Probing the Inhibitor versus Chaperone Properties of sp²-Iminosugars towards Human ß-Glucocerebrosidase: A Picomolar Chaperone for Gaucher Disease.

A series of sp²-iminosugar glycomimetics differing in the reducing or nonreducing character, the configurational pattern (d-gluco or l-ido), the architecture of the glycone skeleton, and the nature of the nonglycone substituent has been synthesized and assayed for their inhibition properties towards commercial glycosidases. On the basis of their affinity and selectivity towards GH1 ß-glucosidases, reducing and nonreducing bicyclic derivatives having a hydroxylation profile of structural complementarity with d-glucose and incorporating an N'-octyl-isourea or -isothiourea segment were selected for further evaluation of their inhibitory/chaperoning potential against human glucocerebrosidase (GCase). The 1-deoxynojirimycin (DNJ)-related nonreducing conjugates behaved as stronger GCase inhibitors than the reducing counterparts and exhibited potent chaperoning capabilities in Gaucher fibroblasts hosting the neuronopathic G188S/G183W mutation, the isothiourea derivative being indeed one of the most efficient chaperone candidates reported up to date (70% activity enhancement at 20 pM). At their optimal concentration, the four selected compounds promoted mutant GCase activity enhancements over 3-fold; yet, the inhibitor/chaperoning balance became unfavorable at much lower concentration for nonreducing as compared to reducing derivatives.

Tn Antigen Mimics Based on sp(2)-Iminosugars with Affinity for an anti-MUC1 Antibody.

The first examples of amino acid (Ser/Thr)-sp(2)-iminosugar glycomimetic conjugates featuring an α-O-linked pseudoanomeric linkage are reported. The key synthetic step involves the completely diastereoselective α-glycosylation of Ser/Thr due to strong stereoelectronic and conformational bias imposed by the bicyclic sp(2)-iminosugar scaffold. Mucin-related glycopeptides incorporating these motifs were recognized by the monoclonal antibody (mAb) scFv-SM3, with activities depending on both the hydroxylation pattern (Glc/Gal/GlcNAc/GalNAc) of the sp(2)-iminosugar and the peptide aglycone structure (Ser/Thr).

New castanospermine glycoside analogues inhibit breast cancer cell proliferation and induce apoptosis without affecting normal cells.

sp²-Iminosugar-type castanospermine analogues have been shown to exhibit anti-tumor activity. However, their effects on cell proliferation and apoptosis and the molecular mechanism at play are not fully understood. Here, we investigated the effect of two representatives, namely the pseudo-S- and C-octyl glycoside 2-oxa-3-oxocastanospermine derivatives SO-OCS and CO-OCS, on MCF-7 and MDA-MB-231 breast cancer and MCF-10A mammary normal cell lines. We found that SO-OCS and CO-OCS inhibited breast cancer cell viability in a concentration- and time-dependent manner. This effect is specific to breast cancer cells as both molecules had no impact on normal MCF-10A cell proliferation. Both drugs induced a cell cycle arrest. CO-OCS arrested cell cycle at G1 and G2/M in MCF-7 and MDA-MB-231 cells respectively. In MCF-7 cells, the G1 arrest is associated with a reduction of CDK4 (cyclin-dependent kinase 4), cyclin D1 and cyclin E expression, pRb phosphorylation, and an overexpression of p21(Waf1/Cip1). In MDA-MB-231 cells, CO-OCS reduced CDK1 but not cyclin B1 expression. SO-OCS accumulated cells in G2/M in both cell lines and this blockade was accompanied by a decrease of CDK1, but not cyclin B1 expression. Furthermore, both drugs induced apoptosis as demonstrated by the increased percentage of annexin V positive cells and Bax/Bcl-2 ratio. Interestingly, in normal MCF-10A cells the two drugs failed to modify cell proliferation, cell cycle progression, cyclins, or CDKs expression. These results demonstrate that the effect of CO-OCS and SO-OCS is triggered by both cell cycle arrest and apoptosis, suggesting that these castanospermine analogues may constitute potential anti-cancer agents against breast cancer.

