Dirigent isoflavene-forming PsPTS2: 3D structure, stereochemical, and kinetic characterization comparison with pterocarpan-forming PsPTS1 homolog in pea.

Pea phytoalexins (-)-maackiain and (+)-pisatin have opposite C6a/C11a configurations, but biosynthetically how this occurs is unknown. Pea dirigent-protein (DP) PsPTS2 generates 7,2'-dihydroxy-4',5'-methylenedioxyisoflav-3-ene (DMDIF), and stereoselectivity toward four possible 7,2'-dihydroxy-4',5'-methylenedioxyisoflavan-4-ol (DMDI) stereoisomers was investigated. Stereoisomer configurations were determined using NMR spectroscopy, electronic circular dichroism, and molecular orbital analyses. PsPTS2 efficiently converted cis-(3R,4R)-DMDI into DMDIF 20-fold faster than the trans-(3R,4S)-isomer. The 4R-configured substrate's near ß-axial OH orientation significantly enhanced its leaving group abilities in generating A-ring mono-quinone methide (QM), whereas 4S-isomer's α-equatorial-OH was a poorer leaving group. Docking simulations indicated that the 4R-configured ß-axial OH was closest to Asp51, whereas 4S-isomer's α-equatorial OH was further away. Neither cis-(3S,4S)- nor trans-(3S,4R)-DMDIs were substrates, even with the former having C3/C4 stereochemistry as in (+)-pisatin. PsPTS2 used cis-(3R,4R)-7,2'-dihydroxy-4'-methoxyisoflavan-4-ol [cis-(3R,4R)-DMI] and C3/C4 stereoisomers to give 2',7-dihydroxy-4'-methoxyisoflav-3-ene (DMIF). DP homologs may exist in licorice (Glycyrrhiza pallidiflora) and tree legume Bolusanthus speciosus, as DMIF occurs in both species. PsPTS1 utilized cis-(3R,4R)-DMDI to give (-)-maackiain 2200-fold more efficiently than with cis-(3R,4R)-DMI to give (-)-medicarpin. PsPTS1 also slowly converted trans-(3S,4R)-DMDI into (+)-maackiain, reflecting the better 4R configured OH leaving group. PsPTS2 and PsPTS1 provisionally provide the means to enable differing C6a and C11a configurations in (+)-pisatin and (-)-maackiain, via identical DP-engendered mono-QM bound intermediate generation, which PsPTS2 either re-aromatizes to give DMDIF or PsPTS1 intramolecularly cyclizes to afford (-)-maackiain. Substrate docking simulations using PsPTS2 and PsPTS1 indicate cis-(3R,4R)-DMDI binds in the anti-configuration in PsPTS2 to afford DMDIF, and the syn-configuration in PsPTS1 to give maackiain.

Effect of Roasting Degree on Major Coffee Compounds: A Comparative Study between Coffee Beans with and without Supercritical CO2 Decaffeination Treatment.

Coffee is a beverage that is consumed worldwide, and the demand for decaffeinated coffee has increased in recent years. This study aimed to investigate the effect of roasting conditions on the concentration of physiologically active compounds in coffee beans with and without supercritical CO2 decaffeination treatment. Decaffeination treatment markedly reduced caffeine concentration and slightly reduced trigonelline concentration in the coffee beans, whereas the concentrations of chlorogenic acids (chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid) were largely unchanged. Roasting was performed using a hot-air coffee roaster machine and the coffee beans were treated at different peak temperatures (125-250℃), different hold times at the peak temperature (120-240 s), and different temperature increase times to reach the peak temperature (60-180 s). Roasting conditions such as long hold and long temperature rise times at high temperatures (≥ 225℃) significantly degraded coffee compounds except for caffeine, with similar degradation rates between non-decaffeinated and decaffeinated coffee beans. In contrast, the L* value of decaffeinated coffee decreased with less thermal history compared to that of non-decaffeinated coffee. This allowed for the complete roasting of decaffeinated coffee with a lower thermal history compared to those of non-decaffeinated counterparts, suppressing the degradation of several coffee compounds. For example, comparing the similar L* values between coffee beans with and without decaffeination treatment, it was found that the former tended to contain more chlorogenic acid. Generally, decaffeination results in the loss of physiologically active compounds along with caffeine, which is a major concern. However, this study showed that appropriate control of decaffeination and roasting conditions can limit the degradation of several valuable coffee compounds, such as trigonelline and chlorogenic acid.

Quantification of ustalic acid, a chemotaxonomic marker, in Tricholoma ustale using liquid chromatography-mass spectrometry.

