Isabelle Schmitz-Afonso

Chemical characterization of Titan's tholins: solubility, morphology and molecular structure revisited

In this work Titans atmospheric chemistry is simulated using a capacitively coupled plasma radio frequency discharge in a N(2)-CH(4) stationnary flux. Samples of Titans tholins are produced in gaseous mixtures containing either 2 or 10% methane before the plasma discharge, covering the methane concentration range measured in Titans atmosphere. We study their solubility and associated morphology, their infrared spectroscopy signature and the mass distribution of the soluble fraction by mass spectrometry. An important result is to highlight that the previous Titans tholin solubility studies are inappropriate to fully characterize such a heterogeneous organic matter and we develop a new protocol to evaluate quantitatively tholins solubility. We find that tholins contain up to 35% in mass of molecules soluble in methanol, attached to a hardly insoluble fraction. Methanol is then chosen as a discriminating solvent to characterize the differences between soluble and insoluble species constituting the bulk tholins. No significant morphological change of shape or surface feature is derived from scanning electron microscopy after the extraction of the soluble fraction. This observation suggests a solid structure despite an important porosity of the grains. Infrared spectroscopy is recorded for both fractions. The IR spectra of the bulk, soluble, and insoluble tholins fractions are found to be very similar and reveal identical chemical signatures of nitrogen bearing functions and aliphatic groups. This result confirms that the chemical information collected when analyzing only the soluble fraction provides a valuable insight representative of the bulk material. The soluble fraction is ionized with an atmospheric pressure photoionization source and analyzed by a hybrid mass spectrometer. The congested mass spectra with one peak at every mass unit between 50 and 800 u confirm that the soluble fraction contains a complex mixture of organic molecules. The broad distribution, however, exhibits a regular pattern of mass clusters. Tandem collision induced dissociation analysis is performed in the negative ion mode to retrieve structural information. It reveals that (i) the molecules are ended by methyl, amine and cyanide groups, (ii) a 27 u neutral moiety (most probably HCN) is often released in the fragmentation of tholin anions, and (iii) an ubiquitous ionic fragment at m/z 66 is found in all tandem spectra. A tentative structure is proposed for this negative ion.

Tholinomics—Chemical Analysis of Nitrogen-Rich Polymers

The polymeric composition of Titans tholins--laboratory analogues of Titans aerosols--is elucidated using high-resolution mass spectrometry. This complex organic matter is produced by plasma discharge in a gaseous nitrogen-methane mixture and analyzed with a hybrid linear trap/orbitrap mass-spectrometer. The highly structured mass spectra are treated with tools developed for petroleomics (Kendrick and van Krevelen diagrams), with original adaptations for nitrogen-rich compounds. Our goal is to find the best chemical basis set to describe the compositional space that these polymers occupy, to shed light onto the chemical structure of tholins. We succeeded in assigning the molecules identified in the mass spectra of tholins to a small number of regularly distributed X-(CH(2))(m)(HCN)(n) families, where the balanced copolymer (m = n) is determined to play a central role. Within each family, the polymer lengths n and m present Poisson-type distributions. We also identify the smallest species of a subset of families as linear and cyclic amino nitrile compounds of great astrobiological interest: biguanide, guanidin, acetamidine, aminoacetonitrile, and methylimidazole.

Ligand-Free Pd-Catalyzed and Copper-Assisted C–H Arylation of Quinazolin-4-ones with Aryl Iodides under Microwave Heating

A microwave-assisted method for the palladium-catalyzed direct arylation of quinazolin-4-one has been developed under copper-assistance. This method is applicable to a wide range of aryl iodides and substituted (2H)-quinazolin-4-ones. This protocol provides a simple and efficient way to synthesize biologically relevant 2-arylquinazolin-4-one backbones.

Acylphenols from Myristica crassa as New Acetylcholinesterase Inhibitors

A significant acetylcholinesterase inhibitory activity was observed for the ethyl acetate and methanol extracts from the leaves and the fruits of MYRISTICA CRASSA. Three new dimeric acylphenols, giganteone C ( 5), maingayones B and C ( 6 and 7) were isolated together with the known malabaricones B and C ( 2 and 3) and giganteone A ( 4). Compounds 2 and 3 possess significant inhibitory activity on acetylcholinesterase. LC/MS study was particularly useful to discriminate structures of compounds 6 and 7.

