Synthetic molecular mimics of naturally occurring cyclopentenones exhibit antifungal activity towards pathogenic fungi

The naturally occurring reactive electrophilic species 12-oxo-phytodienoic acid (12-oxo-PDA) is a potent antifungal agent, whereas the plant growth regulator jasmonic acid, which is synthesized from 12-oxo-PDA, is ineffective. To address what structural features of the molecule endow it with antifungal activity, we synthesized a series of molecular mimics of 12-oxo-PDA varying in the length of the alkyl chain at its C-4 ring position. The octyl analogue (4-octyl cyclopentenone) was the most effective at suppressing spore germination and subsequent mycelial growth of a range of fungal pathogens and was particularly effective against Cladosporium herbarum and Botrytis cinerea, with minimum fungicidal concentrations in the range 100-200 µM. Introduction of a carboxyl group to the end of the chain, mimicking natural fatty acids, markedly reduced antifungal efficacy. Electrolyte leakage, indicative of membrane perturbation, was evident in both C. herbarum and B. cinerea exposed to 4-octyl cyclopentenone. Lipid composition analysis of the fungal spores revealed that those species with a high oil content, namely Fusarium oxysporum and Alternaria brassicicola, were less sensitive to 4-octyl cyclopentenone. The comparable hydrophobicity of 4-octyl cyclopentenone and 12-oxo-PDA accounts for the similar spore suppression activity of these two compounds. The relative ease of synthesis of 4-octyl cyclopentenone makes it an attractive compound for potential use as an antifungal agent. © 2011 SGM.

Synthesis and characterisation of imprinted polymeric receptor mimics

Molecularly imprinted polymers (MIPs) are crosslinked polymers containing bespoke functionalised cavities arising from the inclusion of template molecules in the polymerisation mixture and their later extraction. When the polymers are prepared functional polymerisable monomers are included which become part of the polymer matrix and serve to decorate the cavities with functionality appropriate to the template molecules. Overall, binding sites are created which have a memory for the template both in terms of shape and matching functionality. Fluorescent molecularly imprinted polymers have the benefit of a fluorophore in their cavities that may respond to the presence of bound test compound by a change in their fluorescence output. The work presented falls into three main areas. A series of fluorescent MIPs was prepared with a view to generating material capable of mimicking the binding characteristics of the metabolically important cytochrome isoform CYP2D6. The MIPs re-bound their templates and various cross-reactivities were encountered for test compound/drug recognition. One MIP in particular exhibited a rational discrimination amongst the related synthetic templates and was reasonably successful in recognising CYP2D6 substrates from the drug set tested. In order to give some insights into binding modes in MIPs, attempts were made to produce functional monomers containing two or more fluorophores that could be interrogated independently. A model compound was prepared which fitted the dual-fluorophore criteria and which will be the basis for future incorporation into MIPs. A further strand to this thesis is the deliberate incorporation of hydrophobic moieties into fluorescent functional monomers so that the resulting imprinted cavities might be biomimetic in their impersonation of enzyme active sites. Thus the imprinted cavities had specific hydrophobic regions as well as the usual polar functionality with which to interact with binding test compounds.

Sulphonated biomaterials as glycosaminoglycan mimics in wound healing

This chapter considers the available evidence and underlying physicochemical principles that support the proposition that a biomimetic wound dressing based on glycosaminoglycan models offers a potential means of influencing wound bioactivity. Available evidence showing advantages in wound healing for experimental proteoglycanbased dressing materials is described, together with an overview of the bioactive role of sulphated macromolecules. This leads to an assessment of the analogies between the sulphonate group and the sulphate group and an explanation of their unique water binding behaviour. The available information suggests the desirability of an integrated physicochemical, biochemical and biological approach to the design and synthesis of new wound healing biomaterials.

The primary amine metabolite of sibutramine stimulates lipolysis in adipocytes isolated from lean and obese mice and in isolated human adipocytes

