GC-MS detection of chiral markers in cocoa beans of different quality and geographic origin

Fermented cocoa beans (Theobroma cacao L., Sterculiaceae) from different countries of origin (Ecuador, Ghana, Trinidad) and cocoa beans roasted under defined conditions (industrial roasting; 150-220 degrees C for 20 min, dry roasting in conventional oven) were analyzed for their contents of certain chiral hydroxy acids, catechins, and amino acids. Cocoa beans are fermented, dried, and industrially transformed by roasting for the production of chocolate, cocoa powders, and other cocoa-related products. Fermentation and roasting conditions influence the contents of chiral compounds such as hydroxy acids, amino acids, and polyphenols, depending on technological procedures as well as some technical parameters. The aim of this work was to check if the content and nature of the named chiral compounds present both in fermented and roasted cocoa beans could be related to the traditional parameters used to classify the variety of seeds and the degree of fermentation. The extent of racemization of amino acids in fermented cocoa beans was low while it slowly increased during roasting, depending on the temperature applied. L-lactic acid was always higher than the D-form while citric acid was generally the most abundant hydroxy acid detected in beans. A correlation was found between polyphenol content and degree of fermentation, while epimerization of (-)-epicatechin to (+)-catechin was observed during roasting. On the whole, results showed that several chiral compounds could be considered as good quality markers for cocoa seeds and cocoa-related products of different quality and geographic origin.     

Stereoselective pharmacokinetics of ornidazole after intravenous administration of individual enantiomers and the racemate

The pharmacokinetics of ornidazole (ONZ) were investigated following i.v. administration of racemic mixture and individual enantiomers in beagle dogs. Plasma concentrations of ONZ enantiomers were analyzed by chiral high-performance liquid chromatography (HPLC) on a Chiralcel OB-H column with quantification by UV at 310 nm. Notably, the mean plasma levels of (-)-ONZ were higher in the elimination phase than those of (+)-ONZ. (-)-ONZ also exhibited greater t1/2, MRT, AUC(0-t) and smaller CL, than those of its antipode. The area under the plasma concentration-time curve (AUC(0-t)) of (-)-ONZ was about 1.2 times as high as that of (+)-ONZ. (+)-ONZ total body clearance (CL) was 1.4 times than its optical antipode. When given separately, there were significant differences in the values of AUC(0-infinity) and CL between ONZ enantiomers (P < 0.05), indicating that elimination of (+)-ONZ was more rapid than that of (-)-ONZ. No significant differences were found between the estimates of the pharmacokinetic parameters of (+)-ONZ or (-)-ONZ, obtained following administration as the individual and as a racemic mixture. This study demonstrates that the elimination of ONZ enantiomers is stereoselective and chiral inversion and enantiomer/enantiomer interaction do not occur when the enantiomers are given separately and as racemic mixture.     

The LiCl effect on the conformation of lentinan in DMSO

Lentinan (β‐(1→3)‐D ‐glucan) was found to be successfully fractionated by the mixture of dimethyl sulfoxide (DMSO) and lithium chloride (LiCl) as a solvent and acetone as a precipitant. Light scattering and viscosity measurements were made on solutions of fractionated samples in pure DMSO and 0.2M LiCl/DMSO in the range of the molecular weight M_w from 21.7 × 10⁴ to 84.7 × 10⁴. The values of M_w in both solvents were almost the same, but the remarkable difference between the values of intrinsic viscosity [η] demonstrated that the LiCl/DMSO solvent greatly enhances the stiffness of the lentinan backbone. The observed intrinsic viscosity [η] was analyzed by the Yoshizaki‐Nitta‐Yamakawa theory of a worm‐like chain, and the persistence length q and molecular weight per unit contour length M_L were determined roughly as 6.0 nm and 890 g nm^?1 in 0.2M LiCl/DMSO, and 5.1 nm and 890 g nm^?1 in pure DMSO, respectively. This slightly larger persistent length in 0.2M LiCl/DMSO also confirmed the higher stiffness of lentinan enhanced by the LiCl/DMSO solvent. The enhancement of the chain stiffness was ascribed to the electrostatic repulsion because of the hydrogen bonding of the hydroxyl protons of lentinan with the chloride ion, which is in turn associated with the Li⁺(DMSO)_n macrocation complex. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 840–845, 2012.     

