Chemical Glucosylation of Labile Natural Products Using a (2-Nitrophenyl)acetyl-Protected Glucosyl Acetimidate Donor.

The synthesis of (2-nitrophenyl)acetyl (NPAc)-protected glucosyl donors is described that were designed for the neighboring-group assisted glucosylation of base-labile natural products also being sensitive to hydrogenolysis. Glycosylation conditions were optimized using a trichloroacetimidate glucosyl donor, and cyclohexylmethanol and (+)-menthol as model acceptors. The approach was then extended to a one-pot procedure for the synthesis of 1,2-trans-glycosides. This method was finally applied for improved synthesis of the masked mycotoxin T2-O-ß,d-glucoside.

UDP-Glucosyltransferases from Rice, Brachypodium, and Barley: Substrate Specificities and Synthesis of Type A and B Trichothecene-3-O-ß-d-glucosides.

Trichothecene toxins are confirmed or suspected virulence factors of various plant-pathogenic Fusarium species. Plants can detoxify these to a variable extent by glucosylation, a reaction catalyzed by UDP-glucosyltransferases (UGTs). Due to the unavailability of analytical standards for many trichothecene-glucoconjugates, information on such compounds is limited. Here, the previously identified deoxynivalenol-conjugating UGTs HvUGT13248 (barley), OsUGT79 (rice) and Bradi5g03300 (Brachypodium), were expressed in E. coli, affinity purified, and characterized towards their abilities to glucosylate the most relevant type A and B trichothecenes. HvUGT13248, which prefers nivalenol over deoxynivalenol, is also able to conjugate C-4 acetylated trichothecenes (e.g., T-2 toxin) to some degree while OsUGT79 and Bradi5g03300 are completely inactive with C-4 acetylated derivatives. The type A trichothecenes HT-2 toxin and T-2 triol are the kinetically preferred substrates in the case of HvUGT13248 and Bradi5g03300. We glucosylated several trichothecenes with OsUGT79 (HT-2 toxin, T-2 triol) and HvUGT13248 (T-2 toxin, neosolaniol, 4,15-diacetoxyscirpenol, fusarenon X) in the preparative scale. NMR analysis of the purified glucosides showed that exclusively ß-D-glucosides were formed regio-selectively at position C-3-OH of the trichothecenes. These synthesized standards can be used to investigate the occurrence and toxicological properties of these modified mycotoxins.

Assessing Almond and Peanut Allergens Using Commercially Available Immunoanalytical Kits and LC-MS/MS: A Case Study.

With an ever-increasing allergic population and an emerging market for allergen-free foods, accurate detection of allergens in foods has never been more important. Although ELISA-based methods are the most widely used for detection of allergens in food, there is a need for the development of orthogonal approaches. A commercial ELISA detected a relatively high concentration of peanut and almond in an allergen-free product. However, another commercial ELISA declared a low peanut concentration and was negative for almond. Further testing using a commercial almond lateral-flow device confirmed the results from the second ELISA kit and demonstrated that the positive detection of almond was due to cross-reactivity. An MS method was used for final confirmation that the reported results were negative for both almond and peanut.

Determination of cyclopiazonic acid in white mould cheese by liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) using a novel internal standard.

For the determination of cyclopiazonic acid (CPA) in food and feed samples a simple and accurate LC-MS/MS method, which does not require extensive sample clean-up steps, was developed and validated. A fully carbon-13-labelled internal standard was used to compensate for matrix effects. Briefly, the samples were extracted with 1% formic acid in acetonitrile and directly analysed with HPLC-MS/MS. The following MS/MS transitions were used: m/z 337/196 and 337/182 for cyclopiazonic acid; m/z 357/210 and 357/191 for (13)C20-cyclopiazonic acid. Applying this optimised method, LODs down to 0.2µgkg(-1) and LOQs down to 0.5µgkg(-1) were determined in the validated matrices. The focus of this study was testing different types of white mould cheese but other complex samples could also be analyzed successfully with this method. It was interesting to find out, that in some commercially available white mould cheese, high concentrations of CPA (up to 3700µgkg(-1)) could be found.