Sibutramine is a satiety-inducing serotonin-noradrenaline reuptake inhibitor that acts predominantly via its primary and secondary metabolites. This study investigates the possibility that sibutramine and/or its metabolites could act directly on white adipose tissue to increase lipolysis. Adipocytes were isolated by a collagenase digestion procedure from homozygous lean (+/+) and obese-diabetic ob/ob mice, and from lean nondiabetic human subjects. The lipolytic activity of adipocyte preparations was measured by the determination of glycerol release over a 2-hour incubation period. The primary amine metabolite of sibutramine M2, caused a concentration-dependent stimulation of glycerol release by murine lean and obese adipocytes (maximum increase by 157 ± 22 and 245 ± 1696, respectively, p < 0.05). Neither sibutramine nor its secondary amine metabolite M1 had any effect on lipolytic activity. Preliminary studies indicated that M2-induced lipolysis was mediated via a beta-adrenergic action. The non-selective beta-adrenoceptor antagonist propranolol (10-6M) strongly inhibited M2-stimulated lipolysis in lean and obese murine adipocytes. M2 similarly increased lipolysis by isolated human omental and subcutaneous adipocytes (maximum increase by 194 ± 33 and 136 ± 4%, respectively, p < 0.05) with EC50 values of 12 nM and 3 nM, respectively. These results indicate that the sibutramine metabolite M2 can act directly on murine and human adipose tissue to increase lipolysis via a pathway involving beta-adrenoceptors. © Georg Thieme Verlag KG Stuttgart.

Reactions of singlet oxygen and singlet oxygen mimics with dicyclopropylethylenes: Comparison of reactivity and mechanisms

An extensive study of the reaction pathways of 1,1-dicyclopropyl ethylene, cis- and trans- 1,2-dicyclopropylethylenes has been undertaken with different electrophiles 4-methyl-1,2,4-triazoline-3,5-dione (MTAD), tetracyanoethylene (TCNE), and singlet oxygen $\rm(\sp1O\sb2).$ Comparison of reactivity and reaction mechanisms among the electrophiles is investigated. Singlet oxygen exhibits significantly lower reactivity compared to the other electrophiles. MTAD and TCNE react with dicyclopropylethylenes to produce predominantly $\sp{\prime\prime}2+2\sp{\prime\prime}$ adducts and a small amount of the "ene" adducts. The $\sp{\prime\prime}2+2\sp{\prime\prime}$ is the major product presumably because of the high activation energy leading to the highly strained "ene" products. Solvent trapping studies provide strong evidence of a "stepwise" mechanism, involving a zwitterionic or aziridinium imide as an intermediate from the study of the reactions products of dicyclopropylethylenes and MTAD. ^

The development of an optimized system of narcotic and explosive contraband mimics for calibration and training of biological detectors

Current commercially available mimics contain varying amounts of either the actual explosive/drug or the chemical compound of suspected interest by biological detectors. As a result, there is significant interest in determining the dominant chemical odor signatures of the mimics, often referred to as pseudos, particularly when compared to the genuine contraband material. This dissertation discusses results obtained from the analysis of drug and explosive headspace related to the odor profiles as recognized by trained detection canines. Analysis was performed through the use of headspace solid phase microextraction in conjunction with gas chromatography mass spectrometry (HS-SPME-GC-MS). Upon determination of specific odors, field trials were held using a combination of the target odors with COMPS. Piperonal was shown to be a dominant odor compound in the headspace of some ecstasy samples and a recognizable odor mimic by trained detection canines. It was also shown that detection canines could be imprinted on piperonal COMPS and correctly identify ecstasy samples at a threshold level of approximately 100ng/s. Isosafrole and/or MDP-2-POH show potential as training aid mimics for non-piperonal based MDMA. Acetic acid was shown to be dominant in the headspace of heroin samples and verified as a dominant odor in commercial vinegar samples; however, no common, secondary compound was detected in the headspace of either. Because of the similarities detected within respective explosive classes, several compounds were chosen for explosive mimics. A single based smokeless powder with a detectable level of 2,4-dinitrotoluene, a double based smokeless powder with a detectable level of nitroglycerine, 2-ethyl-1-hexanol, DMNB, ethyl centralite and diphenylamine were shown to be accurate mimics for TNT-based explosives, NG-based explosives, plastic explosives, tagged explosives, and smokeless powders, respectively. The combination of these six odors represents a comprehensive explosive odor kit with positive results for imprint on detection canines. As a proof of concept, the chemical compound PFTBA showed promise as a possible universal, non-target odor compound for comparison and calibration of detection canines and instrumentation. In a comparison study of shape versus vibration odor theory, the detection of d-methyl benzoate and methyl benzoate was explored using canine detectors. While results did not overwhelmingly substantiate either theory, shape odor theory provides a better explanation of the canine and human subject responses.

Reactions of singlet oxygen and singlet oxygen mimics with dicyclopropylethylenes: comparison of reactivity and mechanisms

An extensive study of the reaction pathways of 1,1- dicyclopropyl ethylene, cis- and trans- 1,2-dicyclopropylethylenes has been undertaken with different electrophiles 4-methyl-1,2,4- triazoline-3,5-dione (MTAD), tetracyanoethylene (TCNE), and singlet oxygen (102). Comparison of reactivity and reaction mechanisms among the electrophiles is investigated. Singlet oxygen exhibits significantly lower reactivity compared to the other electrophiles. MTAD and TCNE react with dicyclopropylethylenes to produce predominantly "2+2" adducts and a small amount of the "ene" adducts. The "2+2" is the major product presumably because of the high activation energy leading to the highly strained "ene" products. Solvent trapping studies provide strong evidence of a "stepwise" mechanism, involving a zwitterionic or aziridinium imide as an intermediate from the study of the reactions products of dicyclopropylethylenes and MTAD.

