BACKGROUND Since the work of Sabatier 100 year ago, chemists have turned almost exclusively to metals to activate H 2 by weakening or cleaving its central bond. This paradigm changed with a 2006 report of a metal-free molecule that reversibly activated H 2 across sterically encumbered Lewis acidic boron and Lewis basic phosphorus sites. Shortly thereafter, similar reactions were mediated by systems described as “frustrated Lewis pairs” (FLPs) that were derived from simple combinations of electron donors and acceptors in which steric demands precluded dative bond formation. The variety of such systems has since been expanded to include a wide range of donors and acceptors. Moreover, FLP reactivity has been shown to result when equilibria governing the formation of Lewis acid-base adducts provides access to free acid and base. Mechanistic studies have demonstrated that the FLP activation of H 2 proceeds via a mechanism directly analogous to the Piers mechanism for borane-mediated hydrosilylation of ketones, first described in 1996. Nonetheless, the discovery of these metal-free reactions of H 2 has prompted considerable interest in this concept and its application to various chemical systems. ADVANCES The application of FLP reactivity with H 2 to metal-free hydrogenation catalysis rapidly led to reductions of polar substrates. Over the past decade, the range of reducible substrates has been expanded to a variety of unsaturated compounds, including imines, enamines, olefins, polyaromatics, alkynes, ketones, and aldehydes. Efforts have also extended this technology to asymmetric hydrogenations, with a number of recent systems achieving high selectivity. Early on, it was recognized that the reactivity of FLPs was not limited to H 2 . FLPs have shown the capacity to capture and react with a variety of small molecules, including olefins, alkynes, CO 2 , SO 2 , NO, CO, N 2 O, and N -sulfinyltolyllamines ( p -tol)NSO ( p -tol, para -tolyl). This has led to metal-free strategies for CO and CO 2 reduction and SO generation and new avenues to transient, persistent, or stable radicals. FLP chemistry has been further extended to new strategies in synthetic organic chemistry, including FLP-mediated approaches to hydroamination, hydroboration, cyclization, and boration reactions. Because transition metals may also be acidic or basic, the reactivity of FLP systems in which one or both of the constituents are metal centers has been reported. Further, metal components can also be ancillary fragments for ligand-based FLP chemistry, or they can act as a scaffold, allowing the cooperative action of an FLP and a metal center on a substrate. In related developments, the notion of FLPs has been applied to the design of model systems for the active sites of the [Ni-Fe], [Fe-Fe], or [Fe] hydrogenase enzymes. Reaching beyond main-group and organic chemistry into polymers and materials chemistry, FLP catalysts have been used to prepare lactone-derived polymers, cyanamide oligomers, and Te-containing heterocycles for applications in photoactive materials. In addition, heterogeneous FLP hydrogenation catalysts have emerged, and the concept also provides a new mechanistic perspective on CO 2 reduction on the surface of indium oxide nanocrystals. OUTLOOK Applications of FLP chemistry to metal-free reductions, asymmetric hydrogenations, C–C bond formation, and C–H bond functionalization are continuing to evolve. Such advances offer strategies for reduced costs and the elimination of toxic contaminants that will undoubtedly garner interest from the synthetic chemistry communities in academia and industry. The expanding range of main-group and transition metal–based FLPs continues to demonstrate the generality of this concept and its broadening utility. However, the innovative synthetic strategies, reactivity, and new perspectives derived from the application of this simple concept to other areas of chemistry are perhaps the most exciting prospect.

http://science.sciencemag.org/content/sci/354/6317/aaf7229.full.pdf

The broadening reach of frustrated Lewis pair chemistry

This paper describes the first validated method for the determination of 39 mycotoxins in wheat and maize using a single extraction step followed by liquid chromatography with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS) without the need for any clean-up. The 39 analytes included A- and B-trichothecenes (including deoxynivalenol-3-glucoside), zearalenone and related derivatives, fumonisins, enniatins, ergot alkaloids, ochratoxins, aflatoxins and moniliformin. The large number and the chemical diversity of the analytes required the application of the positive as well as the negative ion ESI mode in two consecutive chromatographic runs of 21 min each. The solvent mixture acetonitrile/water/acetic acid 79 + 20 + 1 (v/v/v) has been determined as the best compromise for the extraction of the analytes from wheat and maize. Raw extracts were diluted 1 + 1 and were injected without any clean-up. Ion-suppression effects due to co-eluting matrix components were negligible in the case of wheat, whereas significant signal suppression for 12 analytes was observed in maize, causing purely proportional systematic errors. Method performance characteristics were determined after spiking blank samples on multiple levels in triplicate. Coefficients of variation of the overall process of <5.1% and <3.0% were obtained for wheat and maize, respectively, from linear calibration data. Limits of detection ranged from 0.03 to 220 microg/kg. Apparent recoveries (including both the recoveries of the extraction step and matrix effects) were within the range of 100 +/- 10% for approximately half of the analytes. In extreme cases the apparent recoveries dropped to about 20%, but this could be compensated for to a large extent by the application of matrix-matched standards to correct for matrix-induced signal suppression, as only a few analytes such as nivalenol and the fumonisins exhibited incomplete extraction. For deoxynivalenol and zearalenone, the trueness of the method was confirmed through the analysis of certified reference materials.