Determining and characterizing hapten loads for carrier proteins by MALDI-TOF MS and MALDI-TOF/RTOF MS.

The increasing number of bioconjugates used for bioanalytical purposes and in pharmaceutical industries has led to an increasing demand for robust quality control of products derived from covalently linking small molecules to proteins. Here we report, for the first time, a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)-based method to determine the quantity and location of the hapten zearalenone (ZEN) introduced to the carrier protein conalbumin (Con). This bioconjugate is of special interest because of its application in lateral flow immunoassays commercially available for fast testing of food and feed for the presence of ZEN, a common contaminant of all major cereal grains worldwide. Mass spectrometry (MS) analysis of the intact protein turned out to be highly reproducible allowing for the determination of the average hapten load of the carrier protein. In that way an easy and fast method to screen for changes in ZEN load after bioconjugate synthesis was established. For a more detailed hapten load characterization, measurements at the peptide level were of importance. Systematic studies, implementing post-source decay (PSD) and high- and low-energy collision-induced dissociation (CID), showed characteristic fragmentation pattern for three model peptides carrying between one and three lysines (the primary target for the ZEN modification) besides other, less obvious modification sites (serine, arginine and the N-terminus). By this, indicative reporter ions (m/z 203 and 316) and neutral losses (Δm/z 373 and 317) for the ZEN modification in general, plus immonium ions (m/z 87, 142 and 159) for the lysine modification in particular were identified. Based on these findings, proteolytic peptides, tentatively assigned to be modified, were unequivocally confirmed to be affected by bioconjugation. For a protein carrying on average only 2-3 modifications per molecule 29 Lys out of 59 potential modifications sites were actually modified. Considerations taking the protein structure into account showed that the affected Lys were predominantly located on the protein's surface.

Isolation and structure elucidation of pentahydroxyscirpene, a trichothecene Fusarium mycotoxin.

Pentahydroxyscirpene, a novel trichothecene-type compound, was isolated from Fusarium-inoculated rice. The structure of pentahydroxyscirpene was elucidated by 1D and 2D NMR spectroscopy and X-ray single-crystal diffraction. The conformation in solution was determined by NOESY experiments supported by quantum chemical calculations. In vitro toxicity tests showed that pentahydroxyscirpene inhibits protein synthesis as do other trichothecenes.

Stable isotopic labelling-assisted untargeted metabolic profiling reveals novel conjugates of the mycotoxin deoxynivalenol in wheat.

An untargeted screening strategy for the detection of biotransformation products of xenobiotics using stable isotopic labelling (SIL) and liquid chromatography-high resolution mass spectrometry (LC-HRMS) is reported. The organism of interest is treated with a mixture of labelled and non-labelled precursor and samples are analysed by LC-HRMS. Raw data are processed with the recently developed MetExtract software for the automated extraction of corresponding peak pairs. The SIL-assisted approach is exemplified by the metabolisation of the Fusarium mycotoxin deoxynivalenol (DON) in planta. Flowering ears were inoculated with 100 µg of a 1 + 1 (v/v) mixture of non-labelled and fully labelled DON. Subsequent sample preparation, LC-HRMS measurements and data processing revealed a total of 57 corresponding peak pairs, which originated from ten metabolites. Besides the known DON and DON-3-glucoside, which were confirmed by measurement of authentic standards, eight further DON-biotransformation products were found by the untargeted screening approach. Based on a mass deviation of less than ±5 ppm and MS/MS measurements, one of these products was annotated as DON-glutathione (GSH) conjugate, which is described here for the first time for wheat. Our data further suggest that two DON-GSH-related metabolites, the processing products DON-S-cysteine and DON-S-cysteinyl-glycine and five unknown DON conjugates were formed in planta. Future MS/MS measurements shall reveal the molecular structures of the detected conjugates in more detail.