Extending the Scope of the C-Functionalization of Cyclam via Copper(I)-Catalyzed Alkyne-Azide Cycloaddition to Bifunctional Chelators of Interest.

Cyclam, known for its potent chelation properties, is explored for diverse applications through selective N-functionalization, offering versatile ligands for catalysis, medical research, and materials science. The challenges arising from N-alkylation, which could decrease the coordination properties, are addressed by introducing a robust C-functionalization method. The facile two-step synthesis proposed here involves the click chemistry-based C-functionalization of a hydroxyethyl cyclam derivative using Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC). Boc-protecting groups prevent undesired copper coordination, resulting in compounds with a wide range of functionalities. The optimized synthesis conditions enable C-functional cyclams to be obtained easily and advantageously, with high application potential in the previously cited fields. The methodology has been extended to trehalose-based Siamese twin amphiphiles, enabling efficient gene delivery applications.

Trehalose-polyamine/DNA nanocomplexes: impact of vector architecture on cell and organ transfection selectivity.

A novel family of precision-engineered gene vectors with well-defined structures built on trehalose and trehalose-based macrocycles (cyclotrehalans) comprising linear or cyclic polyamine heads have been synthesized through procedures that exploit click chemistry reactions. The strategy was conceived to enable systematic structural variations and, at the same time, ensuring that enantiomerically pure vectors are obtained. Notably, changes in the molecular architecture translated into topological differences at the nanoscale upon co-assembly with plasmid DNA, especially regarding the presence of regions with short- or long-range internal order as observed by TEM. In vitro and in vivo experiments further evidenced a significant impact on cell and organ transfection selectivity. Altogether, the results highlight the potential of trehalose-polyamine/pDNA nanocomplex monoformulations to achieve targeting transfection without the need for any additional cell- or organ-sorting component.

Pharmacological Chaperones for the Treatment of α-Mannosidosis.

α-Mannosidosis (AM) results from deficient lysosomal α-mannosidase (LAMAN) activity and subsequent substrate accumulation in the lysosome, leading to severe pathology. Many of the AM-causative mutations compromise enzyme folding and could be rescued with purpose-designed pharmacological chaperones (PCs). We found that PCs combining a LAMAN glycone-binding motif based on the 5 N,6 O-oxomethylidenemannojirimycin (OMJ) glycomimetic core and different aglycones, in either mono- or multivalent displays, elicit binding modes involving glycone and nonglycone enzyme regions that reinforce the protein folding and stabilization potential. Multivalent derivatives exhibited potent enzyme inhibition that generally prevailed over the chaperone effect. On the contrary, monovalent OMJ derivatives with LAMAN aglycone binding area-fitting substituents proved effective as activity enhancers for several mutant LAMAN forms in AM patient fibroblasts and/or transfected MAN2 B1-KO cells. This translated into a significant improvement in endosomal/lysosomal function, reverting not only the primary LAMAN substrate accumulation but also the additional downstream consequences such as cholesterol accumulation.

The Impact of Heteromultivalency in Lectin Recognition and Glycosidase Inhibition: An Integrated Mechanistic Study.

The vision of multivalency as a strategy limited to achieve affinity enhancements between a protein receptor and its putative sugar ligand (glycotope) has proven too simplistic. On the one hand, binding of a glycotope in a dense glycocalix-like construct to a lectin partner has been shown to be sensitive to the presence of a third sugar entity (heterocluster effect). On the other hand, several carbohydrate processing enzymes (glycosidases and glycosyltransferases) have been found to be also responsive to multivalent presentations of binding partners (multivalent enzyme inhibition), a phenomenon first discovered for iminosugar-type inhibitory species (inhitopes) and recently demonstrated for multivalent carbohydrate constructs. By assessing a series of homo- and heteroclusters combining α-d-glucopyranosyl-related glycotopes and inhitopes, it was shown that multivalency and heteromultivalency govern both kinds of events, allowing for activation, deactivation or enhancement of specific recognition phenomena towards a spectrum of lectin and glycosidase partners in a multimodal manner. This unified scenario originates from the ability of (hetero)multivalent architectures to trigger glycosidase binding modes that are reminiscent of those harnessed by lectins, which should be considered when profiling the biological activity of multivalent architectures.