Optimization of a liquid chromatography ion mobility-mass spectrometry method for untargeted metabolomics using experimental design and multivariate data analysis.

High-resolution mass spectrometry coupled with pattern recognition techniques is an established tool to perform comprehensive metabolite profiling of biological datasets. This paves the way for new, powerful and innovative diagnostic approaches in the post-genomic era and molecular medicine. However, interpreting untargeted metabolomic data requires robust, reproducible and reliable analytical methods to translate results into biologically relevant and actionable knowledge. The analyses of biological samples were developed based on ultra-high performance liquid chromatography (UHPLC) coupled to ion mobility - mass spectrometry (IM-MS). A strategy for optimizing the analytical conditions for untargeted UHPLC-IM-MS methods is proposed using an experimental design approach. Optimization experiments were conducted through a screening process designed to identify the factors that have significant effects on the selected responses (total number of peaks and number of reliable peaks). For this purpose, full and fractional factorial designs were used while partial least squares regression was used for experimental design modeling and optimization of parameter values. The total number of peaks yielded the best predictive model and is used for optimization of parameters setting.

Chronic consumption of Annona muricata juice triggers and aggravates cerebral tau phosphorylation in wild-type and MAPT transgenic mice

In the pathogenesis of tauopathies, genetic and environmental factors have been identified. While familial clustering led to the identification of mutations in MAPT encoding the microtubule‐associated protein tau, the high incidence of a sporadic tauopathy endemic in Guadeloupe was linked to the plant‐derived mitochondrial complex I inhibitor annonacin. The interaction of both factors was studied in the present work in a realistic paradigm over a period of 12 months. Mice over‐expressing either human wild‐type tau or R406W mutant tau as well as non‐transgenic mice received either regular drinking water or commercially available tropical fruit juice made of soursop (Annona muricata L.) as dietary source of neurotoxins. HPLC‐MS analysis of this juice identified several Annonaceous acetogenins, mainly annonacin (16.2 mg/L), and 41 isoquinoline alkaloids (18.0 mg/L, mainly asimilobine and reticuline). After 12 month of juice consumption, several brain regions showed an increased number of neurons with phosphorylated tau in the somatodendritic compartment of R406W mice and, to a much lesser extent, of non‐transgenic mice and mice over‐expressing human wild‐type tau. Moreover, juice drinking was associated with a reduction in synaptophysin immunoreactivity, as well as an increase in 3‐nitrotyrosine (3NT) reactivity in all three genotypes. The increase in 3NT suggests that Annona muricata juice promotes the generation of reactive nitrogen species. This study provides first experimental evidence that long‐lasting oral ingestion of a widely consumed environmental factor can induce somatodendritic accumulation of hyperphosphorylated tau in mice expressing rodent or human wild‐type tau, and can accelerate tau pathology in R406W‐MAPT transgenic mice.

Development of HPLC-Orbitrap method for identification of N-bearing molecules in complex organic material relevant to planetary environments

Although the Cassini Spacecraft and the Huygens lander provided numerous information about Titan atmospheric chemistry and the formation of its aerosols, the exact composition of these aerosols still remains unknown. A fruitful proxy to investigate these aerosols is the use of laboratory experiments that allow producing and studying analogs of Titan aerosol, the so-called tholins. Even when produced in the laboratory, unveiling the exact composition of the aerosol remains problematic due to the high complexity of the material. Numerous advances have been recently made using high-resolution mass spectrometry (HRMS) (0020, 0027 and 0011) that allowed the separation of isobaric compounds and a robust identification of chemical species composing tholins regarding their molecular formulae. Nevertheless isomeric species cannot be resolved by a simple mass measurement. We propose here an analysis of tholins by high performance liquid chromatography (HPLC) coupled to HRMS to unveil this isomeric ambiguity for some of the major tholins compounds. By comparing chromatograms obtained when analyzing tholins and chemical standards, we strictly identified seven molecules in our tholins samples: melamine, cyanoguanidine, 6-methyl-1,3,5-triazine-2,4-diamine, 2,4,6-triaminopyrimidine, 3-amino-1,2,4-triazole, 3,5-Dimethyl-1,2,4-triazole and 2,4-diamino-1,3,5-triazine. Several molecules, including hexamethylenetriamine (HMT) were not present at detectable levels in our sample. The use for the first time of a coupled HPLC-HRMS technique applied to tholins study demonstrated the interest of such a technique compared to single high-resolution mass spectrometry for the study of tholins composition.