The development of methods for the detection and qualification of toxic substances in mushrooms is a rapidly growing research area in forensic toxicology. This study aimed to determine liquid chromatographic and mass spectrometric conditions applicable to the analysis of ustalic acid (UA) in Tricholoma ustale. We used HPLC coupled with single quadrupole mass spectrometry (Q-MS) with electrospray ionization in its negative ion mode to analyze UA. We performed HPLC separations on a C18 reversed-phase column with gradient elution using mobile phases containing water, acetonitrile, and formic acid. The MS showed that UA formed the deprotonated molecular ion [M-H]- at m/z 337, which was sufficient for the quantitative analysis of the compound. The average recovery rates of UA from four edible mushrooms (Flammulina velutipes, Pleurotus eryngii, Lentinula edodes, and Grifola frondosa) to which 10.0 µg/g of UA was added were 108%, 104%, 108%, and 107%, respectively, and the relative standard deviation values ranged from 4.1 to 6.4%. Quantitative analysis of UA in three systematically collected individual mushrooms of T. ustale revealed 41.9-155.7 ppm in each dry material. We also explored the fragmentation behaviors of UA in triple quadrupole mass spectrometry and the proposed structures for the product ions. The data suggest that conventional Q-MS with authenticated UA would be able to identify this compound in T. ustale when used for the immediate inspection of incidences of poisoning. Confirmation of the presence of UA in T. ustale would ultimately allow for the chemotaxonomic discrimination of Tricholoma species.

Structures of Cyclic Organosulfur Compounds From Garlic (Allium sativum L.) Leaves.

Five new cyclic organosulfur compounds, foliogarlic disulfanes A1 (1), A2 (2), and A3 (3) and foliogarlic trisulfane A1 (4) and A2 (5), were isolated from the leaves of Allium sativum (garlic). The chemical structures of these compounds were elucidated on the basis of physicochemical evidence including Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS). Compounds 1-5 were obtained as complex compounds with disulfane or trisulfane and tetrahydro-2H-difuro[3,2-b:2',3'-c]furan-5(5aH)-one. In addition, the hypothetical biosynthetic pathways of these compounds were suggested.

Vitamin K3 thio-derivative: a novel specific apoptotic inducer in the doxorubicin-sensitive and -resistant cancer cells.

Vitamin K3, also known as menadione, is a synthetic lipid-soluble 2-methyl-1,4- naphthoquinone analogs of vitamin K. The vitamin K derivatives exhibit potent cytotoxicity against several cancer cell lines through ROS induction and mitochondrial dysfunction. We investigated vitamin K3-inspired derivatives as potential apoptotic inducers and analyzed their mechanisms beyond apoptosis. The cytotoxicity of a panel of vitamin K3 analogs was screened against 10 doxorubicin-sensitive and -resistant cancer cell lines overexpressing ATP-binding cassette transporters (P-glycoprotein, ABCB5, BCRP) or oncogenes (ΔEGFR) or with knockout of tumor suppressors (p53), Cell cycle arrest, apoptosis, cell migration, and microtubule formation were further investigated. The online tool SwissTargetPrediction was utilized for target prediction. Among the screened compounds, one vitamin K3 thio-derivative (No. 45, VKT-1) exhibited the most potent cytotoxicity specifically against both drug-sensitive and -resistant cancer cell lines. In addition, VKT-1 arrested the cells at the G2/M phase and induced apoptosis as detected by flow cytometry. As predicted by SwissTargetPrediction, VKT-1 targeted microtubule-associated tau protein. Indeed, VKT-1 dramatically inhibited cell migration and microtubule formation in vitro. In conclusion, the synthetic vitamin K3 thio-derivative (VKT-1) inhibited doxorubicin-sensitive and -resistant tumor cells by cell arrest, apoptosis induction, as well as, migration inhibition, and microtubule deterioration of U2OS-GFP-α-tubulin cells.

Biofunctional Effects of Thiohemiaminal-Type Dimeric Sesquiterpene Alkaloids from Nuphar Plants.

Dimeric sesquiterpene thioalkaloids from the rhizomes of Nuphar pumilum exhibited immunosuppressive effects using a sheep erythrocyte plaque forming cell (PFC) assay, as well as an anti-metastasis effect, and rapid apoptosis-inducing effects in tumor cell lines. In particular, dimeric sesquiterpene thioalkaloids with a hydroxy group (6-hydroxythiobinupharidine, 6,6'-dihydroxythiobinupharidine, 6-hydroxythionuphlutine B) showed substantial effects, whereas dimeric sesquiterpene thioalkaloids lacking the hydroxy group (thiobinupharidine, thionuphlutine B, 6'-hydroxythionuphlutine B, neothiobinupharidine, thionuphlutine B ß-sulfoxide, neothiobinupharidine ß-sulfoxide) and monomeric sesquiterpene alkaloids (nupharidine, 7-epideoxynupharidine, nupharolutine) showed weak activity. In this review, we summarize our studies of the biofunctional effects of these alkaloids.

