Benzimidazole carbamate residues in milk: Detection by Surface Plasmon Resonance-biosensor, using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method for extraction

A surface plasmon resonance (SPR) biosensor screening assay was developed and validated to detect 11 benzimidazole carbamate (BZT) veterinary drug residues in milk. The polyclonal antibody used was raised in sheep against a methyl 5(6)-[(carboxypentyl)-thio]-2-benzimidazole carbamate protein conjugate. A sample preparation procedure was developed using a modified QuEChERS method. BZT residues were extracted from milk using liquid extraction/partition with a dispersive solid phase extraction clean-up step. The assay was validated in accordance with the performance criteria described in 2002/657/EC. The limit of detection of the assay was calculated from the analysis of 20 known negative milk samples to be 2.7 mu g kg(-1). The detection capability (CC beta) of the assay was determined to be 5 mu g kg(-1) for 11 benzimidazole residues and the mean recovery of analytes was in the range 81-116%. A comparison was made between the SPR-biosensor and UPLC-MS/MS analyses of milk samples (n = 26) taken from cows treated different benzimidazole products, demonstrating the SPR-biosensor assay to be fit for purpose. (C) 2009 Elsevier B.V. All rights reserved.

P3 SAR exploration of biphenyl carbamate based Legumain inhibitors

This Letter describes the further development and SAR exploration of a novel series of Legumain inhibitors. Based upon a previously identified Legumain inhibitor from our group, we explored the SAR of the carbamate phenyl ring system to probe the P3 pocket of the enzyme. This led to the identification of a sub-nanomolar inhibitor of Legumain.



Graphical abstract

Genetic and morphological characterization of Cladobotryum species causing cobweb disease of mushrooms

Cladobotryum dendroides (= Dactylium dendroides) has hitherto been regarded as the major causal agent of cobweb disease of the cultivated mushroom, Agaricus bisporus. Nucleotide sequence data for the internal transcribed spacer (ITS) regions of four Cladobotryum/Hypomyces species reported to be associated with cobweb disease, however, indicate that the most common pathogen is now C. mycophilum. This cobweb pathogen varies somewhat in conidial septation from published descriptions of C. mycophilum and lacks the distinctive colony odor. ITS sequencing revealed minor nucleotide variation which split isolates of the pathogen into three subgroups, two comprising isolates that were sensitive to methylbenzimidazole carbamate (MBC) fungicides and one comprising MBC-resistant isolates. The MBC-resistant isolates, which were only obtained from Ireland and Great Britain, clustered together strongly in randomly amplified polymorphic DNA (RAPD) PCR analysis, suggesting that they may be clonal. The MBC-sensitive isolates were more diverse. A RAPD fragment of 800 to 900 bp, containing a microsatellite and found in the MBC-resistant isolates, also indicated their clonal nature; the microsatellites of these isolates contained the same number of GA repeats. Smaller, polymorphic microsatellites, similarly comprising GA repeats, in the MBC-sensitive isolates in general correlated with their geographic origin.

Identification of benzimidazole resistance in Cladobotryum dendroides using a PCR-based method

Cladobotryutn dendroides, causal agent of cobweb disease of Agaricus bisporus, has become increasingly resistant to methylbenzimidazole carbamate (MBC) fungicides following the extensive use of MBC in cultivated mushroom production in Ireland. Of 38 isolates of C. dendroides obtained from Irish mushroom units, 34 were resistant to carbendazim. Primers based on conserved regions of the -tubulin gene were used to amplify and sequence a portion of the -tubulin gene in C. dendroides. A point mutation was detected at codon 50 in isolates resistant to benzimidazole fungicides, causing an amino acid substitution from tyrosine to cysteine. Species-specific PCR primers were designed to amplify the region of the -tubulin gene containing this substitution. The point mutation removed an Ace I restriction site in the -tubulin gene sequence of resistant isolates. Digestion of the PCR product with Ace I thus provides a rapid diagnostic test to differentiate sensitive and resistant isolates of this fungus. EMBL accession number: YI2256.

Exploring molecular recognition pathways within a family of gelators with different hydrogen bonding motifs

We report the synthesis of a family of gelators in which alkyl chains are connected to the amino groups of L-lysine methyl ester using a range of different hydrogen bonding linking groups (carbamate, amide, urea, thiourea and diacylhydrazine) using simple synthetic methodology based on isocyanate or acid chloride chemistry. The ability of these compounds to gelate organic solvents such as toluene or cyclohexane can be directly related to the ability of the linking group to form intermolecular hydrogen bonds. In general terms, the ability to structure solvents can be considered as: thiourea <carbamate <amide <urea similar to diacylhydrazine. This process has been confirmed by thermal measurements, scanning electron microscopy (SEM) and infrared and circular dichroism spectroscopies. By deprotecting the methyl ester group, we have demonstrated that a balance between hydrophobic and hydrophilic groups is essential-if the system has too much hydrophilicity (e. g., diacylhydrazine, urea) it will not form gels due to low solubility in the organic media. However, the less effective gelators based on amide and carbamate linkages are enhanced by converting the methyl ester to a carboxylic acid. Furthermore, subsequent mixing of the acid with a second component (diaminododecane) further enhances the ability to form networks, and, in the case of the amide, generates a two-component gel, which can immobilise a wide range of solvents of industrial interest including petrol and diesel (fuel oils), olive oil and sunflower oil (renewable food oils) and ethyl laurate, isopropyl myristate and isopropyl palmitate (oils used in pharmaceutical formulation). The gels are all thermoreversible, and may therefore be useful in controlled release/formulation applications.

The addition of CO2 to four superbase ionic liquids: a DFT study

The addition of carbon dioxide to four superbase ionic liquids, [P3333][Benzim], [P3333][124Triz], [P3333][123Triz] and [P3333][Bentriz] was studied using a molecular DFT approach involving anions alone and individual ion pairs. Intermolecular bonding within the individual ion pairs is characterised by a number of weak hydrogen bonds, with the superbase anion geometrically arranged so as to maximize interactions between the heterocyclic N atoms and the cation. The pairing energies show no correlation to the observed CO2 adsorption capacity. Addition of CO2 to the anion alone clearly resulted in the formation of a covalently-bound carbamate function with the strength of binding correlated to experimental capacity. In the ion pair however the cation significantly alters the nature of the bonding such that the overall cohesive energy is reduced. Formation of a strong carbamate function occurs at the expense of weakening the interaction between anion and cation. In the more weakly absorbing ion pairs which contain [123Triz]- and [Bentriz]-, the carbamate-functionalised systems are very close in energy to adducts in which CO2 is more weakly bound, suggesting an equilibrium between the chemi- and physisorbed CO2.