The Development of an Optimized System of Narcotic and Explosive Contraband Mimics for Calibration and Training of Biological Detectors

Current commercially available mimics contain varying amounts of either the actual explosive/drug or the chemical compound of suspected interest by biological detectors. As a result, there is significant interest in determining the dominant chemical odor signatures of the mimics, often referred to as pseudos, particularly when compared to the genuine contraband material. This dissertation discusses results obtained from the analysis of drug and explosive headspace related to the odor profiles as recognized by trained detection canines. Analysis was performed through the use of headspace solid phase microextraction in conjunction with gas chromatography mass spectrometry (HS-SPME-GC-MS). Upon determination of specific odors, field trials were held using a combination of the target odors with COMPS. Piperonal was shown to be a dominant odor compound in the headspace of some ecstasy samples and a recognizable odor mimic by trained detection canines. It was also shown that detection canines could be imprinted on piperonal COMPS and correctly identify ecstasy samples at a threshold level of approximately 100ng/s. Isosafrole and/or MDP-2-POH show potential as training aid mimics for non-piperonal based MDMA. Acetic acid was shown to be dominant in the headspace of heroin samples and verified as a dominant odor in commercial vinegar samples; however, no common, secondary compound was detected in the headspace of either. Because of the similarities detected within respective explosive classes, several compounds were chosen for explosive mimics. A single based smokeless powder with a detectable level of 2,4-dinitrotoluene, a double based smokeless powder with a detectable level of nitroglycerine, 2-ethyl-1-hexanol, DMNB, ethyl centralite and diphenylamine were shown to be accurate mimics for TNT-based explosives, NG-based explosives, plastic explosives, tagged explosives, and smokeless powders, respectively. The combination of these six odors represents a comprehensive explosive odor kit with positive results for imprint on detection canines. As a proof of concept, the chemical compound PFTBA showed promise as a possible universal, non-target odor compound for comparison and calibration of detection canines and instrumentation. In a comparison study of shape versus vibration odor theory, the detection of d-methyl benzoate and methyl benzoate was explored using canine detectors. While results did not overwhelmingly substantiate either theory, shape odor theory provides a better explanation of the canine and human subject responses.

Identification of Spongionella compounds as cyclosporine A mimics

Acknowledgements The research leading to these results has received funding from the following FEDER cofounded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016, and through Axencia Galega de Innovación, Spain, ITC-20133020 SINTOX. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD. From the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEA. We wish to thank the Clínica Losada Arránz, especially Ms. Paula López Arránz for providing the human blood samples for T cells purification. Jon Andoni Sánchez is supported by a fellowship from Plan Galego de Investigación e Crecemento, Xunta de Galicia, Spain.

Seasonal variations in surface metabolite composition of Fucus vesiculosus and Fucus serratus sampled at outer Kiel Fjord (August 2012 - July 2013)

The chemical composition of surface associated metabolites of two Fucus species (Fucus vesiculosus and Fucus serratus) was analysed by means of gas chromatography-mass spectrometry (GC-MS) to describe temporal patterns in chemical surface composition. Method: The two perennial brown macroalgae F. vesiculosus and F. serratus were sampled monthly at Bülk, outer Kiel Fjord, Germany (54°27'21 N / 10°11'57 E) over an entire year (August 2012 - July 2013). Per month and species six non-fertile Fucus individuals were collected from mixed stands at a depth of 0.5 m under mid water level. For surface extraction approx. 50 g of the upper 5-10 cm apical thalli tips were cut off per species. The surface extraction of Fucus was performed according to the protocol of de Nys and co-workers (1998) with minor modifications (see Rickert et al. 2015). GC/EI-MS measurements were performed with a Waters GCT premier (Waters, Manchester, UK) coupled to an Agilent 6890N GC equipped with a DB-5 ms 30 m column (0.25 mm internal diameter, 0.25 mM film thickness, Agilent, USA). The inlet temperature was maintained at 250°C and samples were injected in split 10 mode. He carrier gas flow was adjusted to 1 ml min-1. Alkanes were used for referencing of retention times. For further details (GC-MS sample preparation and analysis) see the related publication (Rickert et al. submitted to PLOS ONE).

