Luc Lebeau | Université de Strasbourg (original) (raw)

Papers by Luc Lebeau

Biomaterials, 2008

A new strategy for the specific immobilization of proteins onto a solid support is presented. The... more A new strategy for the specific immobilization of proteins onto a solid support is presented. The immobilization is highly specific and efficient thanks to functionalized semifluorinated self-assembled monolayers (SAMs), thus reducing non-specific protein adsorption. Protein resistance does not exclusively result from classical steric repulsions and the presence of the both hydrophobic and lipophobic rigid and helical fluorinated cylinder in the depth of the SAMs does play an active role in protein repulsion. That repulsive effect is of higher magnitude than that of polyethylene glycol (PEG) as capping of the semifluorinated SAMs with additional ethylene oxide units provokes an attenuation of protein resistance. Though specific binding capacity of SAMs is intrinsically limited, the fluorinated SAMs described allow sensitive measurements with lower limits of detection and higher signal-to-noise ratios when compared to the best chips so far used in surface plasmon resonance (SPR) analysis.

Bioorganic & Medicinal Chemistry Letters, 2007

A coumarin-based europium chelate ready-to-use for analyte labeling and homogeneous time-resolved... more A coumarin-based europium chelate ready-to-use for analyte labeling and homogeneous time-resolved fluorescence measurements has been designed. Compound 1 displays three functional elements: an azide reactive spacer arm, a coumarin sensitizer, and a seven-coordinate europium complex. That complex can be excited at 370 nm by inexpensive UV-LEDs as a light excitation source.

Organic & Biomolecular Chemistry, 2009

A series of new ligands suitable for the formation of luminescent lanthanide complexes in water i... more A series of new ligands suitable for the formation of luminescent lanthanide complexes in water is described. The chelates are designed for analyte labeling and play the role of fluorescent donor in homogeneous time-resolved fluorescence assays using LEDs as a light source for excitation at 370 nm. Ligands are constructed from a coumarin nucleus, for lanthanide sensitization, and different aminomethylenecarboxy moieties are introduced in positions 7 and 5, 6, or 8 of the sensitizer. A reactive spacer arm under biocompatible conditions (maleimide, azide) is introduced at position 3 for ultimate bioconjugation purposes. The synthesis and characterization of the ligands are described, together with the preparation of their corresponding europium complexes. Photophysical properties of the complexes are investigated in water by means of UV-vis and luminescence spectroscopy.

Pharmaceutical Research, 2013

Biolabile cationic lipids were developed for efficient intracellular delivery of DNA and siRNA. T... more Biolabile cationic lipids were developed for efficient intracellular delivery of DNA and siRNA. The compounds have been designed starting from the membrane lipid DOPC in a way they may loose their cationic charge when exposed to an acidic and/or enzymatic stimulus, such as those met during the journey of a lipoplex in biological media. They demonstrated remarkable efficiency to deliver DNA in various cell lines (BHK-21, Calu-3, NCI-H292, and A549), with no significant cytotoxicity. Furthermore, two of the compounds (carbonate-based DOPC derivatives) revealed able to deliver small interfering RNA in U87Luc and A549Luc cancer cells and to mediate a selective 70-80% knockdown of the stably transfected luciferase gene. The results show that the described bioresponsive cationic lipids have high DNA and siARN delivery activity which is encouraging in view of delivering a therapeutic nucleic acid to pulmonary tissues in vivo.

