Mannose-coated polydiacetylene (PDA)-based nanomicelles: synthesis, interaction with concanavalin A and application in the water solubilization and delivery of hydrophobic molecules.

Carbohydrate-lectin interactions are involved in a number of relevant biological events including fertilization, immune response, cell adhesion, tumour cell metastasis, and pathogen infection. Lectins are also tissue specific, making carbohydrates not only promising drug candidates but also excellent low molecular weight ligands for active drug delivery system decorations. In order for these interactions to be effective multivalency is essential, as the interaction of a lectin with its cognate monovalent carbohydrate epitope usually takes place with low affinity. Unlike the covalent approach, supramolecular self-assembly of glyco-monomers mediated by non-covalent forces allows accessing multivalent systems with diverse topology, composition, and assembly dynamics in a single step. In order to fine-tune the size and sugar adaptability of spherical micelles at the nanoscale for an optimal glycoside cluster effect, herein we report the synthesis of mannose-coated static micelles from diacetylene-based mannopyranosyl glycolipids differing in the length of the poly(ethyleneglycol) (PEG) chains and the oxidation state of the anomeric sulfur atom. The reported shot-gun like synthetic approach for the synthesis of dilution-insensitive micelles is based on the ability of diacetylenic-based neoglycolipids to self-assemble into micelles in water and to undergo an easy photopolymerization by a simple irradiation at 254 nm. The affinity of the obtained 6 nanosystems was assessed by enzyme-linked lectin assay (ELLA) using the mannose-specific concanavalin A lectin as a model receptor. Relative binding potency enhancements, compared to methyl α-d-mannopyranoside used as control, from 20-, to 29- to 300-fold on a sugar molar basis were observed for micelles derived from sulfonyl-, sulfinyl- and thioglycoside monomers with a tatraethyleneglycol spacer, respectively, indicative of a significant cluster glycoside effect. Moreover, pMic1 micelles are able to solubilize and slowly liberate lipophilic clinically relevant drugs, and show the enhanced cytotoxic effect of docetaxel toward prostate cancer cells. These findings highlight the potential of mannose-coated photopolymerized micelles pMic1 as an efficient nanovector for active delivery of cytotoxic hydrophobic molecules.

Tuning of glyconanomaterial shape and size for selective bacterial cell agglutination.

Multivalent glycosystems are potential candidates for anti-adhesive therapy, a non-lethal approach against the ever increasing antibiotic resistance of pathogenic bacteria. In order to fine-tune the glyconanomaterial size and shape for selective bacterial cell agglutination, herein we report the synthesis of sugar-coated dynamic and polymeric 3D-micelles and 1D-carbon nanotubes. The reported shot-gun like synthetic approach is based on the ability of diacetylenic-based neoglycolipids to self-assemble into micelles in water and hierarchically self-assemble into hemimicelles on a single-walled carbon nanotube surface. The affinity of the nanosystems was preliminarily assessed by enzyme-linked lectin assay (ELLA) using the mannose-specific Concanavalin A lectin as a model receptor. Relative binding potency enhancements, compared to methyl α-d-mannopyranoside used as control, from 10- to 25- to 2340-folds in sugar molar basis were observed when passing from 3D dynamic micelles to static micelles, to 1D-mannose coated carbon nanotubes, respectively, indicative of a significant cluster glycoside effect. Importantly, these results were confirmed in vivo showing that the 1D-glyconanoring-coated carbon nanotubes efficiently and selectively regulate the agglutination and proliferation of the enterobacteria Escherichia coli type 1 fimbriae. These findings highlight the potential of sugar coated nano-materials as novel and effective tools in the control of bacterial pathogenesis.

Synthesis of 1D-glyconanomaterials by a hybrid noncovalent-covalent functionalization of single wall carbon nanotubes: a study of their selective interactions with lectins and with live cells.

To take full advantage of the remarkable applications of carbon nanotubes in different fields, there is a need to develop effective methods to improve their water dispersion and biocompatibility while maintaining their physical properties. In this sense, current approaches suffer from serious drawbacks such as loss of electronic structure together with low surface coverage in the case of covalent functionalizations, or instability of the dynamic hybrids obtained by non-covalent functionalizations. In the present work, we examined the molecular basis of an original strategy that combines the advantages of both functionalizations without their main drawbacks. The hierarchical self-assembly of diacetylenic-based neoglycolipids into highly organized and compacted rings around the nanotubes, followed by photopolymerization leads to the formation of nanotubes covered with glyconanorings with a shish kebab-type topology exposing the carbohydrate ligands to the water phase in a multivalent fashion. The glyconanotubes obtained are fully functional, and able to establish specific interactions with their cognate receptors. In fact, by taking advantage of this selective binding, an easy method to sense lectins as a working model of toxin detection was developed based on a simple analysis of TEM images. Remarkably, different experimental settings to assess cell membrane integrity, cell growth kinetics and cell cycle demonstrated the cellular biocompatibility of the sugar-coated carbon nanotubes compared to pristine single-walled carbon nanotubes.

