It has been amply constated that sugar ligand multivalency increases lectin-binding avidities dramatically, thereby modulating the capacity of carbohydrates to participate in supramolecular recognition processes involving transfer of biological information. The importance of this concept, the multivalent or glycoside cluster effect, in cell biology in general and in the glycosciences in particular is reflected in the ever-growing number of papers in the field. An impressive range of glycoarchitectures has been conceived to imitate the glycan coating of cells (the glycocalyx) in order to target complementary lectin receptors. However, these models rarely address the heterogeneity and the fluidity of the densely glycosylated cell membrane. They also disregard the impact that high-density nanosized arrangements could have in their interactions with the whole spectrum of carbohydrate-interacting proteins, among which glycosidases are notable representatives. For many years it has been tacitly assumed that:(i) efficient recognition by lectins generally requires high densities of the putative primary ligand and (ii) the mechanisms governing binding of a carbohydrate motif by a lectin or a glycosidase are totally disparate.Notwithstanding, an increasing amount of evidence seriously questions this paradigm. First, it was shown that secondary ''innocent'' ligands can play important roles in the recognition of heteroglycocluster constructs by lectins through synergistic or antagonistic contributions, a phenomenon termed the heterocluster effect. Second, the existence of multivalent effects in the inhibition of certain glycosidases by glycomimetic-and, even more disturbing, glyco-coated architectures (multivalent enzyme inhibition) was demonstrated. These observations call for a generalized multivalent effect governing the supramolecular chemistry of carbohydrate or glycomimetic structures in a biological context, with (hetero)multivalency acting as a multimodal switcher to drive the encoded information through different pathways. In this Feature Article we review the advancements made in the last few years in our understanding of the mechanisms underpinning the generalized multivalent effect, with an emphasis on the potential risks and opportunities derived from (hetero)multivalency-elicited promiscuity.

Instilling segregated cationic and lipophilic domains with an angular disposition in a trehalose-based trifaceted macrocyclic scaffold allows engineering patchy molecular nanoparticles leveraging directional interactions that emulate those controlling self-assembling processes in viral capsids. The resulting trilobular amphiphilic derivatives, featuring a Mickey Mouse architecture, can electrostatically interact with plasmid DNA (pDNA) and further engage in hydrophobic contacts to promote condensation into transfectious nanocomplexes. Notably, the topology and internal structure of the cyclooligosaccharide/pDNA co-assemblies can be molded by fine-tuning the valency and characteristics of the cationic and lipophilic patches, which strongly impacts the transfection efficacy in vitro and in vivo. Outstanding organ selectivities can then be programmed with no need of incorporating a biorecognizable motif in the formulation. The results provide a versatile strategy for the construction of fully synthetic and perfectly monodisperse nonviral gene delivery systems uniquely suited for optimization schemes by making cyclooligosaccharide patchiness the focus.

Owing to its roles in human health and disease, the modification of nuclear, cytoplasmic, and mitochondrial proteins with Olinked N-acetylglucosamine residues (O-GlcNAc) has emerged as a topic of great interest. Despite the presence of O-GlcNAc on hundreds of proteins within cells, only two enzymes regulate this modification. One of these enzymes is O-GlcNAcase (OGA), a dimeric glycoside hydrolase that has a deep active site cleft in which diverse substrates are accommodated. Chemical tools to control OGA are emerging as essential resources for helping to decode the biochemical and cellular functions of the O-GlcNAc pathway. Here we describe rationally designed bicyclic thiazolidine inhibitors that exhibit superb selectivity and picomolar inhibition of human OGA. Structures of these inhibitors in complex with human OGA reveal the basis for their exceptional potency and show that they extend out of the enzyme active site cleft. Leveraging this structure, we create a high affinity chemoproteomic probe that enables simple one-step purification of endogenous OGA from brain and targeted proteomic mapping of its post-translational modifications. These data uncover a range of new modifications, including some that are less-known, such as O-ubiquitination and N-formylation. We expect that these inhibitors and chemoproteomics probes will prove useful as fundamental tools to decipher the mechanisms by which OGA is regulated and directed to its diverse cellular substrates. Moreover, the inhibitors and structures described here lay out a blueprint that will enable the creation of chemical probes and tools to interrogate OGA and other carbohydrate active enzymes.