Comprehensive metabolite profiling of Plantaginis Semen using ultra high performance liquid chromatography with electrospray ionization quadrupole time-of-flight tandem mass spectrometry coupled with elevated energy technique

Plantaginis Semen is commonly used in traditional medicine to treat edema, hypertension, and diabetes. The commercially available Plantaginis Semen in China mainly comes from three species. To clarify the chemical composition and distinct different species of Plantaginis Semen, we established a metabolite profiling method based on ultra high performance liquid chromatography with electrospray ionization quadrupole time-of-flight tandem mass spectrometry coupled with elevated energy technique. A total of 108 compounds, including phenylethanoid glycosides, flavonoids, guanidine derivatives, terpenoids, organic acids, and fatty acids, were identified from Plantago asiatica L., P. depressa Willd., and P. major L. Results showed significant differences in chemical components among the three species, particularly flavonoids. This study is the first to provide a comprehensive chemical profile of Plantaginis Semen, which could be involved into the quality control, medication guide, and developing new drug of Plantago seeds.

Treatment of lean and diet-induced obesity (DIO) mice with a novel stable obestatin analogue alters plasma metabolite levels as detected by untargeted LC–MS metabolomics

Introduction: Obestatin is a controversial gastrointestinal peptide purported to have metabolic actions.

Objectives: This study investigated whether treatment with a stable obestatin analogue (PEG-OB(Cys10, Cys13)) changed plasma metabolite levels firstly in lean and subsequently in diet-induced obesity (DIO) C57BL6/J mice.

Methods: Untargeted LC-HRMS metabolomics experiments were carried out in ESI + mode with plasma extracts from both groups of animals. Data were normalised, multivariate and univariate statistical analysis performed and metabolites of interest putatively identified.

Results: In lean mice, 39 metabolites were significantly changed by obestatin treatment and the majority of these were increased, including various C16 and C18 moieties of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and monoacylglycerol, along with vitamin A, vitamin D3, tyrosine, acetylcarnitine and 2α-(hydroxymethyl)-5α-androstane-3β,17β-diol. Decreased concentrations of glycolithocholic acid, 3-dehydroteasterone and various phospholipids were observed. In DIO mice, 25 metabolites were significantly affected and strikingly, the magnitudes of changes here were generally much greater in DIO mice than in lean mice, and in contrast, the majority of metabolite changes were decreases. Four metabolites affected in both groups included glycolithocholic acid, and three different long-chain (C18) phospholipid molecules (phosphatidylethanolamine, platelet activating factor (PAF), and monoacylglycerol). Metabolites exclusively affected in DIO mice included various phosphatidylcholines, lysophosphatidylcholines and fatty acyls, as well as creatine and oxidised glutathione.

Conclusion: This investigation demonstrates that obestatin treatment affects phospholipid turnover and influences lipid homeostasis, whilst providing convincing evidence that obestatin may be acting to ameliorate diet-induced impairments in lipid metabolism, and it may influence steroid, bile acid, PAF and glutathione metabolism.

Liver glucose-6-phosphatase proteins in suckling and weaned grey seal pups: structural similarities to other mammals and relationship to nutrition, insulin signalling and metabolite levels

Phocid seals have been proposed as models for diabetes because they exhibit limited insulin response to glucose, high blood glucose and increasing insulin resistance when fasting. Liver glucose-6-phosphatase (G6Pase) catalyses the final step in glucose production and is central to glucose regulation in other animals. G6Pase comprises a translocase (SLC37A4) and a catalytic subunit (G6PC). G6PC and SLC37A4 expression and activity are normally regulated by nutritional state and glucostatic hormones, particularly insulin, and are elevated in diabetes. We tested the hypotheses that (1) grey seal G6PC and SLC37A4 cDNA and predicted protein sequences differ from other species’ at functional sites, (2) relative G6Pase protein abundances are lower during feeding than fasting and (3) relative G6Pase protein abundances are related to insulin, insulin receptor phosphorylation and key metabolite levels. We show that G6PC and partial SLC37A4 cDNA sequences encode proteins sharing 82–95 % identity with other mammals. Seal G6PC contained no differences in sites responsible for activity, stability or subcellular location. Several substitutions in seal SLC37A4 were predicted to be tolerated with low probability, which could affect glucose production. Suckling pups had higher relative abundance of both subunits than healthy, postweaned fasting pups. Furthermore, relative G6PC abundance was negatively related to glucose levels. These findings contrast markedly with the response of relative hepatic G6Pase abundance to feeding, fasting, insulin, insulin sensitivity and key metabolites in other animals, and highlight the need to understand the regulation of enzymes involved in glucose control in phocids if these animals are to be informative models of diabetes.