Efficient transfection of hepatocytes mediated by mRNA complexed to galactosylated cyclodextrins.

In this study, we aimed at specific targeting of polycationic amphiphilic cyclodextrins (paCDs) to HepG2 cells via the asialoglycoprotein receptor (ASGPr). The transfection efficiencies of paCDs modified with galactose moieties were evaluated. In preliminary experiments, attempts to transfect HepG2 cells with pDNA complexed with different modified paCDs resulted in very low transfection levels. In additional series of experiments, we found out that nucleic acid/cyclodextrin complexes (CDplexes) were efficiently taken up by the cells and that photochemical internalization, which facilitates release from endosomes, did not improve transfection. Further experiments showed that pDNA can be readily released from the CDplexes when exposed to negatively charged vesicles. These observations imply that the lack of transfection cannot be attributed to a lack of internalization, release of CDplexes from the endosomal compartment, or release of free pDNA from the CDplexes. This in turn suggests that the nuclear entry of the pDNA represents the main limiting factor in the transfection process. To verify that HepG2 cells were transfected with targeted CDplexes containing mRNA, which does not require entry into the nucleus for being translated. With mRNA encoding the green fluorescent protein, fractions of GFP-positive cells of up to 31% were obtained. The results confirmed that the galactosylated complexes are specifically internalized via the ASGPr.

Design and synthesis of a "click" high-mannose oligosaccharide mimic emulating Man8 binding affinity towards Con A.

A dendritic "click" mannooligomer mimicking the high-mannose oligosaccharide Man(8) has been designed by replacing some of the inner mannopyranosyl subunits with triazole moieties; evaluation of its binding affinity towards the mannose-specific lectin concanavalin A revealed striking similarities between the "click" mimic and the natural Man(8).

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.

Copper(II)-complex directed regioselective mono-p-toluenesulfonylation of cyclomaltoheptaose at a primary hydroxyl group position: an NMR and molecular dynamics-aided design.

Interactions between cyclomaltoheptaose (ß-cyclodextrin, ßCD) and p-toluenesulfonyl chloride (TsCl) were investigated using MD simulations, both in vacuum, approximating the hydrophobic environment of the CD cavity, and with water as a solvent. In both cases, the minimum energy adiabatic paths, and the mean force potentials (MFP) for the insertion of TsCl along a reaction coordinate perpendicular to the CD plane, were calculated for the two possible orientations of TsCl. The results show a preferred entry of TsCl in the CD cavity with the sulfonyl chloride group pointing to the primary hydroxyls rim. In each orientation, two energy minima for the complex are detected in vacuum that reflect the H-H contacts between host and guest observed by NMR spectroscopy (ROESY, NOESY). These separate minima collapsed into a single broader minimum, when the solvent was introduced in the simulations. The resulting association constant between TsCl and ßCD (K(a) ≈ 100 M(-1)) is in good agreement with the NMR results (K(a) = 102 ± 12 M(-1)) in deuterated water solution at 298 K. Advantage has been taken of the dynamics of the reagent inclusion to set up a one step process involving a transient Cu(2+) chelate at the secondary hydroxyls rim position for the electrophilic monoactivation of ßCD at the primary hydroxyls rim using water as solvent.

Bicyclic (galacto)nojirimycin analogues as glycosidase inhibitors: effect of structural modifications in their pharmacological chaperone potential towards ß-glucocerebrosidase.

