Antoine Leydier

Structuring detergents for extracting and stabilizing functional membrane proteins.

Background Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. Methodology/Principal Findings Anionic calix[4]arene based detergents (C4Cn, n = 1–12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5–24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. Conclusion/Significance These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may contribute to the membrane protein crystallization field.

Uranium Extraction from Phosphoric Acid Using Bifunctional Amido-Phosphonic Acid Ligands

Ligand systems containing amido-phosphonic acid moieties were synthesized for subsequent U(VI) extraction from phosphoric acid solutions. Studies have shown that the efficiency of extraction (distribution ratios and selectivity of U(VI) over Fe(III)) is influenced by the nature of the N-dialkyl substituents and the length and nature of the spacer. A structure-activity approach resulted in the identification of a specific ligand called DEHCBPA that exhibited larger D-values than the corresponding URPHOS reference system. The distribution ratios for U(VI) extraction increased considerably with a branched N-dialkyl chain when a steric hindrance was introduced into the methylene bridge of the amido-phosphonic acid ligands.

Carbamoylalkylphosphonates for Dramatic Enhancement of Uranium Extraction from Phosphates Ores

We describe here the full synthesis of a novel family of bifunctional ligands and present a complete study of their extraction properties in regards to an aqueous phosphoric acid solution containing uranium. We developed a high yielding synthesis of amido phosphonate ligands and focused our investigation on the effect of steric hindrance on the methylene bridge between the two functions. These new bifunctional ligands were found to extract selectively hexavalent uranium U(VI) with high distribution coefficient (D) and selectivity towards iron Fe (III) in 5 M phosphoric acid solution. From a structure-activity approach a specific ligand called DEHCNPB has been put forward in regard to the outstanding results obtained for the selective extraction, and quantitative recovery of uranium compared to the URPHOS reference system.

Solid lipid nanoparticles (SLNs) derived from para-acyl-calix[9]-arene: preparation and stability

A study of the parameters relating to the preparation of para-acyl-calix[9]arene-based solid lipid nanoparticles (SLNs) has been undertaken. Dynamic light scattering and electron microscopy have shown that the particle size varies between 85 and 215 nm depending on the acyl chain length. Parameters, including the organic solvent, amphiphile concentration and the presence of a co-surfactant affect the size of the SLNs obtained significantly. In contrast, stirring speed and solution viscosity have no effect. The ionic strength of the suspension has been shown to affect SLN stability in a salt-dependent manner. Ultrasonic and ultraviolet and 80°C treatment of the SLN suspensions have no effect on the SLN stability. The SLNs are unstable with respect to freezing–defreezing cycles, but can be reconstituted using mono- or disaccharides as cryoprotectants. Importantly, the temporal stability of these suspensions in water has been shown to be superior to 6 months. With regard to protein interactions, no SLN aggregation was observed in the presence of human serum albumin, with formation of a monolayer of albumin on the surface of the SLNs. Encapsulation was shown using acridine as a fluorescent probe.

Trianionic calix[4]arene monoalkoxy derivatives: synthesis, solid-state structures and self-assembly properties

The synthesis of a series of novel tris[(carboxy)methyl]-monoalkoxy-trihydroxycalix[4]arenes is described. Treatment of the tris[(dimethylamino)methyl]-monoalkoxy-trihydroxycalix[4]arene derivatives with iodomethane, followed by sodium cyanide as a nucleophile, gave the corresponding tris[(cyano)methyl]-monoalkoxy-trihydroxycalix[4]arenes in excellent yield, and these latter compounds were converted by hydrolysis to their tris[(carboxy)methyl]-monoalkoxy derivatives. The solid-state structures of four of the intermediate tris[(cyano)methyl]-monoalkoxy-trihydroxycalix[4]arenes show inclusion of an acetonitrile guest in the ethoxy derivative and intermolecular inclusion of the hydrophobic terminal region of the alkoxy groups for longer chain lengths. The packing of both structural types show 1-D inclusion polymer behaviour. At pH values above 5.5, all the trianionic tris[(carboxy)methyl]-monoalkoxy-trihydroxycalix[4]arene derivatives showed self-assembly to yield micellar systems, for which there was a clear odd–even effect in the observed surface tensions at the critical micelle concentrations.

Development of a new solvent extraction process based on butyl-1-[N,N-bis(2-ethylhexyl)carbamoyl]nonyl phosphonic acid for the selective recovery of uranium(VI) from phosphoric acid

ABSTRACT The promising new extractant molecule butyl-1-[N,N-bis(2-ethylhexyl)carbamoyl]nonyl phosphonic acid (DEHCNPB) was designed and used to develop a new solvent extraction process for the selective recovery of uranium from phosphoric acid. This bifunctional extracting molecule shows high affinity and selectivity for uranium(VI) versus iron(III) and the other elements present in phosphoric acid (Al, Ti, V, etc.). Batch equilibrium experiments were first carried out to determine the stoichiometries of the different complexes formed with uranium(VI) and iron(III) in organic phase and to optimize the different steps of the process at laboratory scale. These experimental data were then used to develop a chemical model to simulate uranium(VI) and iron(III) extraction from phosphoric acid, which was implemented in the PAREX simulation code. A flowsheet was calculated and tested in laboratory-scale mixer-settlers on a genuine phosphoric acid industrial solution. The continuous counter-current test was very successful and showed the possibility to recover more than 95% of uranium decontaminated from the main impurities. A Fe/U ratio of 0.03% was measured in the uranium product, which confirms the high potential of this new solvent for the further production of nuclear-grade uranium from phosphate ores.

Magnesium dependent complexation of tri-anionic calix[4]arene detergents by the nucleotide binding domain 1 (NBD1) of multidrug resistance protein MRP1

The tri-carboxylatomethylene-mono-alkoxy calix[4]arenes have been shown by fluorescence spectroscopy to bind to the NBD1 domain of MRP1 protein in a magnesium dependent manner. The observed associations constants are of the same order as that observed for ATP–Mg, the natural substrate for this protein.

Uranium adsorption from sulfuric acid media using silica materials functionalised with amide and phosphorous ligands

Various functionalised silica materials have been investigated as uranium extractants from sulfuric acid solutions. Silica was grafted with zero, one or two amido and phosphonate groups either directly or by peptide coupling. The role of the residual functions (silanol, amine and amide) was evaluated. Our results show that the uranium extraction efficiency of materials bearing residual silanol or amide groups may be affected by the presence of molybdenum. Comparing the performance of materials with different numbers of phosphonic acids on the phosphonate group revealed that only those with a monoacid function extract uranium efficiently. Among these materials, those with a diamido phosphonate ligand demonstrated the highest selectivity for uranium versus iron. Adjusting the length and steric hindrance of the alkyl chains of the amido and the phosphonate group, the best extraction capacity and selectivity versus iron were achieved with a butyl group on the phosphonate and an ethyl group on the ternary amide of the diamido phosphonate ligand. Finally, the [ligand]/[U] ratio was found not to vary with the alkyl chain at the end of the amido group or with the bridge length between the carbamoyl monophosphonate extracting group and the amidopropyl anchor to the silica, suggesting that the extraction mechanism remains unchanged.