sp2-Iminosugar azobenzene O-glycosides: Light-sensitive glycosidase inhibitors with unprecedented tunability and switching factors.

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 sp2-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 ß-glucosidase isoenzymes. The most effective derivative reached over a 103-fold higher inhibitory potency towards human ß-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 ß-glucosidase enzymes in a reversible manner, thus expanding the versatility and potential therapeutic applications of this approach.

Iminosugar-Dihydroazulenes as Mutant L444P Glucocerebrosidase Enhancers.

A remarkable enhancer of human glucocerebrosidase enzyme (GCase) was identified among a set of dihydroazulene-tagged iminosugars. An unprecedented 3.9-fold increase in GCase activity was detected on fibroblasts bearing the homozygous L444P mutation, which is frequently associated with neuronopathic Gaucher forms, and which commonly results refractory to chaperone-induced refolding.

Synthesis of the Purported Structure of Glyphaeaside C and Proposed Revisions to the Structures of the Glyphaeaside Alkaloids.

The 10 glyphaeaside alkaloids isolated from the roots of Glyphaea brevis were originally purported as piperidine-based 1-C-alkylated iminosugars, with the A-, B-, and C-type glyphaeasides bearing l-DFJ, DGJ, and DNJ ring configurations, respectively. Subsequent investigations have revealed glyphaeaside C as being a pyrrolidine-based iminosugar with a DMDP ring configuration via total synthesis of the revised structure. In this work, side chain diastereomers of the originally purported structure of glyphaeaside C (10) and two related α-1-C-alkylated DNJ derivatives were synthesized from a common precursor, which was prepared in turn via stereoselective Grignard addition to a protected d-glycosylamine, followed by a reductive amination-cyclization sequence. Glycosidase inhibitory activity studies revealed general structure 10 as having potent inhibition against various α-glucosidases and weak inhibition against almond ß-glucosidase in agreement with similar DNJ-based iminosugars and in contrast to natural glyphaeaside C, suggesting that the (1,2-dihydroxy-3-phenyl)propyl moiety does not play a particularly vital role in the inhibitory modes of action of either compound. Furthermore, the absolute configuration of natural glyphaeaside C was proposed as that of d-DMDP, and the structures of the A- and B-type glyphaeasides were revised as 1-deoxy-DALDP and DALDP derivatives, respectively, based on interpretation of their reported NMR spectroscopic data.

Structural variation of the 3-acetamido-4,5,6-trihydroxyazepane iminosugar through epimerization and C-alkylation leads to low micromolar HexAB and NagZ inhibitors.

We report the synthesis of seven-membered iminosugars derived from a 3S-acetamido-4R,5R,6S-trihydroxyazepane scaffold and their evaluation as inhibitors of functionally related exo-N-acetylhexosaminidases including human O-GlcNAcase (OGA), human lysosomal ß-hexosaminidase (HexAB), and Escherichia coli NagZ. Capitalizing on the flexibility of azepanes and the active site tolerances of hexosaminidases, we explore the effects of epimerization of stereocenters at C-3, C-5 and C-6 and C-alkylation at the C-2 or C-7 positions. Accordingly, epimerization at C-6 (L-ido) and at C-5 (D-galacto) led to selective HexAB inhibitors whereas introduction of a propyl group at C-7 on the C-3 epimer furnished a potent NagZ inhibitor.

Introduction of C-alkyl branches to L-iminosugars changes their active site binding orientation.

L-ido-Deoxynojirimycin (L-ido-DNJ) itself showed no affinity for human lysosomal acid α-glucosidase (GAA), whereas 5-C-methyl-L-ido-DNJ showed a strong affinity for GAA, comparable to the glucose analog DNJ, with a Ki value of 0.060 µM. This excellent affinity for GAA and enzyme stabilization was observed only when methyl and ethyl groups were introduced. Docking simulation analysis revealed that the alkyl chains of 5-C-alkyl-L-ido-DNJs were stored in three different pockets, depending on their length, thereby the molecular orientation was changed. Comparison of the binding poses of DNJ and 5-C-methyl-L-ido-DNJ showed that they formed a common ionic interaction with Asp404, Asp518, and Asp616, but both the binding orientation and the distance between the ligand and each amino acid residue were different. 5-C-Methyl-L-ido-DNJ dose-dependently increased intracellular GAA activity in Pompe patient fibroblasts with the M519V mutation and also promoted enzyme transport to lysosomes. This study provides the first example of a strategy to design high-affinity ligands by introducing alkyl branches into rare sugars and L-sugar-type iminosugars to change the orientation of binding.

Discovery of human hexosaminidase inhibitors by in situ screening of a library of mono- and divalent pyrrolidine iminosugars.