Development and validation of a liquid chromatography/tandem mass spectrometric method for the determination of 39 mycotoxins in wheat and maize.

The aim of this review is to give a comprehensive overview of the current knowledge on plant metabolites of mycotoxins, also called masked mycotoxins. Mycotoxins are secondary fungal metabolites, toxic to human and animals. Toxigenic fungi often grow on edible plants, thus contaminating food and feed. Plants, as living organisms, can alter the chemical structure of mycotoxins as part of their defence against xenobiotics. The extractable conjugated or non-extractable bound mycotoxins formed remain present in the plant tissue but are currently neither routinely screened for in food nor regulated by legislation, thus they may be considered masked. Fusarium mycotoxins (deoxynivalenol, zearalenone, fumonisins, nivalenol, fusarenon-X, T-2 toxin, HT-2 toxin, fusaric acid) are prone to metabolisation or binding by plants, but transformation of other mycotoxins by plants (ochratoxin A, patulin, destruxins) has also been described. Toxicological data are scarce, but several studies highlight the potential threat to consumer safety from these substances. In particular, the possible hydrolysis of masked mycotoxins back to their toxic parents during mammalian digestion raises concerns. Dedicated chapters of this article address plant metabolism as well as the occurrence of masked mycotoxins in food, analytical aspects for their determination, toxicology and their impact on stakeholders.

/pdf/masked-mycotoxins-a-review-1uo7ljn5jx.pdf

Masked mycotoxins: A review

One of the exciting movements in microbial sciences has been a refocusing and revitalization of efforts to mine the fungal secondary metabolome. The magnitude of biosynthetic gene clusters (BGCs) in a single filamentous fungal genome combined with the historic number of sequenced genomes suggests that the secondary metabolite wealth of filamentous fungi is largely untapped. Mining algorithms and scalable expression platforms have greatly expanded access to the chemical repertoire of fungal-derived secondary metabolites. In this Review, I discuss new insights into the transcriptional and epigenetic regulation of BGCs and the ecological roles of fungal secondary metabolites in warfare, defence and development. I also explore avenues for the identification of new fungal metabolites and the challenges in harvesting fungal-derived secondary metabolites. In this Review, Nancy P. Keller describes the classification and genetics of secondary metabolites and biosynthetic gene clusters, cluster regulation and the ecological roles of secondary metabolites, and she explores avenues for the discovery of new fungal metabolites and current challenges in their identification.

/pdf/fungal-secondary-metabolism-regulation-function-and-drug-538bmyv3wm.pdf

Fungal secondary metabolism: regulation, function and drug discovery.

Mycotoxins are fungal metabolites commonly occurring in food, which pose a health risk to the consumer. Maximum levels for major mycotoxins allowed in food have been established worldwide. Good agricultural practices, plant disease management, and adequate storage conditions limit mycotoxin levels in the food chain yet do not eliminate mycotoxins completely. Food processing can further reduce mycotoxin levels by physical removal and decontamination by chemical or enzymatic transformation of mycotoxins into less toxic products. Physical removal of mycotoxins is very efficient: manual sorting of grains, nuts, and fruits by farmers as well as automatic sorting by the industry significantly lowers the mean mycotoxin content. Further processing such as milling, steeping, and extrusion can also reduce mycotoxin content. Mycotoxins can be detoxified chemically by reacting with food components and technical aids; these reactions are facilitated by high temperature and alkaline or acidic conditions. Detoxification of mycotoxins can also be achieved enzymatically. Some enzymes able to transform mycotoxins naturally occur in food commodities or are produced during fermentation but more efficient detoxification can be achieved by deliberate introduction of purified enzymes. We recommend integrating evaluation of processing technologies for their impact on mycotoxins into risk management. Processing steps proven to mitigate mycotoxin contamination should be used whenever necessary. Development of detoxification technologies for high-risk commodities should be a priority for research. While physical techniques currently offer the most efficient post-harvest reduction of mycotoxin content in food, biotechnology possesses the largest potential for future developments.