A molecular-diversity-oriented approach for the preparation of bicyclic sp(2)-iminosugar glycomimetics related to nojirimycin and galactonojirimycin is reported. The synthetic strategy takes advantage of the ability of endocyclic pseudoamide-type atoms in five-membered cyclic iso(thio)ureas and guanidines to undergo intramolecular nucleophilic addition to the masked carbonyl group of monosaccharides. The stereochemistry of the resulting hemiaminal stereocenter is governed by the anomeric effect, with a large preference for the axial (pseudo-α) orientation. A library of compounds differing in the stereochemistry at the position equivalent to C-4 in monosaccharides (D-gluco and D-galacto), the heterocyclic core (cyclic isourea, isothiourea or guanidine) and the nature of the exocyclic nitrogen substituent (apolar, polar, linear or branched) has been thus prepared and the glycosidase inhibitory activity evaluated against commercial glycosidases. Compounds bearing lipophilic substituents behaved as potent and very selective inhibitors of ß-glucosidases. They further proved to be good competitive inhibitors of the recombinant human ß-glucocerebrosidase (imiglucerase) used in enzyme replacement therapy (ERT) for Gaucher disease. The potential of these compounds as pharmacological chaperones was assessed by measuring their ability to inhibit thermal-induced denaturation of the enzyme in comparison with N-nonyl-1-deoxynojirimycin (NNDNJ). The results indicated that amphiphilic sp(2)-iminosugars within this series are more efficient than NNDNJ at stabilizing ß-glucocerebrosidase and have a strong potential in pharmacological chaperone (PC) and ERT-PC combined therapies.

Comparative studies on lectin-carbohydrate interactions in low and high density homo- and heteroglycoclusters.

A versatile synthetic procedure to construct series of high- and low-density homo- and heteroglycoclusters is reported. The binding properties of these synthetic multivalent glycoconjugates to concanavalin A (Con A), a model lectin, have been assessed by using a range of competitive and non-competitive binding assays including enzyme-linked lectin assays (ELLA), isothermal titration microcalorimetry (ITC) and surface plasmon resonance (SPR). In all cases, highly dense glycoclusters showed a substantial amplification of the lectin-binding strength in comparison with low-density counterparts. Interestingly, highly-dense glycoligand presentations, regardless of their homo- or heteroglycoligand pattern, furnished similar Con A binding properties, supporting the existence of a synergic effect (heterocluster effect) due to secondary interactions of "non-active" structural motifs in the presence of a certain density of "active" glycoligands.

Insights in cellular uptake mechanisms of pDNA-polycationic amphiphilic cyclodextrin nanoparticles (CDplexes).

It is generally recognized that the major obstacle to efficient gene delivery is cellular internalization and endosomal escape of the DNA. Recently, we have developed a modular strategy for the preparation of well-defined polycationic amphiphilic cyclodextrins (paCDs) capable of complexing and compacting DNA into homogeneous nanoparticles (<70nm). Since paCDs resemble both cationic polymers and cationic lipids, it is conceivable that the corresponding pDNA-paCD nanoparticles (CDplexes) might use the cell internalization and endosomal escape mechanisms described for both lipoplexes and polyplexes. To verify this hypothesis, we have now investigated the uptake and transfection efficiencies of CDplexes in the presence of several inhibitors of endocytosis, namely chlorpromazine, genistein, dynasore and methylated beta-cyclodextrin (MbCD). Our data show that CDplexes obtained from paCD 1, which ranks among the most efficient paCD gene vectors reported up to date, are internalized by both clathrin-dependent (CDE) and clathrin-independent endocytosis (CIE), both processes being cholesterol- and dynamin-dependent. We observed that the largest fraction of gene complexes is taken up via CDE, but this fraction is less relevant for transfection. The smaller fraction that is internalized via the CIE pathway is predominantly responsible for successful transfection.

Study of the conformational and self-aggregation properties of 2I,3I-O-(o-xylylene)-per-O-Me-alpha- and -beta-cyclodextrins by fluorescence and molecular modeling.

