Etienne Sonveaux

The organic chemistry underlying DNA synthesis

Abstract The procedures used today for oligo-DNA synthesis are reviewed. The main applications of oligonucleotides in biological studies, the protecting group chemistry, the coupling procedures, and the methods for isolating and characterizing products are described. Contents . Introduction. Uses of DNA Oligomers. Established methods of synthesis and general strategies. Types of solid supports. Protection of the nucleic bases. Thymidine. Deoxycytidine. Deoxyadenosine. Deoxyguanosine . Protection of the deoxyribose. Phosphorylation and phosphitylation. The coupling step. The final deprotection step. Purification methods in DNA synthesis. Characterization of oligonucleotides. Conclusion. References.

Synthesis of new phosphonate inhibitors of serine proteases

Abstract Analogues of phenylalanine and lysine esters were synthesized, and their inhibitory power tested in vitro respectively on chymotrypsin, trypsin and urokinase.

Biophysical studies and intracellular destabilization of pH-sensitive liposomes

We examined changes in membrane properties upon acidification of dioleoylphosphatidylethanolamine/cholesterylhemisuccinate liposomes and evaluated their potential to deliver entrapped tracers in cultured macrophages. Membrane permeability was determined by the release of entrapped calcein or hydroxypyrene-1,3,6-trisulfonic acid (HPTS)-p-xylene-bis-pyridinium bromide (DPX); membrane fusion, by measuring the change in size of the liposomes and the dequenching of octadecylrhodamine-B fluorescence; and change in lipid organization, by31P nuclear magnetic resonance spectroscopy. Measurement of cell-associated fluorescence and confocal microscopy examination were made on cells incubated with liposomes loaded with HPTS or HPTS-DPX. The biophysical studies showed (i) a lipid reorganization from bilayer to hexagonal phase progressing from pH 8.0 to 5.0, (ii) a membrane permeabilization for pH<6.5, (iii) an increase in the mean diameter of liposomes for pH<6.0, and (iv) a mixing of liposome membranes for pH<5.7. The cellular studies showed (i) an uptake of the liposomes that were brought from pH 7.5–7.0 to 6.5–6.0 and (ii) a release of ∼15% of the endocytosed marker associated with its partial release from the vesicles (diffuse localization). We conclude that the permeabilization and fusion of pH-sensitive liposomes occur as a consequence of a progressive lipid reorganization upon acidification. These changes may develop intracellular after phagocytosis and allow for the release of the liposome content in endosomes associated with a redistribution in the cytosol.

Is the bacteriophage lambda lysozyme an evolutionary link or a hybrid between the C and V-type lysozymes? Homology analysis and detection of the catalytic amino acid residues.

The relationship between the bacteriophage lambda lysozyme (lambda L) and the C and V-type lysozymes has been investigated by sequence alignment, secondary structure prediction and pattern recognition methods. The alignment of the amino terminal part of lambda L with that of V-type lysozymes suggests that Glu19 is a residue essential for catalysis. Its mutation to Gln leads to a completely inactive enzyme. In the alignment of the sequence of lambda L with those of the C-type lysozymes a strongly homologous fragment of about 30 amino acid residues is detected. Taking into consideration this observation and the published structural alignments between C and V-type lysozymes, a repetition of the beta-sheet motif in lambda L is proposed. The multiple alignment draws the attention to a possible catalytic role for Asp34 that would be positioned in the middle of the second strand of the beta-sheet as in the C-type lysozymes. This role is confirmed by mutagenesis. The implications of these observations in terms of the evolutionary relationship between lambda L and the other lysozymes is discussed.

Flexible Aglycone Residues in Duplex Dna

Nucleotide analogues with a primary amide function attached by a polymethylene chain to C1 of 2-deoxy-D-ribofuranose were synthesized and incorporated into short double helices. The thermodynamic stability of these duplexes was studied. A higher melting temperature was observed when a hydrophobic hydrocarbon chain was attached to C1, instead of the more hydrophilic amide group.

Cell Handling, Membrane-Binding Properties and Membrane-Penetration Modeling Approaches of Pivampicillin and Phthalimidomethylampicillin, Two Basic Esters of Ampicillin, in Comparison with Chloroquine and Azithromycin

AbstractPurpose. The purpose of this work was to examine and understand the cellular pharmacokinetics of two basic esters of ampicillin, pivaloyloxymethyl (PIVA) and phthalimidomethyl (PIMA), in comparison with lysosomotropic drugs (chloroquine, azithromycin). Methods. Cell culture studies (J774 macrophages) were undertaken to study uptake and release kinetics and to assess the influence of concentration, pH, proton ionophore (monensin), and MRP and P-gp inhibitors (probenecid, gemfibrozil, cyclosporin A, GF 120918). Equilibrium dialysis with liposomes were performed to directly asses the extent of drug binding to bilayers. Conformational analysis modeling of the drug penetration in bilayers was conducted to rationalize the experimental observations. Results. PIVA and PIMA showed properties in almost complete contrast with those of chloroquine and azithromycin, i.e., fast apparent accumulation and fast release at 4°C as well as at 37°C, saturation of uptake (apparent Kd 40 μM), no influence of monensin, MRP, or P-gp inhibitors; tight binding to liposomes (Kd approx. 40 μM); and sharp increase in calculated free energy when forced in the hydrophobic domain. Conclusions. Although they are weak organic bases, PIVA and PIMA show none of the properties of lysosomotropic agents. We hypothesize that they remain locked onto the pericellular membrane and may never penetrate cells as such in significant amounts.

Ester prodrugs of ampicillin tailored for intracellular accumulation

Seven new ester prodrugs of ampicillin with hydrolysis half-lives ranging from 65 to 308 min were synthesized. The cellular accumulation of two of them (in J774 mouse macrophages) and their activities against intracellular Staphylococcus aureus were determined in comparison with the pivaloyloxymethylester of ampicillin (pivampicillin) and ampicillin. The esters accumulated extensively and were more active than ampicillin in this in vitro system

Syntheses and hydrolysis of basic and dibasic ampicillin esters tailored for intracellular accumulation.

Readily hydrolysable basic and dibasic esters of ampicillin were synthesised by alkylation of the carboxylate function of ampicillin to obtain prodrugs that may accumulate in cells and allow for an intracellular delivery of ampicillian (Fan et al., Bioorg. Med. Chem. Lett. 1997, 7, 3107). We found that the beta-lactam ring cleavage and the hydrolysis of the ester function were competitive reactions. The prerequisite for biological activity of compounds of this type is therefore that ester hydrolysis proceeds faster than ring opening. Some synthesised compounds show promise as prodrugs since they displayed a reasonable stability and regenerate large quantities of bioactive ampicillin in broth.

A High-field Nmr-study of 2'-deoxyribo-c-nucleosides

Abstract A rule to establish the structure of alpha and beta isomers of C-nucleosides, based on 1H-1H coupling constants is proposed and checked.

Aldehydo-oligonucleotides for bioconjugation

The synthesis of a new phosphoramidite building block with a masked aldehyde function is described. It was incorporated in an oligodeoxyribonucleotide. Coupling to amino derivatives by reductive amination was performed in high yield after unmasking the aldehyde function.