/pdf/impact-of-food-processing-and-detoxification-treatments-on-2974len175.pdf

Impact of food processing and detoxification treatments on mycotoxin contamination.

Deoxynivalenol (DON) is a toxic secondary metabolite produced by molds of the Fusarium genus, which are able to infect cereal crops in the field. Concerning its rate of occurrence and mean concentration, DON is one of the most important mycotoxins in cereal commodities. Its toxic effects range from causing diarrhea, vomiting, and gastro-intestinal inflammation to noncompetitive inhibition of the biosynthesis of proteins in eukaryotic cells. To study the stability of DON under food-processing conditions such as cooking or baking, we performed model heating experiments and screened the residue for degradation products. Heating of DON and 3-acetyldeoxynivalenol (3-AcDON), especially under alkaline conditions, gave a mixture of compounds, which were isolated and structurally elucidated by NMR and MS experiments. Three of these compounds were already known (norDON A, norDON B, and norDON C), while four were new and named 9-hydroxymethyl DON lactone, norDON D, norDON E, and norDON F. The significance of the DON degradation products was checked by analyzing commercially available food samples. norDON A, B, and C were detected in 29-66% of the samples in mean concentrations ranging from 3 to 15 microg/kg. Furthermore, cell culture experiments using IHKE cells showed that the compounds that were detected in food samples are less cytotoxic in the formazan dye cytotoxicity assay compared to DON. Whereas DON revealed a median effective concentration (EC50) at 1.1 micromol/L, all other compounds did not show any significant effect up to 100 micromol/L. These findings indicate that the degradation of DON under thermal treatment might reduce the toxicity of DON contaminated food.

Thermal degradation of the Fusarium mycotoxin deoxynivalenol.

An improved "dilute and shoot" LC-MS/MS multibiomarker approach was used to monitor urinary excretion of 23 mycotoxins and their metabolites in hu- man populations from Asia (Bangladesh), Europe (Ger- many), and the Caribbean region (Haiti). Deoxynivalenol (DON), deoxynivalenol-3-glucuronide (DON-3-GlcA), T-2-toxin (T-2), HT-2-toxin (HT-2), HT-2-toxin-4- glucuronide (HT-2-4-GlcA), fumonisin B1 (FB1), afla- toxins (AFB1 ,A FB2 ,A FG1, AFG2 ,A FM1), zearalenone (ZEA), zearalanone (ZAN), their urinary metabolites α- zearalanol (α-ZEL) and β-zearalanol (β-ZEL), and cor- responding 14-O-glucuronic acid conjugates (ZEA-14- GlcA, ZAN-14-GlcA, β-ZEL, α/β-ZEL-14-GlcA), och- ratoxin A (OTA), and ochratoxin alpha (OTα )a s well as enniatin B (EnB) and dihydrocitrinone (DH-CIT) were among these compounds. Eight urinary mycotoxin biomarkers were detected (AFM1, DH-CIT, DON, DON-GLcA, EnB, FB1 ,O TA, andα-ZEL). DON and DON-GlcA were exclusively detected in urines from Germany and Haiti whereas urinary OTA and DH-CIT concentrations were significantly higher in Bangladeshi samples. AFM1 was present in samples from Bangladesh and Haiti only. Exposure was estimated by the calcula- tion of probable daily intakes (PDI), and estimates sug- gested occasional instances of toxin intakes that exceed established tolerable daily intakes (TDI). The detection of individual mycotoxin exposure by biomarker-based approaches is a meaningful addition to the classical mon- itoring of the mycotoxin content of the food supply.

A comparative study of the human urinary mycotoxin excretion patterns in Bangladesh, Germany, and Haiti using a rapid and sensitive LC-MS/MS approach.

Scope

In this study, the exposure of a German population (n = 101) to mycotoxins was assessed using an LC-MS/MS urinary multibiomarker approach. Food consumption of the participants was documented with a food frequency questionnaire to correlate mycotoxin exposure with individual nutritional habits.