Two libraries of mono- and dimeric pyrrolidine iminosugars were synthesized by CuAAC and (thio)urea-bond-forming reactions from the respective azido/aminohexylpyrrolidine iminosugar precursors. The resulting monomeric and dimeric compounds were screened for inhibition of ß-N-acetylglucosaminidase from Jack beans, the plant ortholog of human lysosomal hexosaminidases. A selection of the best inhibitors of these libraries was then evaluated against human lysosomal ß-N-acetylhexosaminidase B (hHexB) and human nucleocytoplasmic ß-N-acetylglucosaminidase (hOGA). This evaluation identified a potent (nM) and selective monomeric inhibitor of hOGA (compound 7A) that showed a 6770-fold higher affinity for this enzyme than for hHexB. The corresponding dimeric derivative (compound 9D) further remarkably improved the selectivity in the inhibition of hOGA (2.7 × 104 times more selective for hOGA over hHexB) and the inhibition potency (by one order of magnitude). Docking studies were performed to explain the selectivity of inhibition observed in compound 7A.

Multivalent Pyrrolidine Iminosugars: Synthesis and Biological Relevance.

Recently, the strategy of multivalency has been widely employed to design glycosidase inhibitors, as glycomimetic clusters often induce marked enzyme inhibition relative to monovalent analogs. Polyhydroxylated pyrrolidines, one of the most studied classes of iminosugars, are an attractive moiety due to their potent and specific inhibition of glycosidases and glycosyltransferases, which are associated with many crucial biological processes. The development of multivalent pyrrolidine derivatives as glycosidase inhibitors has resulted in several promising compounds that stand out. Herein, we comprehensively summarized the different synthetic approaches to the preparation of multivalent pyrrolidine clusters, from total synthesis of divalent iminosugars to complex architectures bearing twelve pyrrolidine motifs. Enzyme inhibitory properties and multivalent effects of these synthesized iminosugars were further discussed, especially for some less studied therapeutically relevant enzymes. We envision that this comprehensive review will help extend the applications of multivalent pyrrolidine iminosugars in future studies.

Bambus[4,6]urils as Dual Scaffolds for Multivalent Iminosugar Presentation and Ion Transport: Access to Unprecedented Glycosidase-Directed Anion Caging Agents.

Bambusurils, BU[4] and BU[6], were used for the first time as multivalent scaffolds to link glycosidases inhibitors derived from 1-deoxynojirimycin (DNJ). Two linear DNJ ligands having six or nine carbon alkyl azido linkers or a trivalent DNJ dendron were grafted onto octapropargylated BU[4] and dodecapropargylated BU[6] using copper-catalyzed cycloaddition (CuAAC) to yield corresponding neoglycobambus[4] and neoglycobambus[6]urils bearing 8 to 24 iminosugars. The inhibition potencies of neoglycoBU[4], neoglycoBU[6] and neoglycoBU[6] caging anions were evaluated against Jack Bean α-mannosidase and compared to monovalent DNJ derivatives. Strong affinity enhancements per inhibitory head were obtained for the clusters holding trivalent dendrons with inhibitory constants in the nanomolar range (Ki = 24 nM for BU[4] with 24 DNJ units). Interestingly, the anion (bromide or iodide) encapsulated inside the cavity of BU[6] does not modify the inhibition potency of neoglycoBU[6], opening the way to water-soluble glycosidase-directed anion caging agents that may find applications in important fields such as bio(in)organic chemistry or oncology.

Synthesis and Evaluation of Novel Iminosugars Prepared from Natural Amino Acids.

Cyclopropanated iminosugars have a locked conformation that may enhance the inhibitory activity and selectivity against different glycosidases. We show the synthesis of new cyclopropane-containing piperidines bearing five stereogenic centers from natural amino acids l-serine and l-alanine. Those prepared from the latter amino acid may mimic l-fucose, a natural-occurring monosaccharide involved in many molecular recognition events. Final compounds prepared from l-serine bear S configurations on the C5 position. The synthesis involved a stereoselective cyclopropanation reaction of an α,ß-unsaturated piperidone, which was prepared through a ring-closing metathesis. The final compounds were tested as possible inhibitors of different glycosidases. The results, although, in general, with low inhibition activity, showed selectivity, depending on the compound and enzyme, and in some cases, an unexpected activity enhancement was observed.

Kinetic analysis of inhibition of α-glucosidase by leaf powder from Morus australis and its component iminosugars.

