Development of a nonchiral HPLC method with circular dichroism detection for chiral analysis of molybdenum-catalyzed enantioselective synthesis products

The combination of a circular dichroism detector and nonchiral liquid chromatography was used for the chiral analysis of unresolved enantiomers to determine the enantioselectivity of a molybdenum-catalyzed asymmetric allylic alkylation reaction. The CD/UV peak area ratio of the unresolved enantiomers was calculated and compared with that of a reference standard to determine the enantiomeric purity. The limit of quantitation (LOQ) is 20 ng for the chiral ligand and 1 microg for the branched chiral product. The viability of the system depends on the limit of quantitation of the CD response and the linearity range of the CD and UV response.     

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography,Deans Switching and Sulfur-selective Detection

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate interferences, a two dimensional column configuration is used, with a Deans switch employed to transfer hydrogen sulfide from the first to the second column (heart-cutting). Liquid crude samples are first separated on a dimethylpolysiloxane column, and light gases are heart-cut and further separated on a bonded porous layer open tubular (PLOT) column that is able to separate hydrogen sulfide from other light sulfur species. Hydrogen sulfide is then detected with a sulfur chemiluminescence detector, adding an additional layer of selectivity. Following separation and detection of hydrogen sulfide, the system is backflushed to remove the high-boiling hydrocarbons present in the crude samples and to preserve chromatographic integrity. Dissolved hydrogen sulfide has been quantified in liquid samples from 1.1 to 500 ppm, demonstrating wide applicability to a range of samples. The method has also been successfully applied for the analysis of gas samples from crude oil headspace and process gas bags, with measurement from 0.7 to 9,700 ppm hydrogen sulfide.     

Determination of human serum 8-hydroxy-2′-deoxyguanosine (8-OHdG) by HPLC-ECD combined with solid phase extraction (SPE)

Human serum 8-hydroxy-2′-deoxyguanosine (8-OHdG) was measured by HPLC-ECD method combined with solid phase extraction (SPE) developed by our group: (our proprietary kit, named 8-OHdG Pre-treatment Kit (TANITA Corporation)). The major interfering substances and proteins in serum were removed by 8-OHdG Pre-treatment Kit. This measurement method was highly reproducible (CV = 2.2–7.1%) and demonstrated the lower detection limit for control serum sample of less than 10 pg/ml without the sample evaporation. The other hand 8-OHdG concentration in serum for healthy people was in the range of 0–70 pg/ml (25.5 ± 13.8 pg/ml, n = 37). Secondary a relationship between the HPLC-ECD and ELISA methods was investigated. ELISA method could not detect 8-OHdG concentration in serum for healthy people, because the detection limit of 130 pg/ml was higher than the normal range for healthy people. These results show our SPE method has high sensitivity and quantitative accuracy for 8-OHdG analysis.     

Prevention of a systematic underestimation of antioxidant activity in competition assays. The impact of unspecific reactions of the reactive species

In antioxidant competition assays, an antioxidant (A) and a detector compound (D) compete for a reactive species (R). In the evaluation of these assays, it is tacitly assumed that all of R is captured by either D or A. Due to the - by definition - high reactivity of R, unspecific reactions of R are likely to occur and neglecting these reactions will result in a systematic underestimation of antioxidant activity. It was shown that in the standard hydroxyl radical scavenging assay this was indeed the case; the inaccurate mathematical evaluation resulted in an underestimation of antioxidant activity of 25% in this competition assay. The systematic underestimation of antioxidant activity can be prevented by using an adjusted Stern-Volmer equation that takes into account that only part of R is captured by D or A.     

The N-terminal region of the starch-branching enzyme from Phaseolus vulgaris L. is essential for optimal catalysis and structural stability

Starch-branching enzymes (SBEs) play a pivotal role in determining the fine structure of starch by catalyzing the syntheses of alpha-1,6-branch points. They are the members of the alpha-amylase family and have four conserved regions in a central (beta/alpha)8 barrel, including the catalytic sites. Although the role of the catalytic barrel domain of an SBE is known, that of its N- and C-terminal regions remain unclear. We have previously shown that the C-terminal regions of the two SBE isozymes (designated as PvSBE1 and PvSBE2) from kidney bean (Phaseolus vulgaris L.) have different roles in branching enzyme activity. To understand the contribution of the N-terminal region to catalysis, six chimeric enzymes were constructed between PvSBE1 and PvSBE2. Only one enzyme (1Na/2Nb)-II, in which a portion of the N-terminal region of PvSBE2 was substituted by the corresponding region of PvSBE1, retained 6% of the PvSBE2 activity. The N-terminal truncated form (DeltaN46-PvSBE2), lacking 46 N-terminal residues of PvSBE2, lost enzyme activity and stability to proteolysis. To investigate the possible function of this region, three residues (Asp-15, His-24, and Arg-28) among these 46 residues were subjected to site-directed mutagenesis. The purified mutant enzymes showed nearly the same K(m) values as PvSBE2 but had lower V(max) values and heat stabilities than PvSBE2. These results suggest that the N-terminal region of the kidney bean SBE is essential for maximum enzyme activity and thermostability.     

