Elucidation of the environmental reductive metabolism of the herbicide tritosulfuron assisted by electrochemistry and mass spectrometry.

The environmental impact of pesticides and other pollutants is, to a great extent, determined by degradation and accumulation processes. Consequently, degradation pathways of pesticides have to be elucidated before approval by the authorities. In this study, the environmental metabolism of the sulfonylurea-herbicide tritosulfuron was investigated using aerobic soil degradation studies, during which a previously unidentified metabolite was observed using high performance liquid chromatography and mass spectrometry. The new metabolite was formed by reductive hydrogenation of tritosulfuron but the isolated amount and purity of the substance were insufficient to fully elucidate its structure. Therefore, electrochemistry coupled to mass spectrometry was successfully applied to mimic the reductive hydrogenation of tritosulfuron. After demonstrating the general feasibility of electrochemical reduction, the electrochemical conversion was scaled up to the semi-preparative scale and 1.0 mg of the hydrogenated product was synthesized. Similar retention times and mass spectrometric fragmentation patterns proved that the same hydrogenated product was formed electrochemically and in soil studies. Using the electrochemically generated standard, the structure of the metabolite was elucidated by means of NMR spectroscopy, which shows the potential of electrochemistry and mass spectrometry in environmental fate studies.

Controllable Iterative ß-Glucosylation from UDP-Glucose by Bacillus cereus Glycosyltransferase GT1: Application for the Synthesis of Disaccharide-Modified Xenobiotics.

Glycosylation in natural product metabolism and xenobiotic detoxification often leads to disaccharide-modified metabolites. The chemical synthesis of such glycosides typically separates the glycosylation steps in space and time. The option to perform the two-step glycosylation in one pot, and catalyzed by a single permissive enzyme, is interesting for a facile access to disaccharide-modified products. Here, we reveal the glycosyltransferase GT1 from Bacillus cereus (BcGT1; gene identifier: KT821092) for iterative O-ß-glucosylation from uridine 5'-diphosphate (UDP)-glucose to form a ß-linked disaccharide of different metabolites, including a C15 hydroxylated detoxification intermediate of the agricultural herbicide cinmethylin (15HCM). We identify thermodynamic and kinetic requirements for the selective formation of the disaccharide compared to the monosaccharide-modified 15HCM. As shown by NMR and high-resolution MS, ß-cellobiosyl and ß-gentiobiosyl groups are attached to the aglycone's O15 in a 2:1 ratio. Glucosylation reactions on methylumbelliferone and 4-nitrophenol involve reversible glycosyl transfer from and to UDP as well as UDP-glucose hydrolysis, both catalyzed by BcGT1. Collectively, this study delineates the iterative ß-d-glucosylation of aglycones by BcGT1 and demonstrates applicability for the programmable one-pot synthesis of disaccharide-modified 15HCM.

Selective ß-Mono-Glycosylation of a C15-Hydroxylated Metabolite of the Agricultural Herbicide Cinmethylin Using Leloir Glycosyltransferases.

Cinmethylin is a well-known benzyl-ether derivative of the natural terpene 1,4-cineole that is used industrially as a pre-emergence herbicide in grass weed control for crop protection. Cinmethylin detoxification in plants has not been reported, but in animals, it prominently involves hydroxylation at the benzylic C15 methyl group. Here, we show enzymatic ß-glycosylation of synthetic 15-hydroxy-cinmethylin to prepare a putative phase II detoxification metabolite of the cinmethylin in plants. We examined eight Leloir glycosyltransferases for reactivity with 15-hydroxy cinmethylin and revealed the selective formation of 15-hydroxy cinmethylin ß-d-glucoside from uridine 5'-diphosphate (UDP)-glucose by the UGT71E5 from safflower (Carthamus tinctorius). The UGT71E5 showed a specific activity of 431 mU/mg, about 300-fold higher than that of apple (Malus domestica) UGT71A15 that also performed the desired 15-hydroxy cinmethylin mono-glycosylation. Bacterial glycosyltransferases (OleD from Streptomyces antibioticus, 2.9 mU/mg; GT1 from Bacillus cereus, 60 mU/mg) produced mixtures of 15-hydroxy cinmethylin mono- and disaccharide glycosides. Using UDP-glucose recycling with sucrose synthase, 15-hydroxy cinmethylin conversion with UGT71E5 efficiently provided the ß-mono-glucoside (≥95% yield; ∼9 mM) suitable for biological studies.

Mansouramycins A-D, cytotoxic isoquinolinequinones from a marine streptomycete.

Chemical screening of the ethyl acetate extract from the marine-derived Streptomyces sp. isolate Mei37 resulted in five isoquinolinequinones, four new derivatives, mansouramycin A-D (1, 3-5), and the known 3-methyl-7-(methylamino)-5,8-isoquinolinedione (2). Their structures were elucidated by NMR and MS techniques and by comparison with related compounds. Cytotoxicity profiling of the mansouramycins in a panel of up to 36 tumor cell lines indicated significant cytotoxicity of several derivatives, with pronounced selectivity for non-small cell lung cancer, breast cancer, melanoma, and prostate cancer cells.

Bromoalterochromides A and A', unprecedented chromopeptides from a marine Pseudoalteromonas maricaloris strain KMM 636T.

The marine strain Pseudoalteromonas maricaloris KMM 636T was found to produce an inseparable mixture of two brominated yellow main pigments, bromoalterochromide A and A', in a ratio of 3: 1. Both pigments are Thr-Val-Asn-Asn-X pentapeptide lactones, where the amino group of Thr is acylated with 9-(3-bromo-4-hydroxyphenyl)-nona-2,4,6,8-tetraenoic acid, and X is aIle and Leu, respectively. They possess cytotoxic effects on developing eggs of the sea urchin Strongylocentrotus intermedius, but no antibiotic activity.

Modern methods to produce natural-product libraries.

Natural sources offer a wealth of chemically diverse compounds that have been evolutionary preselected to modulate biochemical pathways. Several industrial and academic groups are accessing this source using advanced technology platforms. Methods have been reported to generate large and diverse natural-product libraries optimised for high-throughput screening and for a fast discovery process. In addition to prefractionated and pure natural-product libraries, parallel synthesis gives access to synthetic, semi-synthetic and natural-product-like libraries. Natural-product chemistry and organic synthesis are powerful tools for optimising natural leads and for generating new diversity from natural scaffolds. The amalgamation of both may be expected to become an important strategy in future drug design.