Camille Decroocq

Glycosidase Inhibition with Fullerene Iminosugar Balls: A Dramatic Multivalent Effect†

The electronic and structural properties of fullerene derivatives make them very attractive candidates for the construction of nanostructures that are potentially useful for applications in materials science and biological chemistry. In particular, the C60 hexakis adducts with a Th-symmetrical octahedral addition pattern initially developed by Hirsch and co-workers are unique organic molecules with an appealing compact spherical scaffold for the construction of multifunctional nanomaterials. However, the synthesis of functionalized fullerene hexakis adducts from malonates and C60 is difficult. 4] This major problem limits the applications of such systems and has been recently solved by the development of synthetic methodologies based on the postfunctionalization of easily accessible building blocks of fullerene hexakis adducts. 6] It has been shown that fullerene hexakis adducts that bear 12 peripheral carbohydrate moieties can be prepared in excellent yields by grafting unprotected sugar derivatives onto the fullerene core. Although these fullerene sugar balls are obviously perfectly suited for applications in the field of carbohydrate–lectin interactions, the evaluation of carbohydrate-processing enzyme inhibition with such multivalent derivatives is less obvious. Indeed, among the possible strategies to attain specific potent glycosidase inhibition, the concept of multivalent design has been clearly overlooked. Most enzymes actually have a single, deep active site that is usually less accessible than the shallow binding pockets or grooves on the lectin surfaces. Consequently, a limited number of binding mechanisms, including statistical rebinding, are possible, whereas multivalent ligands may interact with multiple receptors by additional mechanistic options (e.g., the chelate effect, receptor clustering). It is likely that these factors may have hampered interest in projects directed towards the design of multivalent glycosidase inhibitors. In addition, the experimental results obtained to date were not particularly encouraging. Dito tetravalent analogues of 1-deoxynojirimycin, which is a well-known glycosidase inhibitor, generally displayed comparable if not decreased inhibition compared with their monomeric counterparts. The best result reported to date was found for a trivalent iminosugar that showed a sixfold affinity enhancement towards Jack bean a-mannosidase. Herein we report the synthesis of a fullerene hexakis adduct decorated with 12 iminosugar residues. The inhibition profile of this fullerene iminosugar ball has been systematically evaluated against various glycosidases, and dramatic multivalent effects have been observed for the first time. In order to explore the potential of multivalency on glycosidase inhibition with a globular polytopic ligand constructed around the fullerene scaffold, an N-alkyl analogue of 1-deoxynojirimycin was selected as the peripheral ligand. This class of compounds is indeed poorly selective and displays modest to good glycosidase inhibition. It was thus anticipated that these compounds could be excellent models for the examination of the influence of multivalency on inhibition selectivity over a large range of glycosidases. In addition, the alkyl chain on the endocyclic nitrogen atom of the iminosugar is an ideal spacer that may allow for easy grafting onto the central C60 core by means of a cycloaddition reaction. [16] The synthesis of the azide building block is based on the optimization of a strategy reported independently by Overkleeft et al. and Vasella and co-workers. As shown in Scheme 1, the d-hydroxy amide 2 was obtained directly from commercially available tetra-O-benzyl d-glucopyranose (1) in 78% yield by oxidative amidation with iodine in 30% aqueous ammonia (30%). The main advantage of this onepot process is that aldehyde oxidation and C N bond formation are performed in a single synthetic step. Oxidation of the hydroxy group at C5 followed by intramolecular [*] Prof. P. Compain, C. Decroocq, Dr. D. Hazelard Laboratoire de Synth se Organique et Mol cules Bioactives Universit de Strasbourg et CNRS (UMR 7509) Ecole Europ enne de Chimie, Polym res et Mat riaux 25 rue Becquerel, 67087 Strasbourg (France) Fax: (+ 33)3-6885-2754 E-mail: philippe.compain@unistra.fr

The Multivalent Effect in Glycosidase Inhibition: Probing the Influence of Architectural Parameters with Cyclodextrin-based Iminosugar Click Clusters