Multimannosides assembled onto cyclodextrin or cyclopeptide cores exhibit dual specificity towards the macrophage mannose receptor and lysosomal glycosidases.

The late-onset form of Tay-Sachs disease displays when the activity levels of human β-hexosaminidase A (HexA) fall below 10% of normal, due to mutations that destabilise the native folded form of the enzyme and impair its trafficking to the lysosome. Competitive inhibitors of HexA can rescue disease-causative mutant HexA, bearing potential as pharmacological chaperones, but often also inhibit the enzyme O-glucosaminidase (GlcNAcase; OGA), a serious drawback for translation into the clinic. We have designed sp 2 -iminosugar glycomimetics related to GalNAc that feature a neutral piperidine-derived thiourea or a basic piperidine-thiazolidine bicyclic core and behave as selective nanomolar competitive inhibitors of human Hex A at pH 7 with a ten-fold lower inhibitory potency at pH 5, a good indication for pharmacological chaperoning. They increased the levels of lysosomal HexA activity in Tay-Sachs patient fibroblasts having the G269S mutation, the highest prevalent in late-onset Tay-Sachs disease.

A simple and efficient method for the stereoselective synthesis of nojirimycin α- C -glycoside derivatives has been developed using a bicyclic carbamate-type sp 2 -iminosugar, whose preparation on a gram scale has been optimized, as the starting material. sp 2 -iminosugar O -glycosides or anomeric esters serve as excellent precursors of acyliminium cations, which can add nucleophiles, including C -nucleophiles. The stereochemical outcome of the reaction is governed by stereoelectronic effects, affording the target α-anomer with total stereoselectivity. Thus, the judicious combination of C -allylation, carbamate hydrolysis, cross-metathesis, and hydrogenation reactions provides a very convenient entry to iminosugar α- C -glycosides, which have been transformed into N , C -biantennary derivatives by reductive amination or thiourea-forming reactions. The thiourea adducts undergo intramolecular cyclization to bicyclic iminooxazolidine iminosugar α- C -glycosides upon acid treatment, broadening the opportunities for molecular diversity. A preliminary evaluation against a panel of commercial glycosidases validates the approach for finely tuning the inhibitory profile of glycomimetics.

A selective inhibitor of GALA reduces Gb3 levels by over half within Fabry disease patient fibroblasts.

Glycoside hydrolases (GHs) are a class of enzymes with emerging roles in a range of disease. Selective GH inhibitors are sought to better understand their functions and assess the therapeutic potential of modulating their activities. Iminosugars are a promising class of GH inhibitors but typically lack the selectivity required to accurately perturb biological systems. Here, we describe a concise synthesis of iminosugar inhibitors of N‐acetyl‐α‐galactosaminidase (α‐NAGAL), the GH responsible for cleaving terminal α‐N‐acetylgalactosamine residues from glycoproteins and other glycoconjugates. Starting from non‐carbohydrate precursors, this modular synthesis supported the identification of a potent (490 nM) and α‐NAGAL selective (∼200‐fold) guanidino‐containing derivative DGJNGuan. To illustrate the cellular activity of this new inhibitor, we developed a quantitative fluorescence image‐based method to measure levels of the Tn‐antigen, a cellular glycoprotein substrate of α‐NAGAL. Using this assay, we show that DGJNGuan exhibits excellent inhibition of α‐NAGAL within cells using patient derived fibroblasts (EC50=150 nM). Moreover, in vitro and in cell assays to assess levels of lysosomal β‐hexosaminidase substrate ganglioside GM2 show that DGJNGuan is selective whereas DGJNAc exhibits off‐target inhibition both in vitro and within cells. DGJNGuan is a readily produced and selective tool compound that should prove useful for investigating the physiological roles of α‐NAGAL.