General Method for Asymmetric Synthesis of alpha-Methylsulfinyl Ketones: Application to the Synthesis of Optically Pure Oxisuran and Bioisosteres.

The first asymmetric synthesis of oxisuran [1, (methylsulfinyl)methyl 2-pyridyl ketone], a synthetic immunosuppressive drug, is described. Both enantiomers were efficiently synthesized, in optically pure form, using DAG methodology for the key condensation step. Attempts to couple metal enolates of aryl methyl ketones with chiral sulfinyl compounds led to some epimerization at sulfur. This loss of chirality was circumvented by reacting the alpha-lithio derivatives of the N,N-dimethylhydrazones derived from these ketones with either the (R)- or the (S)-methanesulfinate of diacetone D-glucose, to yield the corresponding alpha-(methylsulfinyl)methylhydrazones, with complete inversion of chirality at sulfur. Hydrolysis of the resulting hydrazones with copper(II) chloride gave 1 in optically pure form. The generality of the method was demonstrated by the preparation of the optically active oxisuran analogs, 2-4, in which the pyridyl moiety was replaced by phenyl, furyl, and thienyl moieties, respectively. The optical purities of these products were determined by proton NMR spectroscopy, using chiral shift reagents, following conditions established by the study of racemic mixtures of the beta-keto sulfoxides.

Inositolphosphoglycan mediators structurally related to glycosyl phosphatidylinositol anchors: synthesis, structure and biological activity.

The preparation of the pseudopentasaccharide 1a, an inositol-phosphoglycan (IPG) that contains the conserved linear structure of glycosyl phosphatidylinositol anchors (GPI anchors), was carried out by using a highly convergent 2+3-block synthesis approach which involves imidate and sulfoxide glycosylation reactions. The preferred solution conformation of this structure was determined by using NMR spectroscopy and molecular dynamics simulations prior to carrying out quantitative structure--activity relationship studies in connection with the insulin signalling process. The ability of 1a to stimulate lipogenesis in rat adipocytes as well as to inhibit cAMP dependent protein kinase and to activate pyruvate dehydrogenase phosphatase was investigated. Compound 1a did not show any significant activity, which may be taken as a strong indication that the GPI anchors are not the precursors of the IPG mediators.

NMR investigations of protein-carbohydrate interactions: refined three-dimensional structure of the complex between hevein and methyl beta-chitobioside.

The specific interaction of hevein with GlcNAc-containing oligosaccharides has been analyzed by1H-NMR spectroscopy. The association constants for the binding of hevein to a variety of ligands have been estimated from1H-NMR titration experiments. The association constants increase in the order GlcNAc-alpha(1-->6)-Man < GlcNAc < benzyl-beta-GlcNAc < p-nitrophenyl-beta-GlcNAc < chitobiose < p-nitrophenyl-beta-chitobioside < methyl-beta-chitobioside < chitotriose. Entropy and enthalpy of binding for different complexes have been obtained from van't Hoff analysis. The driving force for the binding process is provided by a negative DeltaH0which is partially compensated by negative DeltaS0. These negative signs indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. NOESY NMR experiments in water solution provided 475 accurate protein proton-proton distance constraints after employing the MARDIGRAS program. In addition, 15 unambiguous protein/carbohydrate NOEs were detected. All the experimental constraints were used in a refinement protocol including restrained molecular dynamics in order to determine the highly refined solution conformation of this protein-carbohydrate complex. With regard to the NMR structure of the free protein, no important changes in the protein nOe's were observed, indicating that carbohydrate-induced conformational changes are small. The average backbone rmsd of the 20 refined structures was 0.055 nm, while the heavy atom rmsd was 0.116 nm. It can be deduced that both hydrogen bonds and van der Waals contacts confer stability to the complex. A comparison of the three-dimensional structure of hevein in solution to those reported for wheat germ agglutinin (WGA) and hevein itself in the solid state has also been performed. The polypeptide conformation has also been compared to the NMR-derived structure of a smaller antifungical peptide, Ac-AMP2.