract, enhanced ABCA1 expression, in vitro, via LXRα in macrophages. In addition, leaf extracts of a traditional Asian edible plant, Nelumbo nucifera (NNE), which contained abundant amounts of quercetin glycosides, significantly elevated plasma HDLC in mice. We are the first to present experimental evidence that Q3GA induced ABCA1 in macrophages, and to provide an alternative explanation to previous studies on arteriosclerosis prevention by quercetin.

N-acetylglutamic acid alleviates oxidative stress based on histone acetylation in plants.

Oxidative stress causes cellular damage and genomic instability through the accumulation of reactive oxygen species (ROS) in plants, resulting in reduced crop production. Chemical priming, which can enhance plant tolerance to environmental stress using functional chemical compounds, is expected to improve agricultural yield in various plants without genetic engineering. In the present study, we revealed that non-proteogenic amino acid N-acetylglutamic acid (NAG) can alleviate oxidative stress damage in Arabidopsis thaliana (Arabidopsis) and Oryza sativa (rice). Exogenous treatment with NAG prevented chlorophyll reduction induced by oxidative stress. The expression levels of ZAT10 and ZAT12, which are regarded as master transcriptional regulators in response to oxidative stress, increased following NAG treatment. Additionally, Arabidopsis plants treated with NAG showed enhanced levels of histone H4 acetylation at ZAT10 and ZAT12 with the induction of histone acetyltransferases HAC1 and HAC12. The results suggest that NAG could enhance tolerance to oxidative stress through epigenetic modifications and contribute to the improvement of crop production in a wide variety of plants under environmental stress.