Highly selective affinity labelling of RNA polymerase B (II) from wheat germ

DNA-dependent RNA polymerase B (II) from wheat germ was modified by incubation with 4-[N-(β-hydroxyethyl)-N-methyl]benzaldehyde esters of AMP, ADP or ATP, followed by reduction with NaBH₄. Reaction of the modified enzyme with [-³²P]UTP in the presence of various DNA templates led to a highly selective affinity labelling of the subunit with M_r 140000 by covalently linked ApU. Labelling was inhibited by 1μg/ml -amanitin.     

Synthesis of the [8] gingerol enantiomers

(+)-(S)-5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-dodecanone 1a commonly named (+)-(S)-[8] gingerol is a natural product known to have cardiotonic activity.^1–5 A total synthesis of both enantiomers is described with details for the first time using a general synthetic scheme which was recently outlined in the literature.⁶ This synthesis relies both on the separation of the diastereoisomers 4a and 4b by simple column chromatography on silica gel and on an HPLC analysis on a chiral phase to determine the optical purity of the enantiomers 8a and 8b of protected [8] gingerol. The gingerol isomers were thus obtained in good chemical yields in greater than 96% enantiomeric excess.     

Tetralones and flavonoids from Pyrola calliantha

Two new tetralones, pyrolones A (1) and B (2), and a new flavonol glycoside, 2'-O-(4-hydroxybenzoyl)hyperin (3), were isolated from Pyrola calliantha (whole plant), together with six structurally related compounds, including 2'-O-galloylhyperin (4), hyperin (5), formononetin (6), quercetin 3-O-alpha-L-arabinopyranoside (7), quercetin 3-O-alpha-L-arabinofuranoside (8), and kaempferol 3-O-beta-D-galactopyranoside (9). The structures and absolute configurations of the new compounds were elucidated on the basis of spectroscopic (UV, ORD, CD, NMR) and mass-spectrometric (HR-ESI-MS) analyses.     

Condensed tannins: co-occurrence of procyanidins,prodelphinidins and profisetinidins in the heartwood of Acacia baileyana

The flavonoids and condensed tannins of the heartwood of Acacia baileyana var. purpurea are described. In conformity with other Acacia species, the hydroxylation pattern of the flavonoids is of the resorcinol type but, in sharp contrast, the tannins are heterogeneous consisting of a mixture of the resorcinol and phtoroglucinol series. Dimeric proanthocyanidins of the phloroglucinol type were absent and this exception to the general observation that they invariably co-occur with the polymers may be explained by the relative nucleophilicity of the aromatic A-rings.     

Biotransformation of the fungistatic compound (R)-(+)-1-(4′-chlorophenyl)propan-1-ol by Botrytis cinerea

The biotransformation of the fungistatic agent (R)-(+)-1-(4′-chlorophenyl)propan-1-ol (1) by the phytopathogen Botrytis cinerea has been studied. The main reaction pathways involved hydroxylations on several positions as well as condensations with secondary metabolites of the fungus. The antifungal activity of compound 1 against B. cinerea has also been determined.     

