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The conventional approach to developing light-sensitive glycosidase activity regulators, involving the combination of a glycomimetic moiety and a photoactive azobenzene module, results in conjugates with differences in glycosidase inhibitory activity between the interchangeable E and Z-isomers at the azo group that are generally below one-order of magnitude. In this study, we have exploited the chemical mimic character of sp 2 -iminosugars to access photoswitchable p- and o-azobenzene α-O-glycosides based on the gluco-configured representative ONJ. Notably, we achieved remarkably high switching factors for glycosidase inhibition, favoring either the E- or Z-isomer depending on the aglycone structure. Our data also indicate a correlation between the isomeric state of the azobenzene module and the selectivity towards α- and β-glucosidase isoenzymes. The most effective derivative reached over a 10 3 -fold higher inhibitory potency towards human β-glucocerebrosidase in the Z as compared with the E isomeric form. This sharp contrast is compatible with ex-vivo activation and programmed self-deactivation at physiological temperatures, positioning it as a prime candidate for pharmacological chaperone therapy in Gaucher disease. Additionally, our results illustrate that chemical tailoring enables the engineering of photocommutators with the ability to toggle inhibition between α- and β-glucosidase enzymes in a reversible manner, thus expanding the versatility and potential therapeutic applications of this approach., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

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.

STD NMR spectroscopy is a powerful ligand-observed NMR tool for screening and characterizing the interactions of small molecules and low molecular weight fragments with a given macromolecule, identifying the main intermolecular contacts in the bound state. It is also a powerful analytical technique for the accurate determination of protein-ligand dissociation constants ( K D ) of medium-to-weak affinity, of interest in the pharmaceutical industry. However, accurate K D determination and epitope mapping requires a long series of experiments at increasing saturation times to carry out a full analysis using the so-called STD NMR build-up curve approach and apply the "initial slopes approximation". Here, we have developed a new protocol to bypass this important limitation, which allows us to obtain initial slopes by using just two saturation times and, hence, to very quickly determine precise protein-ligand dissociation constants by STD NMR.

The "carbohydrate chemical mimicry" exhibited by sp 2 -iminosugars has been utilized to develop practical syntheses for analogs of the branched high-mannose-type oligosaccharides (HMOs) Man 3 and Man 5 . In these compounds, the terminal nonreducing Man residues have been substituted with 5,6-oxomethylidenemannonojirimycin (OMJ) motifs. The resulting oligomannoside hemimimetic accurately reproduce the structure, configuration, and conformational behavior of the original mannooligosaccharides, as confirmed by NMR and computational techniques. Binding studies with mannose binding lectins, including concanavalin A, DC-SIGN, and langerin, by enzyme-linked lectin assay and surface plasmon resonance revealed significant variations in their ability to accommodate the OMJ unit in the mannose binding site. Intriguingly, OMJMan segments demonstrated "in line" heteromultivalent effects during binding to the three lectins. Similar to the mannobiose (Man 2 ) branches in HMOs, the binding modes involving the external or internal monosaccharide unit at the carbohydrate binding-domain exist in equilibrium, facilitating sliding and recapture processes. This equilibrium, which influences the multivalent binding of HMOs, can be finely modulated upon incorporation of the OMJ sp 2 -iminosugar caps. As a proof of concept, the affinity and selectivity towards DC-SIGN and langerin were adjustable by presenting the OMJMan epitope in platforms with diverse architectures and valencies., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

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 (EC 50 =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., (© 2023 Wiley-VCH GmbH.)

β-cyclodextrin (βCyD) derivatives equipped with aromatic appendages at the secondary face exhibit tailorable self-assembling capabilities. The aromatic modules can participate in inclusion phenomena and/or aromatic-aromatic interactions. Supramolecular species can thus form that, at their turn, can engage in further co-assembling with third components in a highly regulated manner; the design of nonviral gene delivery systems is an illustrative example. Endowing such systems with stimuli responsiveness while keeping diastereomeric purity and a low synthetic effort is a highly wanted advancement. Here, we show that an azobenzene moiety can be "clicked" to a single secondary O-2 position of βCyD affording 1,2,3-triazole-linked βCyD-azobenzene derivatives that undergo reversible light-controlled self-organization into dimers where the monomer components face their secondary rims. Their photoswitching and supramolecular properties have been thoroughly characterized by UV-vis absorption, induced circular dichroism, nuclear magnetic resonance, and computational techniques. As model processes, the formation of inclusion complexes between a water-soluble triazolylazobenzene derivative and βCyD as well as the assembly of native βCyD/βCyD-azobenzene derivative heterodimers have been investigated in parallel. The stability of the host-guest supramolecules has been challenged against the competitor guest adamantylamine and the decrease of the medium polarity using methanol-water mixtures. The collective data support that the E -configured βCyD-azobenzene derivatives, in aqueous solution, form dimers stabilized by the interplay of aromatic-aromatic and aromatic-βCyD cavity interactions after partial reciprocal inclusion. Photoswitching to the Z -isomer disrupts the dimers into monomeric species, offering opportunity for the spatiotemporal control of the organizational status by light.

