Reducing the coumarin content of tonka bean extracts using supercritical CO2

By supercritical fluid CO₂ extraction of tonka beans a lipophilic top phase and a crystalline base phase were obtained. These were analysed by gas chromatography-mass spectrometry (GC/MS) and high-performance liquid chromatography (HPLC) and compared with an ethanolic extract of the same batch of tonka beans. It was shown that the three samples differed considerably in their composition and that the bulk of the main compound coumarin was present in the base phase of the CO₂ extraction.     

Anatoxin-a and its metabolites in blue-green algae food supplements from Canada and Portugal

Blue-green algae and spirulina are marketed in health food stores and over the Internet as food supplements in Canada, the United States, and Europe. The reported benefits of consuming these products include improved digestion, strengthening of the immune system, and relief from the symptoms of attention deficit disorder. Some of these products have been found to contain elevated concentrations of microcystins, which are known hepatotoxins. In addition to producing microcystins, Anabaena sp. and Aphanizomenon sp. also produce the potent neurotoxin anatoxin-a. Samples of food supplements containing blue-green algae and spirulina were collected in Portugal and from urban centers across Canada in 2005. Extracts of these supplements were analyzed to determine the presence and concentrations of anatoxin-a and its two main metabolites, dihydroanatoxin-a and epoxyanatoxin-a. Initial analyses were performed using high-performance liquid chromatography (HPLC) with fluorescence detection, and confirmation required the use of LC with tandem mass spectrometry (LC-MS-MS). The HPLC with fluorescence detection indicated no anatoxin-a, but four samples were suspected to contain either dihydroanatoxin-a or epoxyanatoxin-a at 0.1 to 0.2 microg/g. LC-MS-MS results, however, indicated no trace of either transformation product in any sample analyzed. The detection limits for anatoxin-a, dihydroanatoxin-a, and epoxyanatoxin-a were similar for both fluorescence detection (0.2 to 0.3, 0.4 to 1.4, and 0.2 to 1.5 pg on the column, respectively) and mass spectrometry (0.3 to 1.5, 0.3 to 0.8, and 0.5 to 0.8 pg on the column, respectively). Because of the higher specificity of the LC-MS-MS analysis, all tested food supplement samples were considered free of anatoxin-a and its transformation products.     

Size-dependant cellular uptake of dendritic polyglycerol

To study the mechanism of cellular internalization, hyperbranched polyether derivatives consisting of amino-bearing hyperbranched polyglycerols (HPGs) of varied molecular mass and size range are designed and synthesized. HPGs were further fluorescently labelled by conjugating maleimido indocarbocyanine dye (ICC-mal). The conjugates are characterized by UV-vis spectroscopy, fluorescence profile, zeta potential, and dynamic light scattering. The uptake mechanism is studied by fluorescence-activated cell sorting (FACS) analysis, fluorescence spectroscopy, and confocal microscopy with human lung cancer cells A549, human epidermoid carcinoma cells A431, and human umbilical vein endothelial cells (HUVEC) cells. For the first time, the results suggest that the higher-molecular-weight HPGs (40-870 kDa) predominantly accumulate in the cytoplasm much better than their low-molecular-weight counterparts (2-20 kDa). The HPG nanocarriers discussed here have many biomedical implications, particularly for delivering drugs to the targeted site.     

Front Cover: Chemoselective Labeling and Immobilization of Phosphopeptides with Phosphorimidazolide Reagents (ChemBioChem 4/2023)

Protein phosphorylation is one of the most ubiquitous post-translational modifications, regulating numerous essential processes in cells. Accordingly, the large-scale annotation of phosphorylation sites continues to provide central insight into the regulation of signaling networks. The global analysis of the phosphoproteome typically relies on mass spectrometry analysis of phosphopeptides, with an enrichment step necessary due to the sub-stoichiometric nature of phosphorylation. Several affinity-based methods and chemical modification strategies have been developed to date, but the choice of enrichment method can have a considerable impact on the results. Here, we show that a biotinylated, photo-cleavable phosphorimidazolide reagent permits the immobilization and subsequent cleavage of phosphopeptides. The method is capable of the capture and release of phosphopeptides of varying characteristics, and this mild and selective strategy expands the current repertoire for phosphopeptide chemical modification with the potential to enrich and identify new phosphorylation sites in the future.