Mulberry leaves contain iminosugars, such as 1-deoxynojirimycin (1-DNJ), fagomine, and 2-O-α-D-galactopyranosyl deoxynojirimycin (GAL-DNJ) that inhibit α-glucosidase. In this study, we quantified iminosugars in Morus australis leaves and made the kinetic analysis in the hydrolysis of maltose by α-glucosidase. By LC-MS/MS, the concentrations of 1-DNJ, fagomine, and GAL-DNJ in the powdered leaves were 4.0, 0.46, and 2.5 mg/g, respectively, and those in the roasted ones were 1.0, 0.24, and 0.73 mg/g, respectively, suggesting that the roasting process degraded iminosugars. Steady-state kinetic analysis revealed that the powdered and roasted leaves exhibited competitive inhibition. At pH 6.0 at 37ºC, the IC50 values of the extracts from the boiled powdered or roasted leaves were 0.36 and 1.1 mg/mL, respectively. At the same condition, the IC50 values of 1-DNJ, fagomine, and GAL-DNJ were 0.70 µg/mL, 0.18 mg/mL, and 2.9 mg/mL, respectively. These results suggested that in M. australis, 1-DNJ is a major inhibitor of α-glucosidase. ABBREVIATIONS: 1-DNJ: 1-deoxynojirimycin; GAL-DNJ: 2-O-α-D-galactopyranosyl-DNJ.

Toward the Identification of Novel Antimicrobial Agents: One-Pot Synthesis of Lipophilic Conjugates of N-Alkyl d- and l-Iminosugars.

In the effort to improve the antimicrobial activity of iminosugars, we report the synthesis of lipophilic iminosugars 10a-b and 11a-b based on the one-pot conjugation of both enantiomeric forms of N-butyldeoxynojirimycin (NBDNJ) and N-nonyloxypentyldeoxynojirimycin (NPDNJ) with cholesterol and a succinic acid model linker. The conjugation reaction was tuned using the established PS-TPP/I2/ImH activating system, which provided the desired compounds in high yields (94-96%) by a one-pot procedure. The substantial increase in the lipophilicity of 10a-b and 11a-b is supposed to improve internalization within the bacterial cell, thereby potentially leading to enhanced antimicrobial properties. However, assays are currently hampered by solubility problems; therefore, alternative administration strategies will need to be devised.

Structural basis of the inhibition of GH1 ß-glucosidases by multivalent pyrrolidine iminosugars.

The synthesis of multivalent pyrrolidine iminosugars via CuAAC click reaction between different pyrrolidine-azide derivatives and tri- or hexavalent alkynyl scaffolds is reported. The new multimeric compounds, together with the monomeric reference, were evaluated as inhibitors against two homologous GH1 ß-glucosidases (BglA and BglB from Paenibacillus polymyxa). The multivalent inhibitors containing an aromatic moiety in the linker between the pyrrolidine and the scaffold inhibited the octameric BglA (µM range) but did not show affinity against the monomeric BglB, despite the similarity between the active site of both enzymes. A modest multivalent effect (rp/n = 12) was detected for the hexavalent inhibitor 12. Structural analysis of the complexes between the monomeric and the trimeric iminosugar inhibitors (4 and 10) and BglA showed the insertion of the inhibitors at the active site of BglA, confirming a competitive mode of inhibition as indicated by enzyme kinetics. Additionally, structural comparison of the BglA/4 complex with the reported BglB/2F-glucose complex illustrates the key determinants responsible for the inhibitory effect and explains the reasons of the inhibition of BglA and the no inhibition of BglB. Potential inhibition of other ß-glucosidases with therapeutic relevance is discussed under the light of these observations.

Exploring substituent diversity on pyrrolidine-aryltriazole iminosugars: Structural basis of ß-glucocerebrosidase inhibition.

The synthesis of a library of pyrrolidine-aryltriazole hybrids through CuAAC between two epimeric dihydroxylated azidomethylpyrrolidines and differently substituted phenylacetylenes is reported. The evaluation of the new compounds as inhibitors of lysosomal ß-glucocerebrosidase showed the importance of the substitution pattern of the phenyl moiety in the inhibition. Crystallization and docking studies revealed key interactions of the pyrrolidine motif with aminoacid residues of the catalytic site while the aryltriazole moiety extended along a hydrophobic surface groove. Some of these compounds were able to increase the enzyme activity in Gaucher patient fibroblasts, acting as a new type of chemical chaperone for Gaucher disease.

Dual targeting of PTP1B and glucosidases with new bifunctional iminosugar inhibitors to address type 2 diabetes.