In contrast to most lectins, glycosidases may appear to be unpromising targets for multivalent binding because they display only a single active site. To explore the potential of multivalency on glycosidase inhibition, unprecedented cyclodextrin-based iminosugar conjugates have been designed and prepared. The synthesis was performed by way of Cu(I) -catalyzed azide-alkyne cycloaddition reaction under microwave activation between propargylated multivalent β-cyclodextrins and an azide-armed N-alkyl 1-deoxynojirimycin derivative. Evaluation with a panel of glycosidases of this new class of glycomimetic clusters revealed the strongest affinity enhancement observed to date for a multivalent glycosidase inhibitor, with binding enhancement up to four orders of magnitude over the corresponding monovalent ligand for α-mannosidase. These results demonstrate that the multivalency concept extends beyond carbohydrate-lectin recognition processes to glycomimetic-enzyme inhibition.

Iminosugar-based glycopolypeptides: glycosidase inhibition with bioinspired glycoprotein analogue micellar self-assemblies.

Biomimetic nanoparticles prepared by self-assembly of iminosugar-based glycopolypeptides evidenced remarkable multivalency properties when inhibiting α-mannosidase activity. This approach paves the way to obtain biologically active drug delivery systems having glycosidase inhibition potency.

Cyclodextrin-based iminosugar click clusters: the first examples of multivalent pharmacological chaperones for the treatment of lysosomal storage disorders.

The pharmacological chaperone concept has recently raised many hopes for the treatment of inherited diseases that are caused by improperly folded proteins. The most relevant successes have undoubtedly been obtained in the field of glycosphingolipid lysosomal storage disorders (GLSDs), a small group of diseases characterized by a deficiency of the glycosidases involved in the catabolism of glycosphingolipids in the lysosome. Pharmacological chaperone therapy is based on the ability of reversible inhibitors of the deficient enzymes to enhance their residual hydrolytic activity at sub-inhibitory concentrations. The proof-of-concept of this approach was demonstrated in 1999 with 1-deoxygalactonojirimycin (1, Amigal), a potent inhibitor of the galactosidase involved in Fabry disease (Scheme 1). The counterintuitive basis of the pharmacological

The Multivalent Effect in Glycosidase Inhibition: Probing the Influence of Valency, Peripheral Ligand Structure, and Topology with Cyclodextrin‐Based Iminosugar Click Clusters

In view of recent reports of a strong multivalent effect in glycosidase inhibition, a library of β‐CD‐based multivalent iminosugars has been efficiently synthesized by way of CuI‐catalyzed azide–alkyne cycloaddition (CuAAC). In combination with the first application of isothermal titration calorimetry (ITC) experiments to the study of multivalent iminosugar–enzyme interactions, the inhibition properties of these click clusters were evaluated on a panel of glycosidases. The structural parameters that were varied include valency, peripheral ligand structure, and topology. The inhibition results obtained with the iminosugar clusters further highlight the importance of multivalency in the inhibition of α‐mannosidase. Generally, the evaluated multivalent iminosugars displayed comparable thermodynamic signatures of binding towards α‐mannosidase (Jack bean): that is, large negative enthalpies of complexation coupled with small entropies of either sign. In addition, the enthalpy–entropy compensation observed in all tested cases may be attributed to a common mechanism of dissociation for the enzyme–multivalent iminosugar interactions. The measured binding stoichiometries indicated that each iminosugar cluster interacts with no more than one protein molecule.

A systematic investigation of iminosugar click clusters as pharmacological chaperones for the treatment of Gaucher disease.

A series of 18 mono‐ to 14‐valent iminosugars with different ligands, scaffolds, and alkyl spacer lengths have been synthesized and evaluated as inhibitors and pharmacological chaperones of β‐glucocerebrosidase (GCase). Small but significant multivalent effects in GCase inhibition have been observed for two iminosugar clusters. Our study provides strong confirmation that compounds that display the best affinity for GCase are not necessarily the best chaperones. The best chaperoning effect observed for a deprotected iminosugar cluster has been obtained with a tetravalent 1‐deoxynojirimycin (DNJ) analogue (3.3‐fold increase at 10 μM). In addition, our study provides the first evidence of the high potential of prodrugs for the development of potent pharmacological chaperones. Acetylation of a trivalent DNJ derivative, to give the corresponding acetate prodrug, leads to a pharmacological chaperone that produces higher enzyme activity increases (3.0‐fold instead of 2.4‐fold) at a cellular concentration (1 μM) reduced by one order of magnitude.