Symmetry complementarity-guided design of anthrax toxin inhibitors based on ß-cyclodextrin: Synthesis and relative activities of face-selective functionalized polycationic clusters.

Three new series of potential anthrax toxin inhibitors based on the ß-cyclodextrin (ßCD) scaffold were developed by exploiting face-selective Cu(I)-catalyzed azide-alkyne 1,3-cycloadditions, amine-isothiocyanate coupling, and allyl group hydroboration-oxidation/hydroxy → amine replacement reactions. The molecular design follows the "symmetry-complementarity" concept between homogeneously functionalized polycationic ßCD derivatives and protective antigen (PA), a component of anthrax toxin known to form C7-symmetric pores on the cell membrane used by lethal and edema factors to gain access to the cytosol. The synthesis and antitoxin activity of a collection of ßCD derivatives differing in the number, arrangement, and face location of the cationic elements are reported herein. These results set the basis for a structure-activity relationship development program of new candidates to combat the anthrax threat.

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

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.

Polycationic amphiphilic cyclodextrins for gene delivery: synthesis and effect of structural modifications on plasmid DNA complex stability, cytotoxicity, and gene expression.

A molecular-diversity-oriented approach for the preparation of well-defined polycationic amphiphilic cyclodextrins (paCDs) as gene-delivery systems is reported. The synthetic strategy takes advantage of the differential reactivity of primary versus secondary hydroxyl groups on the CD torus to regioselectively decorate each rim with cationic elements and lipophilic tails, respectively. Both the charge density and the hydrophobic-hydrophilic balance can be finely tuned in a highly symmetrical architecture that is reminiscent of both cationic lipids and cationic polymers, the two most prominent types of nonviral gene vectors. The monodisperse nature of paCDs and the modularity of the synthetic scheme are particularly well suited for structure-activity relationship studies. Gel electrophoresis revealed that paCDs self-assemble in the presence of plasmid DNA (pDNA) to provide homogeneous, stable nanoparticles (CDplexes) of 70-150 nm that fully protect pDNA from the environment. The transfection efficiency of the resulting CDplexes has been investigated in vitro on BNL-CL2 and COS-7 cell lines in the absence and presence of serum and found to be intimately dependent on architectural features. Facial amphiphilicity and the presence of a cluster of cationic and hydrogen-bonding centers for cooperative and reversible complexation of the polyanionic DNA chain is crucial to attain high transgene expression levels with very low toxicity profiles. Further enhancement of gene expression, eventually overcoming that of polyplexes from commercial polyethyleneimine (PEI) polymers (22 kDa), is achieved by building up space-oriented dendritic polycationic constructs.

Tailoring beta-cyclodextrin for DNA complexation and delivery by homogeneous functionalization at the secondary face.

An efficient general strategy for the incorporation of functional elements onto the secondary hydroxyl rim of beta-cyclodextrin has been developed and applied to the synthesis of a novel series of C7-symmetric homogeneous macromolecular polycations with improved DNA complexing and delivery properties.

High dopamine transporter selectivity can be displayed by remarkably simple non-nitrogen containing inhibitors.

A series of 2-(3,4-dichlorophenyl)-cyclopent-1-enyl carboxylic acid esters and amides were prepared and tested for binding to the DAT, SERT, and NET. The achiral compounds were easily attained and found to inhibit DAT binding with K(i)-values ranging from 0.095 to 0.00003 mM. Among the compounds tested 2-(3,4-dichlorophenyl)-cyclopent-1-enyl carboxylic acid 2-methylphenyl ester was found to be highly selective with SERT/DAT>7000; NET/DAT>1700, K(i)=60 nM.

Synthesis and biological evaluation of glycosidase inhibitors: gem-difluoromethylenated nojirimycin analogues.

