Carole Chaix

The use of labile base protecting groups in oligoribonucleotide synthesis

Abstract The use of phenoxyacetyl group for the protection of the exocyclic amino function of purine bases and acetyl group for cytosine in oligonucleotide synthesis by the cyanoethylphosphoramidite approach is described. A side reaction - i.e. partial replacement of phenoxyacetyl group of protected guanines by acetyl group - was observed during the capping step. It can be avoided by the use of phenoxyacetic anhydride in place of acetic anhydride.

Viologen-based redox-switchable anion-binding receptors

A series of viologen-based receptors have been synthesized and their anion-binding properties have been investigated by-NMR, UV-visible spectroscopy, electrochemistry and X-ray diffraction analyses. Linking two positively charged viologens through a propyl chain promotes a remarkable chelate-like binding of chlorides revealed by 1H-NMR spectroscopy. Of all the anionic species investigated, only fluoride is detectable by the naked eye and by electrochemical methods. The reduction-triggered formation of a π-dimer from two viologen-based cation radicals was also investigated by electrochemical and spectroelectrochemical methods and by theoretical calculations.

Surface functionalization of oil-in-water nanoemulsion with a reactive copolymer: colloidal characterization and peptide immobilization

Cationic oil-in-water emulsion droplets were prepared using medium chain triglycerides (MCT), phosphatidylcholine/phosphatidylethanolamine phospholipid mixtures (Lipid E-80), Pluronic F68 and poly[N-acryloylmorpholine-co-N-acryloxysuccinimide] (poly[NAM/NAS]) copolymer functionalized with N-(acetyl)spermine and decylamine through chemical reaction with succinimide groups. In addition, 2-(2-pyridyldithio)ethylamine was grafted onto copolymer in order to target the covalent binding of a peptide model. At first, the obtained emulsion was characterized by quasi-elastic light scattering analysis revealing an average droplet diameter around 200 nm and a relatively narrow droplet size distribution. Electrokinetic study and colloidal stability were also performed as a function of both pH and salinity, showing the marginal effect of the Pluronic used as stabilizing agent and confirming the presence of the functionalized copolymer at the interface. Secondly, the immobilization of Influenza hemagglutinin HA-2 derived peptide was carried out on the droplet surface and highlighted the contribution of covalent binding onto the functionalized copolymer to the fixation process.

Oligonucleotide synthesis on maleic anhydride copolymers covalently bound to silica spherical support and characterization of the obtained conjugates

A new route was proposed to make polymer-oligonucleotide conjugates of potential applications in diagnostics. It consisted in direct synthesis of oligonucleotides onto controlled pore glass surface grafted with poly(maleic anhydride-alt-methyl vinyl ether) (P[MAMVE]) or poly(maleic anhydride-alt-ethylene) (P[MAE]). The anhydride moieties were used for both the covalent coupling of the copolymer via ester bond and binding of 5′-dimethoxytrityl thymidine 3′-(6-aminohexyl phosphate) (I) initiator of oligodeoxynucleotide (ODN) synthesis via amide bond. The difference of stability between ester and amide links under basic treatment was used for the selective cleavage of (polymer-oligonucleotide) conjugates after DNA synthesis completion. We succeeded in grafting functionalized copolymer onto silica surface and Polythymidine 26-mer ODN was performed. After concentrated ammonium hydroxide treatment, conjugate crude materials were characterized by size exclusion chromatography coupled to multiangle laser light scattering detection. The number average molecular weight (Mn) for conjugate with P[MAMVE] was abnormally lower than expected and was assigned to polymer degradation using high pH conditions. Such a phenomenon did not occur with P[MAE]-polythymidine conjugate. However, in both cases, parasite ODN synthesis was also evidenced, which was attributed to thymidine phosphoramidite adsorption side reaction during DNA synthesis.

Electrochemical detection of nucleic acids using pentaferrocenyl phosphoramidate α-oligonucleotides

We report the synthesis of α-oligonucleotides exhibiting five phosphoramidate linkages bearing a ferrocenyl (Fc) moiety. Three different linkers: ethyl, propyl and 4-methyl-1-ethyl-1,2,3-triazol between the ferrocene (Fc) residue and the phosphoramidate function have been studied. We demonstrated that these Fc5-α-DNA conjugates exhibit similar or even better stability than the wild type β-DNA when hybridized to their complementary β-DNA. Their electrochemical behavior studied in solution by cyclic voltammetry showed that this original structure leads to a strong difference in the Fc environment when the probe is alone or hybridized with a complementary strand. These preliminary electrochemical analyses are very encouraging and augur a good sensitivity of these probes for DNA detection after grafting on an electrode microsystem.