Biomaterials, 2015

Cationic carbon dots were fabricated by pyrolysis of citric acid and bPEI25k under microwave radi... more Cationic carbon dots were fabricated by pyrolysis of citric acid and bPEI25k under microwave radiation. Various nanoparticles were produced in a 20e30% yield through straightforward modifications of the reaction parameters (stoichiometry of the reactants and energy supply regime). Particular attention was paid to the purification of the reaction products to ensure satisfactory elimination of the residual starting polyamine. Intrinsic properties of the particles (size, surface charge, photoluminescence and quantum yield) were measured and their ability to form stable complexes with nucleic acid was determined. Their potential to deliver plasmid DNA or small interfering RNA to various cell lines was investigated and compared to that of bPEI25k. The pDNA in vitro transfection efficiency of these carbon dots was similar to that of the parent PEI, as was their cytotoxicity. The higher cytotoxicity of bPEI25k/siRNA complexes when compared to that of the CD/siRNA complexes however had marked consequences on the gene silencing efficiency of the two carriers. These results are not fully consistent with those in some earlier reports on similar nanoparticles, revealing that toxicity of the carbon dots strongly depends on their protocol of fabrication. Finally, these carriers were evaluated for in vivo gene delivery through the noninvasive pulmonary route in mice. High transgene expression was obtained in the lung that was similar to that obtained with the golden standard formulation GL67A, but was associated with significantly lower toxicity. Post-functionalization of these carbon dots with PEG or targeting moieties should significantly broaden their scope and practical implications in improving their in vivo transfection efficiency and biocompatibility. Biomaterials j o u r n a l h o m e p a g e : w w w .e l se v i e r. co m/ lo ca t e / b i o m a t e ri a l s http://dx.

Helvetica Chimica Acta, 1995

Chemistry and Physics of Lipids, 1992

Journal of Immunological Methods, 1998

We have developed a competitive enzyme immunoassay suitable for routine monitoring of intracellul... more We have developed a competitive enzyme immunoassay suitable for routine monitoring of intracellular levels of X Ž

European Biophysics Journal, 2001

Working with pure lipidic systems (giant unilamellar vesicles, 10±150 lm in diameter) as models f... more Working with pure lipidic systems (giant unilamellar vesicles, 10±150 lm in diameter) as models for biological membranes, we have considered possible structures of the contact area of two adherent membranes by investigating the diusion of¯uorescent lipid analogues from one vesicle to another. Two bilayers in close contact can almost be seen as a lamellar structure in equilibrium. This is the usual con®guration of two adherent vesicles, in which the interbilayer distance is estimated to be 3 nm. We have increased the attraction between the membranes by either adding depletion forces or by using a trick, inspired from the interaction between nucleic bases in nucleosides (herein adenosine and thymidine). The nucleosides were attached to the polar head of amphiphilic molecules that behave like phospholipids and were incorporated in the model membrane. The extra attraction between two membranes, resulting from base pairing, strongly decreased the interbilayer distance down to about 1 nm. This change of the water content induced lipid rearrangements, which could also be viewed in terms of a phase transition at low water content. These rearrangements were not observed in the case of depletion forces. We conclude that the introduction of an additional attractive force in the system modi®es the equilibrium state, leading to a drastic change in the membrane behavior, which will tentatively be related to hemifusion.

Biophysical Journal, 2001

Photosystem II core complex (PSII CC) absorbs light energy and triggers a series of electron tran... more Photosystem II core complex (PSII CC) absorbs light energy and triggers a series of electron transfer reactions by oxidizing water while producing molecular oxygen. Synthetic lipids with different alkyl chains and spacer lengths bearing functionalized headgroups were specifically designed to bind the Q B site and to anchor this large photosynthetic complex (240 kDa) in order to attempt two-dimensional crystallization. Among the series of different compounds that have been tested, oxygen evolution measurements have shown that dichlorophenyl urea (DCPU) binds very efficiently to the Q B site of PSII CC, and therefore, that moiety has been linked covalently to the headgroup of synthetic lipids. The analysis of the monolayer behavior of these DCPU-lipids has allowed us to select ones bearing long spacers for the anchoring of PSII CC. Oxygen evolution measurements demonstrated that these long-spacer DCPU-lipids specifically bind to PSII CC and inhibit electron transfer. With the use of atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM), it was possible to visualize domains of PSII CC bound to DCPU-lipid monolayers. SNOM imaging has enabled us to confirm that domains observed by AFM were composed of PSII CC. Indeed, the SNOM topography images presented similar domains as those observed by AFM, but in addition, it allowed us to determine that these domains are fluorescent. Electron microscopy of these domains, however, has shown that the bound PSII CC was not crystalline.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2000