In our ongoing program aimed at the design, synthesis, and biological evaluation of novel gem-difluoromethylenated glycosidase inhibitors, gem-4,4-difluoromethylenated iminosugars (5-9) were synthesized. The biological evaluation of these synthetic iminosugars showed that the gem-difluoromethylenyl group generally reduced the inhibition of glycosidases. However, this was not the case at pH 5.0, where the gem-difluoromethylenated iminosugar 6 was a stronger inhibitor than comparable iminosugars 1 and 36, suggesting that the influence of this group is mainly through its effect on the amine. It is proposed that the unprotonated iminosugar is the species preferably bound by beta-glucosidase, due to the lower pK(a) value of iminosugar 6 than of 1 or 36, leaving iminosugars 1 and 36 mostly protonated at pH 5.0, while iminosugar 6 is not. Iminosugar 6 also displayed good and selective inhibition of beta-glucosidase at pH 6.8.

Four orders of magnitude rate increase in artificial enzyme-catalyzed aryl glycoside hydrolysis.

[reaction: see text] (6AR,6DR)-6A,6D-Di-C-cyano-beta-cyclodextrin (1) and 6A,6D-di-C-cyano-alpha-cyclodextrin (2) were synthesized and shown to catalyze hydrolysis of aryl glycosides into glucose and phenol with a reaction following Michaelis-Menten kinetics. At pH 8.0 and 59 degrees C hydrolysis of 4-nitrophenyl alpha-glucopyranoside was catalyzed by 1 with KM = 10.5 +/- 1.5 mM, kcat = 1.42(+/-0.09) x 10(-4) s(-1), and kcat/kuncat = 7922. Catalysis was observed with a concentration of 1 as low as 10 microM. Hydrolysis of the other aryl glycosides containing stereochemical variation in the sugar-moiety and 4-nitro-, 2-nitro-, 2-aldehydo-, and 2,4-dinitro- were also catalyzed by 1 and 2 with kcat/kuncat ranging from 4 to 7100. Hydrolysis of a phenyl beta-d-glucoside or the thioglycoside tolylthio beta-D-glucoside was also catalyzed. From a series of prepared analogues of 1 it was found that the catalysis was associated with the hydroxyl groups alpha to the nitril groups. The monocyanohydrin 6-C-cyano-beta-cyclodextrin (3) was also found to catalyze the hydrolysis of 4-nitrophenyl beta-glucopyranoside with kcat/kuncat = 1356. It was proposed that the cyclodextrin cyanohydrins 1-3 catalyze the hydrolysis by general acid catalysis on the bound substrate.

Artificial glycosyl phosphorylases.

alpha- and beta-Cyclodextrin 6(A),6(D)-diacids (1 and 2), beta-cyclodextrin-6-monoacid (14), beta-cyclodextrin 6(A),6(D)-di-O-sulfate (16) and beta-cyclodextrin-6-heptasulfate (19) were synthesised. Acids 1, 2 and 14 were made from perbenzylated alpha- or beta-cyclodextrin, by diisobutylaluminum hydride (DIBAL)-promoted debenzylation, oxidation and deprotection. Addition of molecular sieves was found to improve the debenzylation reaction. Sulfates 16 and 19 were made by sulfation of the appropriately partially protected derivatives and deprotection. Catalysis of 4-nitrophenyl glycoside cleavage by these cyclodextrin derivatives was studied. Compounds 1, 2 and 16 were found to catalyse the reaction, with the catalysis following Michaelis-Menten kinetics and depending first order on the phosphate concentration. In a phosphate buffer (0.5 M, 59 degrees C, pH 8.0), K(M) varied from 2-10 mM and the k(cat)/k(uncat) ratio from 80-1000 depending on the stereochemistry of the substrate and the catalyst, with 2 being the best catalyst and with the sulfated 16 also displaying catalytic ability. The monoacid 14 and the heptasulfate 19 were not catalytic.

Remarkable supramolecular catalysis of glycoside hydrolysis by a cyclodextrin cyanohydrin.

