Unprecedented reactivity of polyamines with aldehydic DNA modifications: structural determinants of reactivity, characterization and enzymatic stability of adducts.

Apurinic/apyrimidinic (AP) sites, 5-formyluracil (fU) and 5-formylcytosine (fC) are abundant DNA modifications that share aldehyde-type reactivity. Here, we demonstrate that polyamines featuring at least one secondary 1,2-diamine fragment in combination with aromatic units form covalent DNA adducts upon reaction with AP sites (with concomitant cleavage of the AP strand), fU and, to a lesser extent, fC residues. Using small-molecule mimics of AP site and fU, we show that reaction of secondary 1,2-diamines with AP sites leads to the formation of unprecedented 3'-tetrahydrofuro[2,3,4-ef]-1,4-diazepane ('ribodiazepane') scaffold, whereas the reaction with fU produces cationic 2,3-dihydro-1,4-diazepinium adducts via uracil ring opening. The reactivity of polyamines towards AP sites versus fU and fC can be tuned by modulating their chemical structure and pH of the reaction medium, enabling up to 20-fold chemoselectivity for AP sites with respect to fU and fC. This reaction is efficient in near-physiological conditions at low-micromolar concentration of polyamines and tolerant to the presence of a large excess of unmodified DNA. Remarkably, 3'-ribodiazepane adducts are chemically stable and resistant to the action of apurinic/apyrimidinic endonuclease 1 (APE1) and tyrosyl-DNA phosphoesterase 1 (TDP1), two DNA repair enzymes known to cleanse a variety of 3' end-blocking DNA lesions.

Mannose-Coated Fluorescent Lipid Microparticles for Specific Cellular Targeting and Internalization via Glycoreceptor-Induced Phagocytosis.

In this work, we report on the development of mannose-coated fluorescent lipid microparticles to study the role of C-type lectin membrane receptors in phagocytosis. The micrometric droplets of soybean oil-in-water emulsion were functionalized with a tailor-made fluorescent mannolipid. The amphiphilic ligand was built from a mannose unit, a lipid C11 spacer, and a naphthalimide fluorophore. The functionalization of the droplets was monitored by fluorescence microscopy as well as their interaction with concanavalin A, which was used as a model lectin in vitro. The use of a monovalent ligand on the surface of emulsion droplets yielded particles with an affinity approximately 40 times higher than that of free mannose. In cellulo, the coated droplets were shown to be specifically internalized by macrophages in a receptor-dependent phagocytic pathway. The naked droplets, on the other hand, displayed very little internalization because of their low immunogenicity. This work thus brings evidence that C-type lectin membrane receptors may act as phagocytic receptors. The functionalization of the droplets with the tailored amphiphilic fluorescent ligand also provides insights into the development of organic fluorescent particles that may prove useful for developing targeted imaging and delivery tools.

New branched amino acids for high affinity dendrimeric DC-SIGN ligands.

A branched amino acid was synthesized from methyl glucopyranoside; this amino acid presents three amino groups protected by Fmoc and one acid group and can be used in classic peptide synthesis. In parallel, similar azido terminated blocks were synthesized. Successive coupling reaction and deprotection afforded dendrimers with up to 27 azido functional groups. As an example of application, d-mannose and l-fucose residues were linked through CuAAC coupling and resulting glycodendrimers were evaluated in their interaction with DC-SIGN using SPR competition assay.

Candida albicans ß-1,2 mannosyl transferase Bmt3: Preparation and evaluation of a ß (1,2), α (1,2)-tetramannosyl fluorescent substrate.

We describe for the first time the chemical synthesis of a tetramannoside, containing both α (1→2) and ß (1→2) linkages. Dodecylthio (lauryl) glycosides were prepared from odorless dodecyl thiol and used as donors for the glycosylation steps. This tetramannoside, was coupled to a mantyl group, and revealed to be a perfect substrate of ß-mannosyltransferase Bmt3, confirming the proposed specificity and allowing the preparation of a pentamannoside sequence (ß Man (1,2) ß Man (1,2) α Man (1,2) α Man (1,2) α Man) usable as a novel substrate for further elongation studies.

Toward libraries of biotinylated chondroitin sulfate analogues: from synthesis to in vivo studies.

Chondroitin sulfate-E (CS-E) oligosaccharidic analogues (di to hexa) were prepared from lactose. In these compounds, the 2-acetamido group was replaced by a hydroxyl group. This modification speeded up the synthesis, and large oligosaccharides were constructed in a few steps from a lactose-originated block. The protecting groups used were as follows; Fmoc for hydroxyl groups to be glycosylated, allyl group for anomeric position protection, and trichoroacetimidate leaving groups were used to prepare up to octasaccharides. We took advantage of the presence of allyl group to develop a click biotinylation, through its transformation into a 3-azido-2-hydroxyl propyl group in two steps (epoxidation and sodium azide epoxide opening). The biotinylating agent was a water-soluble propargylated and biotinylated triethylene glycol (PEG). By using surface plasmon resonance (SPR), it was shown that the di-, tetra-, and hexasaccharides display a binding affinity and selectivity toward HSF/GSF and CXCL12 similar to that of CS-E. A parallel study confirmed their mimicry of natural compounds, based on the hexasaccharide interaction with Otx2, a homeodomain protein involved in brain maturation, thus validating our simplification approach to synthesize bioactive GAG.

Monogalactopyranosides of fluorescein and fluorescein methyl ester: synthesis, enzymatic hydrolysis by biotnylated ß-galactosidase, and determination of translational diffusion coefficient.

Fluorescein monoglycosides (D-galactopyranoside (FMG) and D-glucopyranoside) and their methyl ester (MFMG) have been prepared from acetobromoglucose/galactose and fluorescein methyl ester in good yields. Enzymatic hydrolysis experiments (using biotinylated ß-galactosidase) of the galacto derivatives have been performed and kinetic parameters were calculated. A 15-20 times increase of the fluorescence intensity has been observed during the hydrolysis. A linear increase of fluorescence has been noted at short time and low concentration of substrate, making these compounds useful and sensitive probes for galactosidases. The magnitude of the Michaelis-Menten constant (K(m)) value for MFMG is higher than that of FMG suggesting a possible conformational change of the fluorogenic substrate. K(m) value for biotinylated ß-Gal with FMG is lower than that for the native enzyme. This observation indicates higher substrate affinity of the biotinylated enzyme in comparison to the native enzyme. Translational diffusion coefficients have been measured, for both fluorogenic substrates and both the products, employing fluorescence correlation spectroscopy. Translational diffusion coefficients for fluorogenic substrates and the enzymatic hydrolysis products have been measured to be similar, in the range of 3.5-4.5×10(-10) m(2) s(-1). Thus an enhancement or retardation of the enzymatic kinetics due to difference in translational mobility of substrate and product is not that apparent.

Synthetic biotinylated tetra ß(1→5) galactofuranoside for in vitro aspergillosis diagnosis.

The synthesis of a tetra ß(1→5) galactofuranoside was achieved using a thioglycoside donor with a methyl tert-butyl phenyl thio leaving group. This tetrasaccharide was conjugated to biotin and validated as antigen with the monoclonal antibody used for clinical detection of Aspergillus fumigatus galactomannan on streptavidin-coated microplates. Then we have shown its ability to detect antibodies associated with A. fumigatus induced disease by using sera from patients with Allergic broncho-pulmonary aspergillosis (ABPA) and correlated the results of antibody detection with those gained with a commercially available diagnostic test.