Collaborative study for the detection of toxic compounds in shellfish extracts using cell-based assays. Part I: screening strategy and pre-validation study with lipophilic marine toxins

Human poisoning due to consumption of seafood contaminated with phycotoxins is a worldwide problem, and routine monitoring programs have been implemented in various countries to protect human consumers. Following successive episodes of unexplained shellfish toxicity since 2005 in the Arcachon Bay on the French Atlantic coast, a national research program was set up to investigate these atypical toxic events. Part of this program was devoted to fit-for-purpose cell-based assays (CBA) as complementary tools to collect toxicity data on atypical positive-mouse bioassay shellfish extracts. A collaborative study involving five laboratories was conducted. The responses of human hepatic (HepG2), human intestinal (Caco2), and mouse neuronal (Neuro2a) cell lines exposed to three known lipophilic phycotoxins-okadaic acid (OA), azaspiracid-1 (AZA1), and pectenotoxin-2 (PTX2)-were investigated. A screening strategy composed of standard operating procedures and a decision tree for dose-response modeling and assay validation were designed after a round of "trial-and-error" process. For each toxin, the shape of the concentration-response curves and the IC(50) values were determined on the three cell lines. Whereas OA induced a similar response irrespective of the cell line (complete sigmoid), PTX2 was shown to be less toxic. AZA1 induced cytotoxicity only on HepG2 and Neuro2a, but not on Caco2. Intra- and inter-laboratory coefficients of variation of cell responses were large, with mean values ranging from 35 to 54 % and from 37 to 48 %, respectively. Investigating the responses of the selected cell lines to well-known toxins is the first step supporting the use of CBA among the panel of methods for characterizing atypical shellfish toxicity. Considering these successful results, the CBA strategy will be further applied to extracts of negative, spiked, and naturally contaminated shellfish tissues.     

Supramolecular chemistry of half-sandwich organometallic building blocks based on RuCl2(p-cymene)Ph2PCH2Y

A new series of neutral organometallic building blocks based on piano-stool ruthenium(II) complexes, RuCl₂(p-cymene)Ph₂PCH₂Y [Y = -NHC₆H₄(2-CO₂H) (2a), -NHC₆H₄(3-CO₂H) (2b), -NHC₆H₃(3-CO₂H)(6-OCH₃) (2c), -NHC₆H₄(4-CO₂H) (2d), -NHC₆H₃(2-CO₂H)(4-OH) (2e), -NHC₆H₃(3-OH)(4-CO₂H) (2f), -NHC₆H₃(2-CO₂H)(5-CO₂H) (2g) and -OH (2h)], were synthesised in high yields (>88%) from {RuCl₂(p-cymene)}₂ and the appropriate phosphines 1a-1h. The new tertiary phosphine 1b was prepared by Mannich condensation of NH₂C₆H₄(3-CO₂H) with Ph₂PCH₂OH in MeOH. Solution NMR (³¹P{¹H}, ¹H), FT-IR and microanalytical data are in full agreement with the proposed structures. Single crystal X-ray studies confirm that, in each case, compounds 2a, 2b and 2d-2h have piano-stool arrangements with typical Ru-P, Ru-Cl and Ru-C_centroid bond lengths. From our crystallographic studies, factors that influence the supramolecular assemblies of these ruthenium(II) complexes include: (i) the type of functional group present, (ii) the geometric disposition of the -N(H)CH₂PPh₂, -CO₂H and -OH groups around the central benzene scaffold, and (iii) the solvents used in the recrystallisations. Hence in isomers 2a and 2b, molecules are associated into head-to-tail dimer pairs through classical intermolecular O-H?O hydrogen bonding. This feature is also observed in isomer 2d but dimer pairs are further associated to give a 1-D chain through assisted intermolecular N-H?Cl hydrogen bonding. The additional 4-hydroxo group in 2e promotes a ladder arrangement via intermolecular O-H?O and O-H?Cl hydrogen bonding. In contrast the isomeric compound 2f does not show head-to-tail O-H?O hydrogen bonding but instead O-H?Cl and N-H?O intermolecular hydrogen bonding is observed. Depending on the choice of solvent (MeOH or DMSO), 2g forms extended networks based on chains (2g · DMSO · 1.5MeOH) or tapes (2g · 3MeOH). In 2h, a single intramolecular O-H?Cl hydrogen bond is observed for each independent molecule. The X-ray structure of one representative tertiary phosphine, 1f, has also been determined.     