Colchicine is a potent antimitotic poison which is well known to prevent microtubule assembly by ... more Colchicine is a potent antimitotic poison which is well known to prevent microtubule assembly by binding tubulin very tightly. Colchicine also possesses anti-inflammatory properties which are not well understood yet. Here we show that colchicine tightly interacts with lipid layers. The physical and biological properties of three different lipid derivatives of colchicine are investigated parallel to those of membrane lipids in the presence of colchicine. Upon insertion in the fatty alkyl chains, colchicine rigidifies the lipid monolayers in a fluid phase and fluidifies rigid monolayers. Similarly X-ray diffraction data show that lecithin^water phases are destabilized by colchicine. In addition, an unexpectedly drastic enhancement of the photoisomerization rate of colchicine into lumicolchicine in the lipid environment is observed and further supports insertion of the alkaloid in membranes. Finally the interaction of colchicine with lipids makes the drug inaccessible to tubulin. The possible in vivo significance of these results is discussed. ß

Biophysical Journal, 2002

It is now well admitted that hydrophobic interactions and hydrogen bonds are the main forces driv... more It is now well admitted that hydrophobic interactions and hydrogen bonds are the main forces driving protein folding and stability. However, because of the complex structure of a protein, it is still difficult to separate the different energetic contributions and have a reliable estimate of the hydrogen bond part. This energy can be quantified on simpler systems such as surfaces bearing hydrogen-bonding groups. Using the surface force apparatus, we have directly measured the interaction energy between monolayers of lipids whose headgroups can establish hydrogen bonds in water: nitrilotriacetate, adenosine, thymidine, and methylated thymidine lipids. From the adhesion energy between the surfaces, we have deduced the energy of a single hydrogen bond in water. We found in each case an energy of 0.5 kcal/mol. This result is in good agreement with recent experimental and theoretical studies made on protein systems showing that intramolecular hydrogen bonds make a positive contribution to protein stabilization.

The Journal of Organic Chemistry, 1998

The synthesis of five enzymatically stable analogues of guanosine diphosphate (GDP) has been carr... more The synthesis of five enzymatically stable analogues of guanosine diphosphate (GDP) has been carried out. The pyrophosphate moiety was mimicked in turn by the malonate, the acetophosphonate, the phosphonoacetate, the methylene-bis-phosphonate, and the imidodiphosphate groups. All the compounds were prepared via the synthesis of a transient fully protected nucleoside diphosphate analogue, and the final deprotection step was achieved by catalytic hydrogenolysis. The biological properties of the compounds have been evaluated toward transducin, the G-protein of the visual photoreceptor. Three guanosine imidodiphosphate derivatives bearing a linker at different positions on the sugar and on the base were then prepared and evaluated, giving some insight into the GDP binding site of transducin.

Analytical Chemistry, 2002

Direct LC/MS/MS methods have recently been developed for measuring triphosphate anabolites of sev... more Direct LC/MS/MS methods have recently been developed for measuring triphosphate anabolites of several nucleosidic reverse transcriptase inhibitor (NRTI) in peripheral blood mononuclear cells (PBMCs) from HIV-positive patients. Whereas AZT is one of the most-used NRTIs, no such method has been developed for AZT-TP, its active anabolite, mainly because of the presence of endogenous nucleotides that interfere with such an assay. In this paper, we first describe the development of two enzyme immunoassays (EIA) of AZT-TP in PBMCs: one directly measuring AZT-TP content; the other, measuring the nucleoside AZT after selective extraction of AZT-TP and dephosphorylation. The precision of these two assays was too low to achieve precise determination of AZT-TP in PBMC samples. Direct LC/MS/MS is not specific enough for AZT-TP, since at least two interfering endogenous nucleotides (same m/z ratio and fragment as well as retention time close to that of AZT-TP) are found in the intracellular medium of PBMCs. The off-line combination of immunoaffinity extraction (IAE) and LC/MS/MS proved to be a successful strategy allowing without dephosphorylation appropriate specificity and sensitivity (limit of quantification established as 9.3 fmol/10(6) cells) to determine AZT-TP in PBMCs from 7 mL of blood of HIV-infected patients. Validation of this IAE-LC/MS/MS method demonstrated CV percent for repeatability and intermediate precision lower than 15%. More than 150 samples/week can be analyzed by one analyst, making this method suitable for routine analysis during clinical studies.