Cyclic sulfur-containing compounds from Allium fistulosum 'Kujou'.

Sulfur-containing compounds, allicin and ajoene, etc., were isolated from Allium species. In a recent study, some sulfur-containing cyclic compounds were isolated from A. sativum, A. cepa, and A. fistulosum. Four new compounds with multiple rings with methyl disulfide or propyl disulfide at the side chain of the 7-position, kujounins A3 (1), B1 (2), B2 (3) and B3 (4), and two new thiolane type compounds with methoxy and methyl sulfoxide moiety at the 2- and 5-positions, and allium sulfoxides A2 (5) and A3 (6), were isolated from the acetone extract of the fresh white parts of Allium fistulosum 'Kujou' with three known compounds, kujounin A1 (7) and A2 (8), and allium sulfoxide A1 (9). The chemical structures of the new compounds were elucidated on the basis of physicochemical evidence. The kujounins had a rare molecular skeleton, which was tetrahydro-2H-difuro[3,2-b:2',3'-c]furan-5(5aH)-one.

Cytotoxicity of sesquiterpene alkaloids from Nuphar plants toward sensitive and drug-resistant cell lines.

Multi-drug resistance (MDR) is a critical problem in cancer chemotherapy. MDR causes the overexpression of ATP-binding cassette (ABC) transporters and mutations in tumor suppressor genes and oncogenes. To tackle this issue, in this study, we focused on Nuphar plants, which have been traditionally used as food. Sesquiterpene alkaloids (1-3) were isolated from N. japonicum and dimeric sesquiterpene thioalkaloids (4-10) were isolated from N. pumilum. P-glycoprotein-overexpressing CEM/ADR5000 cells were cross-resistant to 6,6'-dihydroxythiobinupharidine (10). Using in silico molecular docking, we calculated the binding energies and simulated the interactions of these compounds with the corresponding amino acid residues at the binding site of P-gp. In addition, we investigated the cytotoxicity of these compounds towards cell lines overexpressing other ABC transporters (BCRP, ABCB5), cell lines with a knocked out tumor suppressor gene TP53 or cell lines overexpressing a deletion-activated EGFR oncogene. These cell lines were sensitive or only minimally cross-resistant to these compounds compared with their corresponding wild-type cell lines.

Rare Sulfur-Containing Compounds, Kujounins A1 and A2 and Allium Sulfoxide A1, from Allium fistulosum 'Kujou'.

Three sulfur-containing compounds, kujounins A1 (1) and A2 (2) and allium sulfoxide A1 (3), were isolated from the acetone extract of Allium fistulosum 'Kujou'. Their chemical structures were elucidated on the basis of physicochemical evidence, including X-ray crystallographic data. Compounds 1 and 2 possess three rings and an acetal structure and were obtained as complex compounds having disulfide and monosaccharide moieties. On the other hand, compound 3 has a thiolane skeleton derived from allicin. Naturally occurring compounds 1-3 have rare molecular skeletons. This study is the first to determine the absolute configuration of thiolane-type compounds.

Neolignan and megastigmane glucosides from the aerial parts of Isodon japonicus with cell protective effects on BaP-induced cytotoxicity.

Six neolignan glucosides, named isodonosides I-VI, and a megastigmane glucoside named isodonmegastigmane I, were isolated together with 15 known compounds from the methanolic extract of aerial parts of Isodon japonicus cultivated in Tokushima, Japan. The chemical structures of the compounds were elucidated based on their MS and NMR spectroscopic analysis. The absolute configurations of the neolignan and megastigmane glucosides were determined by derivatizations, by ECD (electronic circular dicroism) Cotton effect approximation, and by the modified Mosher's method. In addition, a significant cell protective effects of neolignan glucosides on benzo[a]pyrene-induced cytotoxicity was found.

Triterpenes with Anti-invasive Activity from Sclerotia of Inonotus obliquus.