Modulation of amyloid-β aggregation by metal complexes with a dual binding mode and their delivery across the blood–brain barrier using focused ultrasound

One of the key hallmarks of Alzheimer''s disease is the aggregation of the amyloid-β peptide to form fibrils. Consequently, there has been great interest in studying molecules that can disrupt amyloid-β aggregation. While a handful of molecules have been shown to inhibit amyloid-β aggregation in vitro, there remains a lack of in vivo data reported due to their inability to cross the blood–brain barrier. Here, we investigate a series of new metal complexes for their ability to inhibit amyloid-β aggregation in vitro. We demonstrate that octahedral cobalt complexes with polyaromatic ligands have high inhibitory activity thanks to their dual binding mode involving π–π stacking and metal coordination to amyloid-β (confirmed via a range of spectroscopic and biophysical techniques). In addition to their high activity, these complexes are not cytotoxic to human neuroblastoma cells. Finally, we report for the first time that these metal complexes can be safely delivered across the blood–brain barrier to specific locations in the brains of mice using focused ultrasound.

We report a series of non-toxic cobalt(iii) complexes which inhibit Aβ peptide aggregation in vitro; these complexes can be safely delivered across the blood–brain barrier in mice using focused ultrasound.     

Synthesis of azafluorenones and related compounds using deprotocupration–aroylation followed by intramolecular direct arylation

The efficiency of the deprotocupration–aroylation of 2-chloropyridine using lithiocuprates prepared from CuX (X=Cl, Br) and LiTMP (TMP=2,2,6,6-tetramethylpiperidido, 2 equiv) was investigated. CuCl was identified as a more suitable copper source than CuBr for this purpose. Different diaryl ketones bearing a halogen at the 2 position of one of the aryl groups were synthesized in this way from azines and thiophenes. These were then involved in palladium-catalyzed ring closure: substrates underwent expected CH-activation-type arylation to afford fluorenone-type compounds, and were also subjected to cyclization reactions leading to xanthones, notably in the presence of oxygen-containing substituents or reagents.     

Synthesis and properties of a functionalized heterobimetallic Re(I)–Gd(III) complex as a potential dual-contrast agent for molecular imaging

In the design of dual-imaging probes, the first functionalized and neutral heterobimetallic Re(I)–Gd(III) complex, highly soluble in aqueous solutions, has been prepared. This system exhibits interesting photophysical properties (λ_em = 578 nm, ? = 1.4%) for optical imaging and substantial higher relaxivity (r₁ = 6.6 mM⁻¹ s⁻¹ at 0.47 T and 37 °C) than the clinically used MRI contrast agents. Moreover, this system incorporates an aromatic ester functionality suitable for bioconjugation.     

Schistosomiasis Chemotherapy

After malaria, schistosomiasis (or bilharzia) is the second most prevalent disease in Africa, and is occurring in over 70 countries in tropical and subtropical regions. It is estimated that 600 million people are at risk of infection, 200 million people are infected, and at least 200 000 deaths per year are associated with the disease. All schistosome species are transmitted through contact with fresh water that is infested with free‐swimming forms of the parasite, which is known as cercariae and produced by snails. When located in the blood vessels of the host, larval and adult schistosomes digest red cells to acquire amino acids for growth and development. Vaccine candidates have been unsuccessful up to now. Against such devastating parasitic disease, the antischistosomal arsenal is currently limited to a single drug, praziquantel, which has been used for more than 35 years. Because the question of the reduction of the activity of praziquantel was raised recently, it is thus urgent to create new and safe antischistosomal drugs that should be combined with praziquantel to develop efficient bitherapies.     