Biochemistry, 2003

The microtubule-dependent kinesin-like protein Eg5 from Homo sapiens is involved in the assembly ... more The microtubule-dependent kinesin-like protein Eg5 from Homo sapiens is involved in the assembly of the mitotic spindle. It shows a three-domain structure with an N-terminal motor domain, a central coiled coil, and a C-terminal tail domain. In vivo HsEg5 is reversibly inhibited by monastrol, a small cell-permeable molecule that causes cells to be arrested in mitosis. Both monomeric and dimeric Eg5 constructs have been examined in order to define the minimal monastrol binding domain on HsEg5. NMR relaxation experiments show that monastrol interacts with all of the Eg5 constructs used in this study. Enzymatic techniques indicate that monastrol partially inhibits Eg5 ATPase activity by binding directly to the motor domain. The binding is noncompetitive with respect to microtubules, indicating that monastrol does not interfere with the formation of the motor-MT complex. The binding is not competitive with respect to ATP. Both enzymology and in vivo assays show that the S enantiomer of monastrol is more active than the R enantiomer and racemic monastrol. Stopped-flow fluorometry indicates that monastrol inhibits ADP release by forming an Eg5-ADP-monastrol ternary complex. Monastrol reversibly inhibits the motility of human Eg5. Monastrol has no inhibitory effect on the following members of the kinesin superfamily: MC5 (Drosophila melanogaster Ncd), HK379 (H. sapiens conventional kinesin), DKH392 (D. melanogaster conventional kinesin), BimC1-428 (Aspergillus nidulans BimC), Klp15 (Caenorhabditis elegans C-terminal motor), or Nkin460GST (Neurospora crassa conventional kinesin). FIGURE 1: Full-length Eg5 protein and Eg5 constructs. (A) Bar diagram of the human Eg5 full-length motor presenting the putative domain structure. (B) Bar diagrams of human Eg5 constructs used in this study.

GBM Annual Spring meeting Mosbach 2005, 2005

Physical Review Letters, 2001

We present a statistical mechanical treatment relating the macroscopic adhesion energy of two sur... more We present a statistical mechanical treatment relating the macroscopic adhesion energy of two surfaces, which can be obtained by micropipette aspiration studies, to the microscopic adhesion energy between individual bonds. The treatment deals with the case of weak reversible bonds, so that the equilibrium partition function has significance. This description is coherent with previous theories. Experiment and theory are compared to probe the nature of weak bonds in membranes, where local equilibria can be obtained. The case of a bead and a vesicle decorated by nucleosides was considered.

Tetrahedron Letters, 2001

The Journal of Organic Chemistry, 2002

Dinucleoside polyphosphates are ubiquitous compounds tightly involved in the regulation of a numb... more Dinucleoside polyphosphates are ubiquitous compounds tightly involved in the regulation of a number of key biological processes. Hydrolysis-resistant analogues of Ap(3)A and Gp(3)G, two important members of that family of nucleotides, have been synthesized. P(1),P(2):P(2),P(3)-Bis-methylene diadenosine and diguanosine triphosphates were prepared from O,O-dialkyl methaneselenophosphonates using an original methodology. Whereas the 2-fold addition of the methanephosphonate anion to the activated phosphorus species cannot be performed, multiple condensation of lithiated methaneselenophosphonate with electrophilic trivalent phosphorus compounds is revealed to be very effective. A one-pot condensation/esterification/oxidation sequence involving O,O-dialkyl methaneselenophosphonates provides a highly efficient route to the PCH(2)PCH(2)P backbone. This new development in selenophosphonate chemistry offers a great potential for further regioselective functionalization of polyphosphate mimics.