The methanolic extract [inhibition (%): 61.2±3.8 (p<0.01) at 100 µg/mL] and its EtOAc-soluble fraction [inhibition (%): 82.5±1.7 (p<0.01) at 100 µg/mL1 from the sclerotia of Inonotus obliquus collected in Japan significantly inhibited invasion of human fibrosarcoma HT1080 cells through matrigel-coated filters. In addition, the methanolic extract significantly inhibited lung tumor formation fifteen days after injection of BI6F10 melanoma cells in mice [inhibition (%) 66.1 ± 12:8 (p < 0.05) at 500 mg/kg/d, p.o.]. Lanostane-type triterpenes were isolated as the common principal constituents from Japanese and Russian . obliquus. Furtheremore, we examine the inhibitory effects of the constituents on the invasion of HT 1080 cells. Interestingly, 3ß-hydroxylanosta-8,24-dien- 21-al [inhibition (%) 37.9 ± 3.0 (p < 0.05) at 30 µM] significantly inhibited the invasion, and no cytotoxic effect at 30 µM was observed.

Analysis of CYP2R1 and CYP26A1 Expression Patterns in Regeneration in Mice with Liver Injury.

Cytochrome P450 enzymes (CYPs) are involved in the metabolism of various substances in the liver and small intestine and show markedly higher expression levels in the liver compared to other organs. The liver exhibits a remarkable capacity to regenerate. After excision of 70% of the liver, the organ can regenerate to its original size in approximately 1 week. Unlike the normal liver, in the injured liver, hepatic stem cells known as oval cells are considered to play an important role in regeneration. However, the role of CYPs in liver regeneration remains unclear. In the present study, we investigated the role of CYPs in the regeneration of injured liver. Liver injury was induced by 4-week repeated doses of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in the diet. Next, both DDC-fed mice and control diet (containing no DDC)-fed mice were subjected to 70% hepatectomy, and the hepatic gene expression patterns measured during regeneration were analyzed. Mice with DDC-induced liver injury expressed the oval cell markers cytokeratin 19 (CK19) and epithelial cell adhesion molecule (EpCAM), and partial hepatectomy increased the expression levels of CYP2R1 and CYP26A1 as well as the hepatoblast marker alpha-fetoprotein (AFP) in these mice. The results of this study suggest that CYP2R1 and CYP26A1 are important in the differentiation of oval cells into hepatoblast-like cells in the injured liver.

Chemical structures of constituents from the whole plant of Bacopa monniera.

Two new dammarane-type triterpene oligoglycosides, bacomosaponins A and B, and three new phenylethanoid glycosides, bacomosides A, B1, and B2, were isolated from the whole plant of Bacopa monniera Wettst. The chemical structures of the new constituents were characterized on the basis of chemical and physicochemical evidence. In the present study, bacomosaponins A and B with acyl groups were obtained from the whole plant of B. monniera. This is the first report of acylated dammarane-type triterpene oligoglycosides isolated from B. monniera. In addition, dammarane-type triterpene saponins significantly inhibited the aggregation of 42-mer amyloid ß-protein.

γ-Lactam alkaloids from the flower buds of daylily.

Four new alkaloids, hemerocallisamines IV-VII, were isolated from the methanol extract of flower buds of daylily. The chemical structures of the new compounds were elucidated on the basis of chemical and physicochemical evidence. The absolute stereochemistry of the hemerocallisamines IV-VI was elucidated by the application of the modified Mosher's method, HPLC analysis, and optical rotation. In the present study, the isolated alkaloids significantly inhibited the aggregation of Aß42 in vitro. This is the first report about bioactive alkaloids with a γ-lactam ring from daylily. In addition, isolated nucleosides showed accelerative effects on neurite outgrowth under the non-fasting condition.

Structure of a Coumaric Acid Analogue with a Monoterpene Moiety from the Flowers of Osmanthusfragrans var. aurantiacus and Evaluation of Cinnamic Acid Analogues as Nitric Oxide Production and Degranulation Inhibitors.

A coumaric acid analogue with a monoterpene moiety named floraosmanol A (1) was isolated from the flowers of Osmanthisfragrans var. aurantiacus. The chemical structure was elucidated on the basis of chemical and physicochemical evidence. Floraosmanol A (1) significantly inhibited nitric oxide .(NO) production in lipopolysaccharide- (LPS) activated RAW264.7 macrophages and the release of P-hexosaminidase as a marker of degranulation from rat basophile leukemia (RBL-2H3) cells. In addition, several cinnamic acid analogues were conjugated with geraniol and 3-methyl-2-buten-1-ol through an ester linkage. The inhibitory effects on NO production and the release of P-hexosaminidase of the synthesized compounds were examined for structure-activity relationships.

Chemical structures of constituents from the flowers of Osmanthus fragrans var. aurantiacus.