Synthesis and evaluation of glucuronic acid derivatives as alkylating agents for the reversible masking of internucleoside groups of antisense oligonucleotides

Abstract 2-Iodoethyl (methyl α- d -glucopyranosid)uronate and 2-iodoethyl (methyl β- d -glucopyranosid)thiouronate were prepared in five steps by an efficient synthetic route starting from d -glucuronic acid. Both compounds were used to alkylate dithymidine phosphorothioate and phosphorodithioate diesters, leading to the corresponding phosphotriesters 12 to 15 . Hydrolytic stability of 12–15 was studied in different biological media. The enzymatic hydrolysis of 12–15 was accompanied by another reaction resulting in formation of the dithymidine phosphodiesters. Several possible mechanisms for these reactions are proposed.

Impact of immobilization support on colorimetric microarrays performances.

We report here a comparison of support materials for colorimetric hybridization assays on microarrays. Four surfaces with various chemistries and architectures (roughness and porosity) were evaluated: (i) bare and (ii) activated polystyrene surfaces classically used for ELISA; (iii) a double-sided adhesive support; and (iv) a porous nitrocellulose/cellulose acetate membrane. Each substrate was functionalized with a microarray of probes and subjected to an enzymatic colorimetric DNA hybridization test. Tests were carried out in a 96-well assembly suitable for automated high-throughput analysis. Colorimetry results, microscopy observations and a chemiluminescence study showed that the test efficiency not only depends on the surface probe density but that the capacity of the material to retain the colored enzymatic product is also a critical parameter. All parameters being considered, the adhesive coated surface proposes the best surface properties for efficient colorimetric microarrays.

Conformational analysis and anion-binding properties of ferrocenyl-calixarene receptors

Novel synthetic approaches toward redox active calixarene-based receptors are described wherein ferrocene fragments have been introduced at the lower rim through anion-binding urea or amide connections. A thorough 1H NMR investigation on macrocycles was performed in order to estimate their hydrogen bonding-driven self-association properties and improve our understanding of the correlation between molecular structures and redox properties. The anion-binding properties of these artificial receptors have also been revealed by NMR spectroscopy and thoroughly investigated by electrochemical methods. We especially assessed the importance of the urea–phosphate bonds in the observed electrochemical response upon studying receptors wherein the ferrocene reporters and binding fragments are intimately associated or fully disconnected through a long alkyl chain. The experimental results clearly showed the utmost importance of ion pairing effects in the electrochemical recognition process accounting for most of the transduction signal in organic apolar media.

Polymer functionalized submicrometric emulsions as potential synthetic DNA vectors.

Triglyceride-based emulsions were first prepared by a solvent displacement procedure which was modified to achieve their functionalization by surface deposition of various amphiphilic comb-like copolymers. These emulsions have been characterized as regards to hydrodynamic particle size and surface charges using dynamic light scattering and electrophoretic mobility measurements. The adsorption isotherms of a polydT15 oligonucleotide and a model plasmid showed that the process was dependent on the nature of the interfaces, the affinity for the nucleic acid increasing with more cationic charges, together with improved accessibility. The binding process was found to proceed according to two regimes: one at low nucleic acid coverage, independent of the initial plasmid concentration, and the second one at high coverage, which was nucleic-acid-concentration dependent. This behavior was considered to occur because of the development of repulsive interactions upon increasing the amount of immobilized nucleic acid. The complexation of plasmid complexed at the interface was finally investigated using the ethidium bromide displacement technique. The level of compaction of plasmid complexed onto the functionalized emulsions was lower than that obtained with the parent free polymer.

Robust Electrografting on Self-Organized 3D Graphene Electrodes

Improving graphene-based electrode fabrication processes and developing robust methods for its functionalization are two key research routes to develop new high-performance electrodes for electrochemical applications. Here, a self-organized three-dimensional (3D) graphene electrode processed by pulsed laser deposition with thermal annealing is reported. This substrate shows great performance in electron transfer kinetics regarding ferrocene redox probes in solution. A robust electrografting strategy for covalently attaching a redox probe onto these graphene electrodes is also reported. The modification protocol consists of a combination of diazonium salt electrografting and click chemistry. An alkyne-terminated phenyl ring is first electrografted onto the self-organized 3D graphene electrode by in situ electrochemical reduction of 4-ethynylphenyl diazonium. Then the ethynylphenyl-modified surface efficiently reacts with the redox probe bearing a terminal azide moiety (2-azidoethyl ferrocene) by means of Cu(I)-catalyzed alkyne-azide cycloaddition. Our modification strategy applied to 3D graphene electrodes was analyzed by means of atomic force microscopy, scanning electron microscopy, Raman spectroscopy, cyclic voltammetry, and X-ray photoelectron spectroscopy (XPS). For XPS chemical surface analysis, special attention was paid to the distribution and chemical state of iron and nitrogen in order to highlight the functionalization of the graphene-based substrate by electrochemically grafting a ferrocene derivative. Dense grafting was observed, offering 4.9 × 10(-10) mol cm(-2) surface coverage and showing a stable signal over 22 days. The electrografting was performed in the form of multilayers, which offers higher ferrocene loading than a dense monolayer on a flat surface. This work opens highly promising perspectives for the development of self-organized 3D graphene electrodes with various sensing functionalities.