Ronald A. Glabonjat

Quantification of Arsenolipids in the Certified Reference Material NMIJ 7405-a(Hijiki) using HPLC/Mass Spectrometry after Chemical Derivatization

Arsenic-containing lipids (arsenolipids) are novel natural products recently shown to be widespread in marine animals and algae. Research interest in these arsenic compounds lies in their possible role in the membrane chemistry of organisms and, because they occur in many popular seafoods, their human metabolism and toxicology. Progress has been restricted, however, by the lack of standard arsenolipids and of a quantitative method for their analysis. We report that the certified reference material CRM 7405-a (Hijiki) is a rich source of arsenolipids, and we describe a method based on HPLC-ICPMS/ESMS to quantitatively measure seven of the major arsenolipids present. Sample preparation involved extraction with DCM/methanol, a cleanup step with silica, and conversion of the (oxo)arsenolipids originally present to thio analogues by brief treatment with H2S. Compared to their oxo analogues, the thioarsenolipids showed much sharper peaks on reversed-phase HPLC, which facilitated their resolution and quantification. The compounds were determined by HPLC-ICPMS and HPLC-ESMS, which provided both arsenic-selective detection and high resolution molecular mass detection of the arsenolipids. In this way, the concentrations of two arsenic-containing hydrocarbons and five arsenosugar phospholipids are reported in the CRM Hijiki. This material may serve as a convenient source of characterized arsenolipids to delineate the presence of these compounds in seafoods and to facilitate research in a new era of arsenic biochemistry.

Biosynthesis of the Enterotoxic Pyrrolobenzodiazepine Natural Product Tilivalline

Abstract The nonribosomal enterotoxin tilivalline was the first naturally occurring pyrrolobenzodiazepine to be linked to disease in the human intestine. Since the producing organism Klebsiella oxytoca is part of the intestinal microbiota and the pyrrolobenzodiazepine causes the pathogenesis of colitis it is important to understand the biosynthesis and regulation of tilivalline activity. Here we report the biosynthesis of tilivalline and show that this nonribosomal peptide assembly pathway initially generates tilimycin, a simple pyrrolobenzodiazepine with cytotoxic properties. Tilivalline results from the non‐enzymatic spontaneous reaction of tilimycin with biogenetically generated indole. Through a chemical total synthesis of tilimycin we could corroborate the predictions made about the biosynthesis. Production of two cytotoxic pyrrolobenzodiazepines with distinct functionalities by human gut resident Klebsiella oxytoca has important implications for intestinal disease.

Arsenobetaine in Seawater: Depth Profiles from Selected Sites in the North Atlantic

Arsenic occurs in marine waters, typically at concentrations of 1-2 μg As kg-1, primarily as the inorganic species arsenate. Marine animals, however, contain extremely high levels of arsenic (typically 2000-20 000 μg As kg-1 wet mass), most of which is present as arsenobetaine, an organic form of arsenic that has never been found in seawater. We report a method based on ion-exchange preconcentration and HPLC/mass spectrometry to measure arsenobetaine in seawater, and apply the method to samples of seawater collected at various depths from seven sites in the North Atlantic. Arsenobetaine was detected in most samples at levels ranging from 0.5 to 10 ng As kg-1, and was found at depths down to 4900 m. Furthermore, we report the presence of 15 additional organoarsenicals in seawater, 14 of which had never been detected in marine waters. The arsenobetaine depth profile was related, albeit weakly, to that of chlorophyll; this relationship probably reflects arsenobetaines release to water from marine animals associated with the euphotic zone rather than its direct biosynthesis by primary producers. Future application of the new method for seawater analysis will shed new light on the biogeochemical cycle of marine arsenic.

A 2-O-methylriboside unknown outside the RNA world contains arsenic

Lipid-soluble arsenic compounds, also called arsenolipids, are ubiquitous marine natural products of currently unknown origin and function. In our search for clues about the possible biological roles of these compounds, we investigated arsenic metabolism in the unicellular green alga Dunaliella tertiolecta, and discovered an arsenolipid fundamentally different from all those previously identified; namely, a phytyl 5-dimethylarsinoyl-2-O-methyl-ribofuranoside. The discovery is of particular interest because 2-O-methylribosides have, until now, only been found in RNA. We briefly discuss the significance of the new lipid in biosynthesis and arsenic biogeochemical cycling.

Mono-acyl arsenosugar phospholipids in the edible brown alga Kombu (Saccharina japonica)

Twenty one arsenolipids, including eight new compounds (AsSugPL 692, AsSugPL 706, AsSugPL 720, AsSugPL 734, AsSugPL 742, AsSugPL 746, AsSugPL 748, and AsSugPL 776) were identified in the edible brown alga Kombu, Saccharina japonica, by means of HPLC coupled with elemental and molecular mass spectrometry. The hitherto undescribed compounds are all mono-acyl arsenosugar phospholipids, differing from previously reported natural arsenic-containing phospholipids by containing only one fatty acid on the glycerol group. Collectively, this new group of mono-acyl compounds constituted about 30% of total lipid arsenic; other significant groups were the di-acyl arsenosugar phospholipids (50%) and arsenic hydrocarbons (20%). The origin and relevance of the mono-acyl arsenosugar phospholipids in Kombu, a commercial seafood product, is briefly discussed.