José L. Jiménez Blanco

Multivalency in heterogeneous glycoenvironments: hetero-glycoclusters, -glycopolymers and -glycoassemblies

Despite efficiently imitating functional ligand presentations in terms of valency and density, most of the reported multivalent carbohydrate prototypes barely reflect the inherent heterogeneity of biological systems, therefore underestimating the potential contribution of synergistic or antagonistic effects to molecular recognition events. To address this question, the design of novel molecular and supramolecular entities displaying different saccharide motifs in a controlled manner is of critical importance. In this review we highlight the current efforts made to synthesize heteromultivalent glycosystems on different platforms (peptides, dendrimers, polymers, oligonucleotides, calixarenes, cyclodextrins, microarrays, vesicles) and to evaluate the influence of heterogeneity in carbohydrate–protein (lectin, antibody) recognition phenomena. Although the number of publications on this topic is limited as compared to the huge volume of reports on homomultivalent sugar displays, the current body of results has already unravelled the existence of new binding mechanisms that operate in heterogeneous environments whose exact biological significance remains to be unveiled.

Mannosyl-coated nanocomplexes from amphiphilic cyclodextrins and pDNA for site-specific gene delivery

Fully homogeneous facial amphiphiles consisting in a cyclodextrin (CD) platform onto which a polycationic cluster and a multi-tail hydrophobic moiety have been installed (polycationic amphiphilic CDs; paCDs) self-organized in the presence of plasmid DNA to form nanometric complexes (CDplexes) which exhibit broad-range transfection capabilities. We hypothesized that biorecognizable moieties located at the hydrophilic rim in the CD scaffold would be exposed at the surface of the corresponding nanoparticles after DNA-promoted aggregation, endowing the system with molecular recognition abilities towards cell receptors. This concept has been demonstrated by developing an efficient synthetic strategy for the preparation of multivalent polycationic glyco-amphiphilic CDs (pGaCDs). Self-assembled nanoparticles obtained from mannosylated pGaCDs and pDNA (average hydrodynamic diameter 80 nm) have been shown to be specifically recognized by mannose-specific lectins, including concanavalin A (Con A) and the human macrophage mannose receptor (MMR). Further macrophage adhesion studies indicated that unspecific binding, probably due to electrostatic interactions with negatively charged cell membrane components, can also operate. The relative specific versus non-specific internalization is dependent on the pGaCD:pDNA proportion, being optimal at a protonable nitrogen/phosphate (N/P) ratio of 5. The resulting GlycoCDplexes were shown to specifically mediate transfection in Raw 264.7 (murine macrophage) cells expressing the mannose-fucose receptor in vitro. FACS experiments confirmed that transfection using these nanoparticles is mannose-dependent, supporting the potential of the approach towards vectorized gene delivery.

Cochlear compression in listeners with moderate sensorineural hearing loss

Psychophysical estimates of basilar membrane (BM) responses suggest that normal-hearing (NH) listeners exhibit constant compression for tones at the characteristic frequency (CF) across the CF range from 250 to 8000 Hz. The frequency region over which compression occurs is broadest for low CFs. This study investigates the extent that these results differ for three hearing-impaired (HI) listeners with sensorineural hearing loss. Temporal masking curves (TMCs) were measured over a wide range of probe (500-8000 Hz) and masker frequencies (0.5-1.2 times the probe frequency). From these, estimated BM response functions were derived and compared with corresponding functions for NH listeners. Compressive responses for tones both at and below CF occur for the three HI ears across the CF range tested. The maximum amount of compression was uncorrelated with absolute threshold. It was close to normal for two of the three HI ears, but was either slightly (at CFs < or =1000 Hz) or considerably (at CFs > or =4000 Hz) reduced for the third ear. Results are interpreted in terms of the relative damage to inner and outer hair cells affecting each of the HI ears. Alternative interpretations for the results are also discussed, some of which cast doubts on the assumptions of the TMC-based method and other behavioral methods for estimating human BM compression.

Building Blocks for Glycopeptide Synthesis. Disaccharide Glycosyl Isothiocyanates

Abstract The reaction of aldose oligosaccharides with aqueous ammonium hydrogencarbonate was revised and optimized for gram scale preparation of glycosylamines, using lactose, cellobiose, maltose, and melibiose as model compounds. The unprotected glycosylamines 1b-4b were transformed into the corresponding hepta-O-acetyl hydrochlorides 1e-4e via diethyl N-glycosylaminomethylenemalonates (1c-4c and 1d-4d). Reaction of le-3e with thiophosgene in a three-phased system led to the target hepta-O-acetylglycosyl isothiocyanates lf-3f in excellent yields. Under the same conditions, the melibiosyl derivative 4e underwent partial hydrolysis to give a mixture of melibiosyl isothiocyanate 4f and the reducing sugar derivative 4h. Treatment of 1f-4f with ammonia resulted in the quantitative formation of N-glycosylthioureas (1g-4g).