Three new megastigmane glycosides named floraosmanosides I-III and a new γ-decalactone named floraosmanolactone I together with 16 known constituents were isolated from the flowers of Osmanthus fragrans var. aurantiacus cultivated in Guangxi Zhuang Autonomous Region, China. The chemical structures of the new compounds were elucidated on the basis of chemical and physicochemical evidence. Among them, ligustroside and (+)-pinoresinol significantly inhibited nitric oxide production in lipopolysaccharide-activated RAW264.7 macrophages.

Structure of diarylheptanoids with antiallergic activity from the rhizomes of Curcuma comosa.

The methanolic extract from the dried rhizomes of Curcuma comosa cultivated in Thailand was found to inhibit the release of ß-hexosaminidase as a maker of degranulation from rat basophil leukemia (RBL-2H3) cells. Two new diarylheptanoids, diarylcomosols IV and V, were isolated from the methanolic extract. The chemical structures of the new compounds were elucidated on the basis of chemical and physicochemical evidence. The isolated diarylheptanoids showed inhibitory activity, and the structural requirements of the active constituents for the inhibition were clarified.

Structural characterization of a carbon monoxide adduct of a heme-DNA complex.

The structure of a carbon monoxide (CO) adduct of a complex between heme and a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), has been characterized using ¹H and ¹³C NMR spectroscopy and density function theory calculations. The study revealed that the heme binds to the 3'-terminal G-quartet of the DNA though a π-π stacking interaction between the porphyrin moiety of the heme and the G-quartet. The π-π stacking interaction between the pseudo-C2-symmetric heme and the C4-symmetric G-quartet in the complex resulted in the formation of two isomers possessing heme orientations differing by 180° rotation about the pseudo-C2 axis with respect to the DNA. These two slowly interconverting heme orientational isomers were formed in a ratio of approximately 1:1, reflecting that their thermodynamic stabilities are identical. Exogenous CO is coordinated to heme Fe on the side of the heme opposite the G-quartet in the complex, and the nature of the Fe-CO bond in the complex is similar to that of the Fe-CO bonds in hemoproteins. These findings provide novel insights for the design of novel DNA enzymes possessing metalloporphyrins as prosthetic groups.

Effect of heme modification on oxygen affinity of myoglobin and equilibrium of the acid-alkaline transition in metmyoglobin.

Functional regulation of myoglobin (Mb) is thought to be achieved through the heme environment furnished by nearby amino acid residues, and subtle tuning of the intrinsic heme Fe reactivity. We have performed substitution of strongly electron-withdrawing perfluoromethyl (CF(3)) group(s) as heme side chain(s) of Mb to obtain large alterations of the heme electronic structure in order to elucidate the relationship between the O(2) affinity of Mb and the electronic properties of heme peripheral side chains. We have utilized the equilibrium constant (pK(a)) of the "acid-alkaline transition" in metmyoglobin in order to quantitatively assess the effects of the CF(3) substitutions for the electron density of heme Fe atom (rho(Fe)) of the protein. The pK(a) value of the protein was found to decrease by approximately 1 pH unit upon the introduction of one CF(3) group, and the decrease in the pK(a) value with decreasing the rho(Fe) value was confirmed by density functional theory calculations on some model compounds. The O(2) affinity of Mb was found to correlate well with the pK(a) value in such a manner that the P(50) value, which is the partial pressure of O(2) required to achieve 50% oxygenation, increases by a factor of 2.7 with a decrease of 1 pK(a) unit. Kinetic studies on the proteins revealed that the decrease in O(2) affinity upon the introduction of an electron-withdrawing CF(3) group is due to an increase in the O(2) dissociation rate. Since the introduction of a CF(3) group substitution is thought to prevent further Fe(2+)-O(2) bond polarization and hence formation of a putative Fe(3+)-O(2)(-)-like species of the oxy form of the protein [Maxwell, J. C.; Volpe, J. A.; Barlow, C. H.; Caughey, W. S. Biochem. Biophys. Res. Commun. 1974, 58, 166-171], the O(2) dissociation is expected to be enhanced by the substitution of electron-withdrawing groups as heme side chains. We also found that, in sharp contrast to the case of the O(2) binding to the protein, the CO association and dissociation rates are essentially independent of the rho(Fe) value. As a result, the introduction of electron-withdrawing group(s) enhances the preferential binding of CO to the protein over that of O(2). These findings not only resolve the long-standing issue of the mechanism underlying the subtle tuning of the intrinsic heme Fe reactivity, but also provide new insights into the structure-function relationship of the protein.