(6AR,6DR)-6A,6D-di-C-cyano-beta-cyclodextrin (3) was synthesized and shown to catalyze hydrolysis of nitrophenyl glycosides with the reaction following Michaelis-Menten kinetics. At pH 7.4 and 25 degrees C, hydrolysis of 4-nitrophenyl-beta-glucopyranoside (2) was catalyzed with KM = 15 mM, kcat = 8.2 x 10-6 s-1, and kcat/kuncat = 1217. Catalysis was observed with concentration of 3 as low as 10 muM. Hydrolysis of the corresponding alpha-glucoside, alpha-galactoside, alpha-mannoside, and 2-nitrophenyl-beta-galactoside was also catalyzed by 3, with kcat/kuncat ranging from 283 to 2147. A series of analogues of 3 was prepared and investigated for catalysis of the hydrolysis of 2: (6AR,6DR)-6A,6D-di-C-propyl-beta-cyclodextrin (9) was not catalytic, while 6A,6D-di-C-cyano-6A,6D-dideoxy-beta-cyclodextrin (12) had a low catalytic activity (kcat/kuncat = 4). A kcat/kuncat = 48 was found for 6A,6D-dialdehydo-beta-cyclodextrin dihydrate (11). It was proposed that 3 acts by general acid catalysis on the bound substrate.

Synthesis of nitrogen-functionalized beta-cycloaltrins.

We report the synthesis of functionalized beta-cycloaltrins having azido groups at C-3, C-6, and both C-3 and C-6 by nucleophilic epoxy ring-opening of per-2,3-anhidro-beta-cyclomannin derivatives. The value of these compounds as templates for further functionalization is exemplified by the conversion of heptakis(3,6-diazido-3,6-dideoxy)-beta-cycloaltrin into the per-3,6-diamino, per-3,6-diacetamido, per-3,6-dichloroacetamido, and per[3,6-bis(N'-ethylureido)] derivatives in good yields.

Diverse motifs of mannoside clustering on a beta-cyclodextrin core.

[reaction: see text] A new method for the synthesis of beta-cyclodextrin-based cluster mannosides by application of the Sonogashira cross-coupling reaction is described. The method allows for the persubstitution of the beta-cyclodextrin at either 2- and 3-positions to give two types of heptavalent clusters, at both 2- and 6-positions to give clusters with 14 mannopyranoside units and at 2-, 3-, and 6-positions to obtain clusters with 21 mannopyranoside ligands.

Dendritic galactosides based on a beta-cyclodextrin core for the construction of site-specific molecular delivery systems: synthesis and molecular recognition studies.

In order to evaluate the ability of multivalent glycosides based on a beta-cyclodextrin core as site-specific molecular carriers, a study on both the inclusion complexation behaviour and lectin binding affinity of branched and hyperbranched beta-cyclodextrins is presented. A series of cluster galactosides constructed on beta-cyclodextrin scaffolds containing seven 1-thio-beta-lactose or beta-lactosylamine bound to the macrocyclic core through different spacer arms were synthesised. In addition, the first synthesis of three first-order dendrimers based on a beta-cyclodextrin core containing fourteen 1-thio-beta-D-galactose, 1-thio-beta-lactose and 1-thio-beta-melibiose residues was performed. Calorimetric titrations performed at 25 degrees C in buffered aqueous solution (pH 7.4) gave the affinity constants and the thermodynamic parameters for the complex formation of these beta-cyclodextrin derivatives with guests sodium 8-anilino-1-naphthalenesulfonate (ANS) and 2-naphthalenesulfonate, and lectin from peanut (Arachis hypogaea) (PNA). The persubstitution of the primary face of the beta-cyclodextrin with saccharides led to a slight increase of the binding constant values for the inclusion complexation with ANS relative to the native beta-cyclodextrin. However, the increase of the steric congestion due to the presence of the saccharide residues on the narrow rim of the beta-cyclodextrin may cause a decrease of the binding ability as shown for sodium 2-naphthalenesulfonate. The spacer arms are not passive elements and influence the host binding ability according to their chemical nature. PNA forms soluble cross-linked complexes with cluster galactosides and lactosides scaffolded on beta-cyclodextrin but not with cluster galactopyranosylamines or melibiose. Both, perbranched and hyperbranched beta-cyclodextrins, form stronger complexes with PNA than the monomeric analogues. However, the use of hyperbranched CDs does not contribute to the improvement of the complex stability relative to heptakis-glycocyclodextrin derivatives. Finally, a titration experiment with PNA and a complex formed by a heptakis lactose beta-cyclodextrin derivative with sodium 2-naphthalenesulfonate showed the formation of a soluble cross-linked complex with stronger affinity constant and higher stoichiometry than those observed for the complex formation of PNA with the same heptakis-lactose beta-cyclodextrin derivative, suggesting the formation of a three component complex.