Glycolipids with TLR4 agonistic properties can serve either as therapeutic agents or as vaccine adjuvants by stimulating the development of proinflammatory responses. Translating them to the clinical setting is hampered by synthetic difficulties, the lack of stability in biological media, and/or a suboptimal profile of balanced immune mediator secretion. Here, we show that replacement of the sugar fragment by an sp 2 -iminosugar moiety in a prototypic TLR4 agonist, CCL-34 , yields iminoglycolipid analogues that retain or improve their biological activity in vitro and in vivo and can be accessed through scalable protocols with total stereoselectivity. Their adjuvant potential is manifested in their ability to induce the secretion of proinflammatory cytokines, prime the maturation of dendritic cells, and promote the proliferation of CD8 + T cells, pertaining to a Th1-biased profile. Additionally, their therapeutic potential for the treatment of asthma, a Th2-dominated inflammatory pathology, has been confirmed in an ovalbumin-induced airway hyperreactivity mouse model.

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.

Selective DC-SIGN targeting vs . langerin might lead to anti-infective agents, given their counteracting effects upon infection by some pathogens. Here we show that multivalent sp 2 -iminosugar-containing mannobioside analogs can achieve total DC-SIGN selectivity by levering the canonic binding mode towards high-mannose oligosaccharide ligands, behaving as factual biomimics.

Inflammatory processes play a central role in the pathogenesis of diabetic nephropathy (DN) in the early stages of the disease. The authors demonstrate that the glycolipid mimetic ( S s )-DS-ONJ is able to abolish inflammation via the induction of autophagy flux and provokes the inhibition of inflammasome complex in ex vivo and in vitro models, using adult kidney explants from BB rats. The contribution of ( S s )-DS-ONJ to reducing inflammatory events is mediated by the inhibition of classical stress kinase pathways and the blocking of inflammasome complex activation. The ( S s ) -DS-ONJ treatment is able to inhibit the epithelial-to-mesenchymal transition (EMT) progression, but only when the IL18 levels are reduced by the treatment. These findings suggest that ( S s )-DS-ONJ could be a novel, and multifactorial treatment for DN.

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., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

A robust strategy is reported to build perfectly monodisperse star polycations combining a trehalose-based cyclooligosaccharide (cyclotrehalan, CT) central core onto which oligoethyleneimine radial arms are installed. The architectural perfection of the compounds is demonstrated by a variety of physicochemical techniques, including NMR, MS, DLS, TEM, and GPC. Key to the strategy is the possibility of customizing the cavity size of the macrocyclic platform to enable/prevent the inclusion of adamantane motifs. These properties can be taken into advantage to implement sequential levels of stimuli responsiveness by combining computational design, precision chemistry and programmed host-guest interactions. Specifically, it is shown that supramolecular dimers implying a trimeric CT-tetraethyleneimine star polycation and purposely designed bis-adamantane guests are preorganized to efficiently complex plasmid DNA (pDNA) into transfection-competent nanocomplexes. The stability of the dimer species is responsive to the protonation state of the cationic clusters, resulting in dissociation at acidic pH. This process facilitates endosomal escape, but reassembling can take place in the cytosol then handicapping pDNA nuclear import. By equipping the ditopic guest with a redox-sensitive disulfide group, recapturing phenomena are prevented, resulting in drastically improved transfection efficiencies both in vivo and in vitro., (© 2022 The Authors. Macromolecular Rapid Communications published by Wiley-VCH GmbH.)

Owing to its roles in human health and disease, the modification of nuclear, cytoplasmic, and mitochondrial proteins with O-linked 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.

sp 2 -Iminosugar glycolipids (sp 2 -IGLs) represent a consolidated family of glycoconjugate mimetics encompassing a monosaccharide-like glycone moiety with a pseudoamide-type nitrogen replacing the endocyclic oxygen atom of carbohydrates and an axially-oriented lipid chain anchored at the pseudoanomeric position. The combination of these structural features makes them promising candidates for the treatment of a variety of conditions, spanning from cancer and inflammatory disorders to parasite infections. The exacerbated anomeric effect associated to the putative sp 2 -hybridized N-atom imparts chemical and enzymatic stability to sp 2 -IGLs and warrants total α-anomeric stereoselectivity in the key glycoconjugation step. A variety of O -, N -, C - and S -pseudoglycosides, differing in glycone configurational patterns and lipid nature, have been previously prepared and evaluated. Here we expand the chemical space of sp 2 -IGLs by reporting the synthesis of α-d-gluco-configured analogs with a bicyclic (5 N ,6 O -oxomethylidene)nojirimycin (ONJ) core incorporating selenium at the glycosidic position. Structure-activity relationship studies in three different scenarios, namely cancer, Leishmaniasis and inflammation, convey that the therapeutic potential of the sp 2 -IGLs is highly dependent, not only on the length of the lipid chain (linear aliphatic C 12 vs. C 8 ), but also on the nature of the glycosidic atom (nitrogen vs. sulfur vs. selenium). The ensemble of results highlights the α-dodecylseleno-ONJ-glycoside as a promising multitarget drug candidate.