Rescue of Functional CFTR Channels in Cystic Fibrosis: A Dramatic Multivalent Effect Using Iminosugar Cluster-Based Correctors

Cystic fibrosis is caused by a mutation in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. N‐butyl 1‐deoxynojirimycin (N‐Bu DNJ), a clinical candidate for the treatment of cystic fibrosis, is able to act as a CFTR corrector by overcoming the processing defect of the mutant protein. To explore the potential of multivalency on CFTR correction activity, a library of twelve DNJ click clusters with valencies ranging from 3 to 14 were synthesized. Significantly, the trivalent analogues were found to be up to 225‐fold more potent than N‐Bu DNJ and up to 1000‐fold more potent than the corresponding monovalent models. These results provide the first description of a multivalent effect for correcting protein folding defects in cells and should have application for the treatment of a number of protein folding disorders. Preliminary mechanistic studies indicated that CFTR correction activity enhancement was not due to a multivalent effect in ER‐glucosidase inhibition or to a different mode of action of the multivalent iminosugars.

Synthesis of Azide-armed α-1-C-Alkyl-imino-d-xylitol Derivatives as Key Building Blocks for the Preparation of Iminosugar Click Conjugates

Azide-armed α-1-C-alkyl-imino-d-xylitol derivatives have been efficiently prepared by way of olefin cross-metathesis in eight to nine steps and in an overall yield of 19% to 26% from 2,3,4-tri-O-benzyl-d-xylopyranose. Optimization of cleavage conditions of N-NAP-protected tertiary amines using DDQ in CH2Cl2-H2O (18:1) is also reported. The iminosugars synthesized will be used as key building blocks in the synthesis of multivalent iminosugars of biological interest by way of Cu(I)-catalyzed azide-alkyne cycloaddition reaction.

Iminosugars as a new class of cholinesterase inhibitors

To further extend the scope of iminosugar biological activity, a systematic structure-activity relationship investigation has been performed by synthesizing and evaluating as cholinesterase inhibitors a library of twenty-three iminoalditols with different substitutions and stereochemistry patterns. These compounds have been evaluated in vitro for the inhibition of cholinesterases (different sources of acetylcholinesterase and butyrylcholinesterase). Some compounds have IC50 values in the micromolar range and display significant inhibition selectivity for butyrylcholinesterase over acetylcholinesterase. These are the first examples of iminosugar-based inhibitors of cholinesterases.

Glucocerebrosidase enhancers for selected Gaucher disease genotypes by modification of α-1-C-substituted imino-D-xylitols (DIXs) by click chemistry.

A series of hybrid analogues was designed by combination of the iminoxylitol scaffold of parent 1C9‐DIX with triazolylalkyl side chains. The resulting compounds were considered potential pharmacological chaperones in Gaucher disease. The DIX analogues reported here were synthesized by CuAAC click chemistry from scaffold 1 (α‐1‐C‐propargyl‐1,5‐dideoxy‐1,5‐imino‐D‐xylitol) and screened as imiglucerase inhibitors. A set of selected compounds were tested as β‐glucocerebrosidase (GBA1) enhancers in fibroblasts from Gaucher patients bearing different genotypes. A number of these DIX compounds were revealed as potent GBA1 enhancers in genotypes containing the G202R mutation, particularly compound DIX‐28 (α‐1‐C‐[(1‐(3‐trimethylsilyl)propyl)‐1H‐1,2,3‐triazol‐4‐yl)methyl]‐1,5‐dideoxy‐1,5‐imino‐D‐xylitol), bearing the 3‐trimethylsilylpropyl group as a new surrogate of a long alkyl chain, with approximately threefold activity enhancement at 10 nM. Despite their structural similarities with isofagomine and with our previously reported aminocyclitols, the present DIX compounds behaved as non‐competitive inhibitors, with the exception of the mixed‐type inhibitor DIX‐28.