Steady-state and time-resolved fluorescence techniques were used to study the behavior of 2I,3I-O-(o-xylylene)-per-O-Me-alpha- and -beta-cyclodextrins in aqueous solution, based on the fluorescence of the bidentate xylylene moiety. Fluorescence decay profiles obtained upon excitation of the xylylene group were fitted to three-exponential decay functions. In addition to a fast component due to stray and/or scattered light, two other components ascribed to the monomer and dimer species, respectively, were identified. The dimer/monomer ratio increases with concentration and decreases with temperature, which is in agreement with an enthalpy-driven association process. The corresponding dimerization equilibrium constants (KD) were obtained from nonlinear regression analysis of the plots of tau against [CD] in the 5-45 degrees C range. A linear van't Hoff analysis for KD allows us to obtain the DeltaH and DeltaS associated to dimer formation. Molecular mechanics as well as molecular dynamics calculations in the presence of water were also employed to study the conformational behavior of such secondary-face-substituted cyclodextrins and rationalize the dimerization processes.

Chemical and enzymatic approaches to carbohydrate-derived spiroketals: di-D-fructose dianhydrides (DFAs).

Di-D-fructose dianhydrides (DFAs) comprise a unique family of stereoisomeric spiro-tricyclic disaccharides formed upon thermal and/or acidic activation of sucrose- and/ or D-fructose-rich materials. The recent discovery of the presence of DFAs in food products and their remarkable nutritional features has attracted considerable interest from the food industry. DFAs behave as low-caloric sweeteners and have proven to exert beneficial prebiotic nutritional functions, favouring the growth of Bifidobacterium spp. In the era of functional foods, investigation of the beneficial properties of DFAs has become an important issue. However, the complexity of the DFA mixtures formed during caramelization or roasting of carbohydrates by traditional procedures (up to 14 diastereomeric spiroketal cores) makes evaluation of their individual properties a difficult challenge. Great effort has gone into the development of efficient procedures to obtain DFAs in pure form at laboratory and industrial scale. This paper is devoted to review the recent advances in the stereoselective synthesis of DFAs by means of chemical and enzymatic approaches, their scope, limitations, and complementarities.

Synthesis, structure, and inclusion capabilities of trehalose-based cyclodextrin analogues (cyclotrehalans).

Concise and efficient strategies toward the synthesis of D2h- and D3h-symmetric cyclodextrin analogues alternating alpha,alpha'-trehalose disaccharide subunits and pseudoamide segments (cyclotrehalans, CTs) are reported. The conformational properties of these cyclooligosaccharides are governed by the rigidity of the alpha,alpha'-trehalose disaccharide repeating unit and the partial double-bond character of the N-(C=X) linkages. In contrast to the typical concave-shaped cavity of cyclodextrins (CDs), CTs feature a convex-shaped hydrophobic cavity in which the beta-face of the monosaccharide subunits is oriented toward the inner side, as supported by NMR and modeling (molecular mechanics and dynamics) studies. In the case of cyclodimeric CTs (CT2s), the existence of intramolecular hydrogen bonds results in collapsed cavities, too small to allow the formation of inclusion complexes with organic molecules. Cyclotrimeric CTs (CT3s) display cavity sizes that are intermediate between those of alphaCD and betaCD, ideally suited for the complexation of complementary guests with ternary symmetry such as adamantane 1-carboxylate (AC). The higher flexibility of the pseudoamide bridges as compared with classical glycosidic linkages endow these glyconanocavities with some conformational adaptability properties, making them better suited than CDs for complexation of angular guests, as seen from comparative inclusion capability experiments against the fluorescent probes 6-p-toluidinonaphthalene-2-sulfonate (TNS; linear) and 8-anilinonaphthalene-1-sulfonate (ANS; angular).

Synthesis of alpha- and beta-glycosyl isothiocyanates via oxazoline intermediates.

A practical synthesis of acylated glycosyl isothiocyanates from sugar oxazolines, by reaction with thiophosgene, is reported. In the absence of any additive, the reaction is governed by the reverse anomeric effect, leading to the equatorially oriented isothiocyanate. However, in the presence of copper(II) chloride, the reaction proceeds preferentially with retention of the configuration at the anomeric center, providing the axial anomer as the major product. Noteworthy, this strategy allows accessing per-O-acetylated glycopyranosyl isothiocyanates with 1,2-cis relative configuration (e.g., the alpha-anomer in the D-gluco and D-galacto series), a problem that was outside the scope of previous methodologies.