Polycationic amphiphilic cyclodextrins as gene vectors: effect of the macrocyclic ring size on the DNA complexing and delivery properties

A collection of homologous monodisperse facial amphiphiles consisting of an α-, β- or γ-cyclodextrin (α, β or γCD) platform exposing a multivalent display of cationic groups at the primary rim and bearing hexanoyl chains at the secondary hydroxyls have been prepared to assess the influence of the cyclooligosaccharide core size in their ability to complex, compact and protect pDNA and in the efficiency of the resulting nanocondensates (CDplexes) to deliver DNA into cells and promote transfection in the presence of serum. All the polycationic amphiphilic CDs (paCDs) were able to self-assemble in the presence of the plasmid and produce transfectious nanoparticles at nitrogen/phosphorous ratios ≥5. CDplexes obtained from βCD derivatives generally exhibited higher transfection capabilities, which can be ascribed to their ability to form inclusion complexes with cholesterol, thereby enhancing biological membrane permeability. The presence of thiourea moieties as well as increasing the number of primary amino groups then favour cooperative complexation of the polyphosphate chain, enhancing the stability of the complex and improving transfection. In the α and γCD series, however, only the presence of tertiary amino groups in the cationic clusters translates into a significant improvement of the transfection efficiency, probably by activating endosome escape by the proton sponge mechanism. This set of results illustrates the potential of this strategy for the rational design and optimisation of nonviral gene vectors.

Dual drug-loaded halloysite hybrid-based glycocluster for sustained release of hydrophobic molecules

A dual drug-loaded HNT–CD glycocluster delivery system based on halloysite nanotubes and carbohydrate functionalized cyclodextrin was developed by a green protocol using solvent-free microwave irradiation. The nanohybrid was employed for concurrent load and release of silibinin and curcumin. The new delivery system was characterized by means of TGA, FT-IR spectroscopy, SEM and DLS. These techniques confirm the successful loading of the two drugs in the system. SEM and DLS measurements highlighted that the nanomaterial preserves a tubular structure with an average hydrodynamic radius of ca. 200 nm. The release of the drugs from the HNT glycocluster was investigated by means of UV-vis spectroscopy at two different pH values simulanting the typical physiological conditions of either gastric or intestinal fluids. Enzyme-linked lectin assays (ELLA) demonstrated that highly mannoside–cyclodextrins HNT entities display high affinity towards mannose selective ConA lectin. Biological assays showed that the new drug delivery system exhibits anti-proliferative activity against the investigated cell lines. Fluorescence microscopy confirmed ELLA results and it showed a high propensity of this drug delivery system to cross cell membranes and to penetrate into the cell nucleus. The results revealed that the synthesized multicavity system is a material of suitable size and nanoarchitecture to transport drugs into living cells.

(Pseudo)amide-linked oligosaccharide mimetics: molecular recognition and supramolecular properties

Summary Oligosaccharides are currently recognised as having functions that influence the entire spectrum of cell activities. However, a distinct disadvantage of naturally occurring oligosaccharides is their metabolic instability in biological systems. Therefore, much effort has been spent in the past two decades on the development of feasible routes to carbohydrate mimetics which can compete with their O-glycosidic counterparts in cell surface adhesion, inhibit carbohydrate processing enzymes, and interfere in the biosynthesis of specific cell surface carbohydrates. Such oligosaccharide mimetics are potential therapeutic agents against HIV and other infections, against cancer, diabetes and other metabolic diseases. An efficient strategy to access this type of compounds is the replacement of the glycosidic linkage by amide or pseudoamide functions such as thiourea, urea and guanidine. In this review we summarise the advances over the last decade in the synthesis of oligosaccharide mimetics that possess amide and pseudoamide linkages, as well as studies focussing on their supramolecular and recognition properties.

Amphiphilic Oligoethyleneimine−β-Cyclodextrin "Click" Clusters for Enhanced DNA Delivery

Monodisperse amphiphilic oligoethyleneimine (OEI)-β-cyclodextrin (βCD) clusters have been prepared, and their potential as gene delivery systems has been evaluated in comparison with a nonamphiphilic congener. The general prototype incorporates tetraethyleneimine segments linked to the primary rim of βCD through either triazolyl or thioureidocysteaminyl connectors. Transfection efficiency data for the corresponding CD:pDNA nanocomplexes (CDplexes) in BNL-CL2 murine hepatocytes evidenced the strong beneficial effect of facial amphiphilicity.

Synthesis, conformational flexibility and preliminary complexation behaviour of α,α′-trehalose-based macrocycles containing thiourea spacers

An efficient synthesis of macrocyclic ligands incorporating two α,α′-trehalose subunits linked through the primary C-6,6′ positions by means of 1,3-thiourea spacers is reported; the Z/E configuration of the N–C(S) bonds is governed by intramolecular hydrogen bonding as well as steric factors.

Influence of intramolecular hydrogen-bonding on the conformational properties of sugar thioureas

Abstract Reaction of deoxyisothiocyanato derivatives of 1,2:3,4-di- O -isopropylidene-α-D-galactopyranose, 1,2:3,5-di- O -isopropylidene-α-D-glucofuranose, and 2,3:4,5-di- O -isopropylidene-β-D-fructopyranose with ammonia afforded the conesponding sugar thioureas. Both the Z and E stereoisomers around the NHC(S) bond were observed in the 1 H and 13 C NMR spectra of the later in the low temperature range, their relative proportions being a function of the sugar configuration. Experimental evidence for the existence of seven-membered NH⋯O intramolecular hydrogen bonds in the E isomers of thioureas has been obtained from DNMR experiments, rotational barrier height calculations, measurements of temperature coefficients for the 1 H chemical shifts of the NH signals, and study of the influence of the solvent polarity in the rotameric ratio.