Synthesis and characterization of model compounds of the lysine tyrosyl quinone cofactor of lysyl oxidase

4-n-Butylamino-5-ethyl-1,2-benzoquinone (1(ox)) has been synthesized as a model compound for the LTQ (lysine tyrosyl quinone) cofactor of lysyl oxidase (LOX). At pH 7, 1(ox) has a lambda(max) at 504 nm and exists as a neutral o-quinone in contrast to a TPQ (2,4,5-trihydroxyphenylalanine quinone) model compound, 4, which is a resonance-stabilized monoanion. Despite these structural differences 1(ox) and 4 have the same redox potential (ca. -180 mV vs SCE). The structure of the phenylhydrazine adduct of 1(ox) (2) is reported, and 2D NMR spectroscopy has been used to show that the position of nucleophilic addition is at C(1). UV-vis spectroscopic pH titration of phenylhydrazine adducts of 1(ox) and 4, 2, and 11, respectively, reveals a similar red shift in lambda(max) at alkaline pH with the same pK(a) (approximately 11.8). In contrast, the red shift in lambda(max) at acidic pH conditions yields different pK(a) values (2.12 for 2 vs -0.28 for 11), providing a means to distinguish LTQ from TPQ. Reactions between in situ generated 4-ethyl-1,2-benzoquinone and primary amines give a mixture of products, indicating that the protein environment must play an essential role in LTQ biogenesis by directing the nucleophilic addition of the epsilon-amino group of a lysine residue to the C(4) position of a putative dopaquinone intermediate. Characterization of a 1,6-adduct between an o-quinone and butylamine (3-n-butylamino-5-ethyl-1,2-benzoquinone, 13) confirms the assignment of LTQ as a 1,4-addition product.     

Enzyme electrokinetics: electrochemical studies of the anaerobic interconversions between active and inactive states of Allochromatium vinosum [NiFe]-hydrogenase

The cycling between active and inactive states of the catalytic center of [NiFe]-hydrogenase from Allochromatium vinosum has been investigated by dynamic electrochemical techniques. Adsorbed on a rotating disk pyrolytic graphite "edge" electrode, the enzyme is highly electroactive: this allows precise manipulations of the complex redox chemistry and facilitates quantitative measurements of the interconversions between active catalytic states and the inactive oxidized form Ni(r) (also called Ni-B or "ready") as functions of pH, H(2) partial pressure, temperature, and electrode potential. Cyclic voltammograms for catalytic H(2) oxidation (current is directly related to turnover rate) are highly asymmetric (except at pH > 8 and high temperature) due to inactivation being much slower than activation. Controlled potential-step experiments show that the rate of oxidative inactivation increases at high pH but is independent of potential, whereas the rate of reductive activation increases as the potential becomes more negative. Indeed, at 45 degrees C, activation takes just a few seconds at -288 mV. The cyclic asymmetry arises because interconversion is a two-stage reaction, as expected if the reduced inactive Ni(r)-S state is an intermediate. The rate of inactivation depends on a chemical process (rearrangement and uptake of a ligand) that is independent of potential, but sensitive to pH, while activation is driven by an electron-transfer process, Ni(III) to Ni(II), that responds directly to the driving force. The potentials at which fast activation occurs under different conditions have been analyzed to yield the potential-pH dependence and the corresponding entropies and enthalpies. The reduced (active) enzyme shows a pK of 7.6; thus, when a one-electron process is assumed, reductive activation at pH < 7 involves a net uptake of one proton (or release of one hydroxide), whereas, at pH > 8, there is no net exchange of protons with solvent. Activation is favored by a large positive entropy, consistent with the release of a ligand and/or relaxation of the structure around the active site.     

The pressure–volume–temperature relationship of cellulose

Pressure–volume–temperature (PVT) measurements of α-cellulose with different water contents, were performed at temperatures from 25 to 180 °C and pressures from 19.6 to 196 MPa. PVT measurements allowed observation of the combined effects of pressure and temperature on the specific volume during cellulose thermo-compression. All isobars showed a decrease in cellulose specific volume with temperature. This densification is associated with a transition process of the cellulose, occurring at a temperature defined by the inflection point T _t of the isobar curve. T _t decreases from 110 to 40 °C with pressure and is lower as moisture content increases. For isobars obtained at high pressures and high moisture contents, after attaining a minimum, an increase in volume is observed with temperature that may be related to free water evaporation. PVT α-cellulose experimental data was compared with predicted values from a regression analysis of the Tait equations of state, usually applied to synthetic polymers. Good correlations were observed at low temperatures and low pressures. The densification observed from the PVT experimental data, at a temperature that decreases with pressure, could result from a sintering phenomenon, but more research is needed to actually understand the cohesion mechanism under these conditions.