Urinary metabolic phenotyping of mucopolysaccharidosis type I combining untargeted and targeted strategies with data modeling

BACKGROUND Application of metabolic phenotyping could expand the pathophysiological knowledge of mucopolysaccharidoses (MPS) and may reveal the comprehensive metabolic impairments in MPS. However, few studies applied this approach to MPS. METHODS We applied targeted and untargeted metabolic profiling in urine samples obtained from a French cohort comprising 19 MPS I and 15 MPS I treated patients along with 66 controls. For that purpose, we used ultra-high-performance liquid chromatography combined with ion mobility and high-resolution mass spectrometry following a protocol designed for large-scale metabolomics studies regarding robustness and reproducibility. Furthermore, 24 amino acids have been quantified using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Keratan sulfate, Heparan sulfate and Dermatan sulfate concentrations have also been measured using an LC-MS/MS method. Univariate and multivariate data analyses have been used to select discriminant metabolites. The mummichog algorithm has been used for pathway analysis. RESULTS The studied groups yielded distinct biochemical phenotypes using multivariate data analysis. Univariate statistics also revealed metabolites that differentiated the groups. Specifically, metabolites related to the amino acid metabolism. Pathway analysis revealed that several major amino acid pathways were dysregulated in MPS. Comparison of targeted and untargeted metabolomics data with in silico results yielded arginine, proline and glutathione metabolisms being the most affected. CONCLUSION This study is one of the first metabolic phenotyping studies of MPS I. The findings might help to generate new hypotheses about MPS pathophysiology and to develop further targeted studies of a smaller number of potentially key metabolites.

Gas-phase reactivity of acylphenols in electrospray and matrix-assisted laser desorption ionization mass spectrometry

Acylphenols from Myristica crassa were identified based on liquid chromatography high-resolution mass spectrometry and liquid chromatography tandem mass spectrometry (MS/MS) experiments. Two types of compound were found in the extract of the plant: monomeric (malabaricone B and C) and dimeric compounds (C–C bonded biphenyl and C–C bonded phenyl–linear carbon chain). Evidence of formation of covalent dimeric ions during the electrospray ionization and matrix-assisted laser desorption ionization (MALDI) processes was established. [2M–3H]− dimeric ions were detected on the mass spectra of each monomeric compound during high-performance liquid chromatography separation. The MS/MS spectra of those species were compared to the MS/MS spectra obtained for the dimeric compounds synthesized by the plant. Fragmentation pathways were studied for the two classes of dimer. The dimeric ions formed in the ion source were C–C bonded biphenyl compounds. Further evidence was obtained from MALDI experiments: increase in the extraction delay time leads to an increase of the dimeric ions relative abundance. Their formation is based on the high reactivity of phenols or phenolate ions which are easily oxidized yielding phenoxy radicals.

Validated Method for Strigolactone Quantification by Ultra High‐Performance Liquid Chromatography – Electrospray Ionisation Tandem Mass Spectrometry Using Novel Deuterium Labelled Standards

INTRODUCTION Strigolactones (SLs) are important plant hormones. They are difficult to analyse because they occur in very small concentrations especially in comparison with other plant hormones and other substances can interfere with their detection. OBJECTIVE To develop a procedure for the extraction, purification and quantification of SLs from plant roots. METHODOLOGY Samples were prepared by extraction of plant root tissues with ethyl acetate. Then the extracts were further purified with silica column chromatography. The natural SLs in the final extracts were quantified using novel deuterium labelled SLs. The results of the methodology were compared with those of the procedure of Yoneyama and coworkers. RESULTS This procedure required about 1-g root samples to detect and quantify simultaneously the SLs (orobanchyl acetate and fabacyl acetate) concentration with high reliability. CONCLUSION A method was developed for determining endogenous fabacyl acetate and orobanchyl acetate in plant tissue based on novel deuterium labelled standards. A method of orobanchol quantification using a synthetic SL GR24 as internal standard was proposed. Copyright