Identification of the major constituents of Hypericum perforatum by LC/SPE/NMR and/or LC/MS

The newly established hyphenated instrumentation of LC/DAD/SPE/NMR and LC/UV/(ESI)MS techniques have been applied for separation and structure verification of the major known constituents present in Greek Hypericum perforatum extracts. The chromatographic separation was performed on a C18 column. Acetonitrile-water was used as a mobile phase. For the on-line NMR detection, the analytes eluted from column were trapped one by one onto separate SPE cartridges, and hereafter transported into the NMR flow-cell. LC/DAD/SPE/NMR and LC/UV/MS allowed the characterization of constituents of Greek H. perforatum, mainly naphtodianthrones (hypericin, pseudohypericin, protohypericin, protopseudohypericin), phloroglucinols (hyperforin, adhyperforin), flavonoids (quercetin, quercitrin, isoquercitrin, hyperoside, astilbin, miquelianin, I3,II8-biapigenin) and phenolic acids (chlorogenic acid, 3-O-coumaroylquinic acid). Two phloroglucinols (hyperfirin and adhyperfirin) were detected for the first time, which have been previously reported to be precursors in the biosynthesis of hyperforin and adhyperforin.     

Continuous administration of polyphenols from aqueous rooibos (Aspalathus linearis) extract ameliorates dietary-induced metabolic disturbances in hyperlipidemic mice

The incidence of obesity and related metabolic diseases is increasing globally. Current medical treatments often fail to halt the progress of such disturbances, and plant-derived polyphenols are increasingly being investigated as a possible way to provide safe and effective complementary therapy. Rooibos (Aspalathus linearis) is a rich source of polyphenols without caloric and/or stimulant components. We have tentatively characterized 25 phenolic compounds in rooibos extract and studied the effects of continuous aqueous rooibos extract consumption in mice. The effects of this extract, which contained 25% w/w of total polyphenol content, were negligible in animals with no metabolic disturbance but were significant in hyperlipemic mice, especially in those in which energy intake was increased via a Western-type diet that increased the risk of developing metabolic complications. In these mice, we found hypolipemiant activity when given rooibos extract, with significant reductions in serum cholesterol, triglyceride and free fatty acid concentrations. Additionally, we found changes in adipocyte size and number as well as complete prevention of dietary-induced hepatic steatosis. These effects were not related to changes in insulin resistance. Among other possible mechanisms, we present data indicating that the activation of AMP-activated protein kinase (AMPK) and the resulting regulation of cellular energy homeostasis may play a significant role in these effects of rooibos extract. Our findings suggest that adding polyphenols to the daily diet is likely to help in the overall management of metabolic diseases.     

Characterization of the Regions Involved in the Calcium-Induced Folding of the Intrinsically Disordered RTX Motifs from the Bordetella pertussis Adenylate Cyclase Toxin

Repeat in toxin (RTX) motifs are nonapeptide sequences found among numerous virulence factors of Gram-negative bacteria. In the presence of calcium, these RTX motifs are able to fold into an idiosyncratic structure called the parallel β-roll. The adenylate cyclase toxin (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough, is one of the best-characterized RTX cytolysins. CyaA contains a C-terminal receptor domain (RD) that mediates toxin binding to the eukaryotic cell receptor. The receptor-binding domain is composed of about forty RTX motifs organized in five successive blocks (I to V). The RTX blocks are separated by non-RTX flanking regions of variable lengths. It has been shown that block V with its N- and C-terminal flanking regions constitutes an autonomous subdomain required for the toxicity of CyaA. Here, we investigated the calcium-induced biophysical changes of this subdomain to identify the respective contributions of the flanking regions to the folding process of the RTX motifs. We showed that the RTX polypeptides, in the absence of calcium, exhibited the hallmarks of intrinsically disordered proteins and that the C-terminal flanking region was critical for the calcium-dependent folding of the RTX polypeptides, while the N-terminal flanking region was not involved. Furthermore, the secondary and tertiary structures were acquired concomitantly upon cooperative binding of several calcium ions. This suggests that the RTX polypeptide folding is a two-state reaction, from a calcium-free unfolded state to a folded and compact conformation, in which the calcium-bound RTX motifs adopt a β-roll structure. The relevance of these results to the toxin physiology, in particular to its secretion, is discussed.