Link between primary and secondary metabolism in the biotransformation of trimethylammonium compounds by escherichia coli

The aim of this work was to understand the steps controlling the process of biotransformation of trimethylamonium compounds into L(-)-carnitine by Escherichia coli and the link between the central carbon or primary and the secondary metabolism expressed. Thus, the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA (pyruvate dehydrogenase, acetyl-CoA synthetase, and ATP:acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (isocitrate dehydrogenase) and glyoxylate (isocitrate lyase) cycles, were followed in batch with both growing and resting cells and during continuous cell growth in stirred-tank and high-cell-density membrane reactors. In addition, the levels of carnitine, crotonobetaine, gamma-butyrobetaine, ATP, NADH/NAD(+), and acetyl-CoA/CoA ratios were measured to determine how metabolic fluxes were distributed in the catabolic system. The results provide the first experimental evidence demonstrating the important role of the glyoxylate shunt during biotransformation of resting cells and the need for high levels of ATP to maintain metabolite transport and biotransformation (2.1 to 16.0 mmol L cellular/mmol ATP L reactor h). Moreover, the results obtained for the pool of acetyl-CoA/CoA indicate that it also correlated with the biotransformation process. The main metabolic pathway operating during cell growth in the high cell-density membrane reactor was that related to isocitrate dehydrogenase (during start-up) and isocitrate lyase (during steady-state operation), together with phosphotransacetylase and acetyl-CoA synthetase. More importantly, the link between central carbon and L(-)-carnitine metabolism at the level of the ATP pool was also confirmed.     

Chemical composition of the epicuticular wax from the fruits of Eucalyptus globulus

The chemical composition of the epicuticular wax from the fruits of Eucalyptus globulus was studied by GC-MS before and after alkaline hydrolysis. The wax had two main components, ursolic acid and tritriacontan-16,18-dione, together with several other triterpenic acids. After alkaline hydrolysis, a large increase in the amounts of triterpenic acids and fatty acids (particularly in hexadecanoic acid) was observed, suggesting that these components were present predominantly in esterified forms in the fruit wax. Six compounds were isolated from the fruits by preparative chromatography, and were identified as 8-desmethyleucalyptin, sesamin, tritriacontan-16,18-dione, ursolic acid, 3beta-hydroxyurs-11-en-13beta(28)-olide (ursolic acid lactone) and 3beta,11alpha-dihydroxyurs-12-en-28-oic acid, the latter of which was identified for the first time.     

Amino acid deletion products resulting from incomplete deprotection of the Boc group from Npi-benzyloxymethylhistidine residues during solid-phase peptide synthesis.

In peptide synthesis, the use of N(alpha)-tert-butyloxycarbonyl-N(pi)-benzyloxymethylhistidine [Boc-His(pi-Bom)] raises the problem of the Bom group generating formaldehyde during the hydrogen fluoride (HF) cleavage reaction. This can lead to modification of the functional groups on amino acids in the peptide chain. Besides this side reaction, the failure of N(alpha)-Boc deprotection from the His(pi-Bom) residue occurs during TFA treatment for the standard solid-phase peptide synthesis (SPPS) even in the case of a non 'difficult sequence'. This gives amino acid deletion products generated at the N-terminus of the His(pi-Bom) residues. Reviewing the removability of the Boc group on amino acid derivatives showed that the group on the His(pi-Bom) residue was much more resistant under the deprotecting conditions than expected. To circumvent this problem, special precautions, i.e. prolonged deprotection steps and/or increased concentrations of TFA, should be taken for a successful SPPS.     

昆明山海棠茎中化合物的分离及其抗炎活性

从昆明山海棠茎的氯仿提取物中分离得到6个化合物,经鉴定为雷公藤内酯甲(Ⅰ)、昆明山海棠二萜内酯(Ⅱ)、雷公藤内酯乙(Ⅲ)、雷酚二萜酸(Ⅳ)、雷公藤碱(Ⅴ)及3-epikatonic acid(Ⅵ)。其中,化合物Ⅲ及Ⅵ为首次从该植物中分离得到,化合物Ⅴ为首次从该植物茎中分离得到。药理实验显示化合物Ⅰ和Ⅱ对大鼠足跖部角叉菜胶炎症模型具有明显的抑制作用。     

Structural characterization of the Acetobacter xylinum endo-beta-1,4-glucanase CMCax required for cellulose biosynthesis