Methods and results

The presence of 23 urinary biomarkers including trichothecenes (deoxynivalenol (DON), DON-3-glucuronide (DON-3-GlcA), T-2 toxin, HT-2 toxin (HT-2, HT-2-toxin-4-glucuronide (HT-2-GlcA), fumonisins (fumonisin B1, fumonisin B2), aflatoxins (aflatoxin B1, aflatoxin G2, aflatoxin B2, aflatoxin M1), zearalenone and derivatives (zearalanone, α-zearalenol, β-zearalenol, zearalenone-14-O-glucuronide, zearalanone-14-O-glucuronide, α-zearalenol-14-O-glucuronide/β-zearalenol-14-O-glucuronide), ochratoxin A, ochratoxin alpha, enniatin B and dihydrocitrinone was evaluated using a validated, sensitive “dilute and shoot”-LC-MS/MS method applying Scheduled MRMTM technology. Six mycotoxins and urinary metabolites were detected (DON, DON-3-GlcA, zearalenone-14-O-glucuronide, T-2 toxin, enniatin B, and dihydrocitrinone) in 87% of the samples in single- or co-occurence. Only DON and DON-3-GlcA were detectable in quantifiable amounts. A provisional mean daily intake of 0.52 μg DON/kg body weight was calculated. No statistical evidence for the correlation of staple food intake and urinary biomarker concentration could be determined.



Conclusion

The results of this study suggest a low everyday exposure of the investigated German population to mycotoxins, but reveal peak exposures above the widely accepted tolerable daily intake to DON in parts of the population.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/mnfr.201400406

Determination of mycotoxin exposure in Germany using an LC‐MS/MS multibiomarker approach

A HPLC-MS/MS-based method for the quantification of nine mycotoxins produced by fungi of the genus Alternaria in various food matrices was developed. The method relies on a single-step extraction, followed by dilution of the raw extract and direct analysis. In combination with an analysis time per sample of 12 min, the sample preparation is cost-effective and easy to handle. The method covers alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), altenuene (ALT), iso-altenuene (isoALT), tentoxin (TEN), altertoxin-I (ATX-I), and the AAL toxins TA1 and TA2. Some Alternaria toxins which are either not commercially available or very expensive, namely AOH, AME, ALT, isoALT, and ATX-I, were isolated as reference compounds from fungal cultures. The method was extensively validated for tomato products, bakery products, sunflower seeds, fruit juices, and vegetable oils. AOH, AME, TeA, and TEN were found in quantifiable amounts and 92.1% of all analyzed samples (n = 96) showed low level contamination with one or more Alternaria toxins. Based on the obtained results, the average daily exposure to Alternaria toxins in Germany was calculated.

Survey of Alternaria toxin contamination in food from the German market, using a rapid HPLC-MS/MS approach

The mycotoxin ochratoxin A is degraded by up to 90% during coffee roasting. In order to investigate this degradation, model heating experiments with ochratoxin A were carried out, and the reaction products were analyzed by HPLC-DAD and HPLC-MS/MS. Two ochratoxin A degradation products were identified, and their structure and absolute configuration were determined. As degradation reactions, the isomerization to 14-(R)-ochratoxin A and the decarboxylation to 14-decarboxy-ochratoxin A were identified. Subsequently, an analytical method for the determination of these compounds in roasted coffee was developed. Quantification was carried out by HPLC-MS/MS and the use of stable isotope dilution analysis. By using this method for the analysis of 15 coffee samples from the German market, it could be shown that, during coffee roasting, the ochratoxin A diastereomer 14-(R)-ochratoxin A was formed in amounts of up to 25.6% relative to ochratoxin A. The decarboxylation product was formed only in traces. For toxicity evaluations, first preliminary cell culture assays were performed with the two new substances. Both degradation products exhibited higher IC50 values and caused apoptotic effects with higher concentrations than ochratoxin A in cultured human kidney epithelial cells. Thus, these cell culture data suggest that the degradation products are less cytotoxic than ochratoxin A.

Benedikt Cramer

Papers

Thermal degradation of the Fusarium mycotoxin deoxynivalenol.

A comparative study of the human urinary mycotoxin excretion patterns in Bangladesh, Germany, and Haiti using a rapid and sensitive LC-MS/MS approach.

Determination of mycotoxin exposure in Germany using an LC‐MS/MS multibiomarker approach

Survey of Alternaria toxin contamination in food from the German market, using a rapid HPLC-MS/MS approach

Identification and in vitro cytotoxicity of ochratoxin A degradation products formed during coffee roasting.