Previous studies have demonstrated that endoglucanase is required for cellulose biosynthesis both in bacteria and plants. However, it has yet to be elucidated how the endoglucanases function in the mechanism of cellulose biosynthesis. Here we describe the crystal structure of the cellulose biosynthesis-related endo-beta-1,47-glucanase (CMCax; EC 3.2.1.4) from the cellulose-producing Gramnegative bacterium, Acetobacter xylinum (= Gluconacetobacter xylinus), determined at 1.65-A resolution. CMCax falls into the glycoside hydrolase family 8 (GH-8), and the structure showed that the overall fold of the CMCax is similar to those of other glycoside hydrolases belonging to GH-8. Structure comparison with Clostridium thermocellum CelA, the best characterized GH-8 endoglucanase, revealed that sugar recognition subsite +3 is completely missing in CMCax. The absence of the subsite +3 leads to significant broadness of the cleft at the cellooligosaccharide reducing-end side. CMCax is known to be a secreted enzyme and is present in the culture medium. However, electron microscopic analysis using immunostaining clearly demonstrated that a portion of CMCax is localized to the cell surface, suggesting a link with other known membrane-anchored endoglucanases that are required for cellulose biosynthesis.     

Kinetic and mechanistic investigation of the selective acidolysis of the C-terminal amide bond of N-acyl-N,alpha,alpha-trialkyl glycine amides.

Accurate rate constants were calculated from HPLC kinetic measurements of the selective acidolysis of the C-terminal amide bond of eight N-acyl-N-(4-methoxybenzyl)-alpha,alpha-trialkyl glycine amides in TFA at 25.00 degrees C. The results were in all cases consistent with a first order behaviour with respect to the substrate and, apparently, also to the acid, and a clear relationship between reactivity and structure could be observed. The data collected also allowed experimental evidence to be obtained for the first time in support of the previously postulated formation of an intermediate oxazolonium salt. In the case of the more crowded species this intermediate compound undergoes slow hydrolytic ring opening, which takes place in competition with cleavage of the N-alkyl group to give another oxazolonium derivative that hydrolysed still more slowly. The stability of the intermediate cyclic compounds may result either from conjugation of the phenyl group with the oxazolonium ring in the case of N-benzoyl derivatives, or from conformational assistance imparted by the bulky amino acid side chains of the alpha,alpha-dialkyl glycine species, or both. The loss of the N-alkyl group also seems to be assisted by the bulkiness of the amino acid side chains, which thus tends to decrease the selectivity of cleavage.     

Isolation and identification of flavonoids, including flavone rotamers, from the herbal drug 'Crataegi folium cum flore' (hawthorn)

Twelve flavonoids, including seven flavones, four flavonols and one flavanone, were isolated from methanolic extract of the herbal drug 'Crataegi folium cum flore' (hawthorn leaves and flowers) by a combination of CC (over Amberlite XAD-7 and Sephadex LH-20) and preparative HPLC. Their structures, including that of the novel flavonol 8-methoxykaempferol 3-O-(6"-malonyl-beta-glucopyranoside), were elucidated by homo- and heteronuclear NMR and electrospray/MS. The 1H- and 13C-NMR of all compounds, including rotameric pairs of five flavone C-glycosides, were assigned. The presence and relative proportion of each rotamer was shown by various NMR experiments, including two-dimensional nuclear Overhauser and exchange spectroscopy, to depend on solvent, linkage position and structure of the C-glycosyl substituent.     

Chemical Constituents from the Heartwood of Haematoxylon campechianum as Protein Tyrosine Kinase Inhibitors

In a previous study, we showed that a series of homoisoflavonoids from the stems of Haematoxylon campechianum possess potent protein tyrosine kinase inhibitory activity. In a further chemical investigation of the heartwood of H. campechianum, three new homoisoflavonoids, epihematoxylol B ( 2 ), 10‐O‐methylhematoxylol B ( 3 ), and 10‐O‐methylepihematoxylol B ( 4 ), were isolated and identified, together with 15 known compounds, including three homoisoflavonoids, three flavonoids, six lignans, and three unsaturated fatty acids. The structures of the new compounds were established on the basis of 1D‐ and 2D‐NMR and other spectroscopic analyses.     

Biochemical Characterization and Substrate Specificity of Autophagin-2 from the Parasite Trypanosoma cruzi

The genome of the parasite Trypanosoma cruzi encodes two copies of autophagy-related cysteine proteases, Atg4.1 and Atg4.2. T. cruzi autophagin-2 (TcAtg4.2) carries the majority of proteolytic activity and is responsible for processing Atg8 proteins near the carboxyl terminus, exposing a conserved glycine. This enables progression of autophagy and differentiation of the parasite, which is required for successful colonization of humans. The mechanism of substrate hydrolysis by Atg4 was found to be highly conserved among the species as critical mutations in the TcAtg4.2, including mutation of the conserved Gly-244 residue in the hinge region enabling flexibility of the regulatory loop, and deletion of the regulatory loop, completely abolished processing capacity of the mutants. Using the positional scanning-substrate combinatorial library (PS-SCL) we determined that TcAtg4.2 tolerates a broad spectrum of amino acids in the P4 and P3 positions, similar to the human orthologue autophagin-1 (HsAtg4B). In contrast, both human and trypanosome Atg4 orthologues exhibited exclusive preference for aromatic amino acid residues in the P2 position, and for Gly in the P1 position, which is absolutely conserved in the natural Atg8 substrates. Using an extended P2 substrate library, which also included the unnatural amino acid cyclohexylalanine (Cha) derivative of Phe, we generated highly selective tetrapeptide substrates acetyl-Lys-Lys-Cha-Gly-AFC (Ac-KKChaG-AFC) and acetyl-Lys-Thr-Cha-Gly-AFC (Ac-KTChaG-AFC). Althoughthese substrates were cleaved by cathepsins, making them unsuitable for analysis of complex cellular systems, they were recognized exclusively by TcAtg4.2, but not by HsAtg4B nor by the structurally related human proteases SENP1, SENP2, and UCH-L3.     

BH(4) (tetrahydrobiopterin)-dependent activation, but not the expression, of inducible NOS (nitric oxide synthase)-2 in proinflammatory cytokine-stimulated, cultured normal human astrocytes is mediated by MEK-ERK kinases

Nitric oxide (NO) from astrocytes is one of the signalers used by the brain's extensive glial-neuronal-vascular network, but its excessive production by pro-inflammatory cytokine-stimulated glial cells can be cytodestructive. Here, we show how three pro-inflammatory cytokines (IL-1beta, TNF-alpha, and IFN-gamma) together stimulated the activation, but not the prior expression, of NOS-2 protein via a mechanism involving MEK-ERKs protein kinases in astrocytes from adult human cerebral temporal cortex. The cytokines triggered a transient burst of p38 MAPK activity and the production of NOS-2 mRNA which were followed by bursts of MEK-ERK activities, synthesis of the NOS-2 co-factor tetrahydrobiopterin (BH(4)), a build-up of NOS-2 protein and from it active NOS-2 enzyme. Selectively inhibiting MEK1/MEK2, but not the earlier burst of p38 MAPK activity, with a brief exposure to U0126 between 24 and 24.5 h after adding the cytokine triad affected neither NOS-2 expression nor NOS-2 protein accumulation but stopped BH(4) synthesis and the assembly of the NOS-2 protein into active NOS-2 enzyme. The complete blockage of active NOS-2 production by the brief exposure to U0126 was bypassed by simply adding BH(4) to the culture medium. Therefore, this cytokine triad triggered two completely separable, tandem operating mechanisms in normal human astrocytes, the first being NOS-2 gene expression and accumulation of NOS-2 protein and the second being the synthesis of the BH(4) factor needed to dimerize the NOS-2 protein into active, NO-making NOS-2 enzyme.