The diffusion of type 2 diabetes (T2D) throughout the world represents one of the most important health problems of this century. Patients suffering from this disease can currently be treated with numerous oral anti-hyperglycaemic drugs, but none is capable of reproducing the physiological action of insulin and, in several cases, they induce severe side effects. Developing new anti-diabetic drugs remains one of the most urgent challenges of the pharmaceutical industry. Multi-target drugs could offer new therapeutic opportunities for the treatment of T2D, and the reported data on type 2 diabetic mice models indicate that these drugs could be more effective and have fewer side effects than mono-target drugs. α-Glucosidases and Protein Tyrosine Phosphatase 1B (PTP1B) are considered important targets for the treatment of T2D: the first digest oligo- and disaccharides in the gut, while the latter regulates the insulin-signaling pathway. With the aim of generating new drugs able to target both enzymes, we synthesized a series of bifunctional compounds bearing both a nitro aromatic group and an iminosugar moiety. The results of tests carried out both in vitro and in a cell-based model, show that these bifunctional compounds maintain activity on both target enzymes and, more importantly, show a good insulin-mimetic activity, increasing phosphorylation levels of Akt in the absence of insulin stimulation. These compounds could be used to develop a new generation of anti-hyperglycemic drugs useful for the treatment of patients affected by T2D.

Synthesis of pentacyclic iminosugars with constrained butterfly-like conformation and their HIV-RT inhibitory activity.

Novel pentacyclic iminosugars 1 and 2 with the constrained butterfly-like conformation were first synthesized by the key intramolecular click reaction from the tricyclic iminosugars fused benzo[e][1,3]thiazin-4-one 3 and 4. The pentacyclic iminosugar was constructed by fusing both benzo[e][1,3]thiazin-4-one and triazolo[5,1-c][1,4]oxazepine scaffolds. Their structures were determined by their 1H, 13C NMR, and HRMS (ESI) spectra and X-ray. The pentacyclic iminosugars 1(a-c), 2(a-b) and their corresponding protected precursors 13(a-c) and 14(a-b) were examined for their HIV reverse transcriptase (RT) inhibitory activities. The result showed that all compounds could effectively inhibit RT activity. Among them, compound 13c was the best one with the IC50 value of RT inhibitory activity of 0.69 µM. Structure-activity relationship analysis suggested that the improvement of the hydrophilicity of the pentacycles was of benefit to their anti-HIV RT activity.

A Fluorescence Polarization Activity-Based Protein Profiling Assay in the Discovery of Potent, Selective Inhibitors for Human Nonlysosomal Glucosylceramidase.

Human nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that controls levels of glycolipids and whose activity is linked to several human disease states. There is a major need to design or discover selective GBA2 inhibitors both as chemical tools and as potential therapeutic agents. Here, we describe the development of a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) assay for the rapid identification, from a 350+ library of iminosugars, of GBA2 inhibitors. A focused library is generated based on leads from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset against the other glucosylceramide metabolizing enzymes, glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic retaining ß-glucosidase, GBA3. Our work, yielding potent and selective GBA2 inhibitors, also provides a roadmap for the development of high-throughput assays for identifying retaining glycosidase inhibitors by FluoPol-ABPP on cell extracts containing recombinant, overexpressed glycosidase as the easily accessible enzyme source.

Iminosugar antivirals: the therapeutic sweet spot.

Many viruses require the host endoplasmic reticulum protein-folding machinery in order to correctly fold one or more of their glycoproteins. Iminosugars with glucose stereochemistry target the glucosidases which are key for entry into the glycoprotein folding cycle. Viral glycoproteins are thus prevented from interacting with the protein-folding machinery leading to misfolding and an antiviral effect against a wide range of different viral families. As iminosugars target host enzymes, they should be refractory to mutations in the virus. Iminosugars therefore have great potential for development as broad-spectrum antiviral therapeutics. We outline the mechanism giving rise to the antiviral activity of iminosugars, the current progress in the development of iminosugar antivirals and future prospects for this field.

Multivalency To Inhibit and Discriminate Hexosaminidases.

A set of multivalent polyhydroxylated acetamidoazepanes based on ethylene glycol, glucoside, or cyclodextrin scaffolds was prepared. The compounds were assessed against plant, mammalian, and therapeutically relevant hexosaminidases. Multimerization was shown to improve the inhibitory potency with synergy, and to fine tune the selectivity profile between related hexosaminidases.

Polyhydroxylated Quinolizidine Iminosugars as Nanomolar Selective Inhibitors of α-Glucosidases.

Polyhydroxylated quinolizidines bearing a hydroxymethyl group at the ring junction were synthesized from a readily available l-sorbose-derived ketonitrone. Evaluated as glycoside hydrolase inhibitors, these quinolizidines revealed to be potent and selective α-glucosidase inhibitors. Quinolizidine 9a is the first quinolizidine-scaffolded iminosugar exhibiting nanomolar inhibition of a glycoenzyme.

Polyhydroxylated azetidine iminosugars: Synthesis, glycosidase inhibitory activity and molecular docking studies.

An efficient and practical strategy for the synthesis of unknown azetidine iminosugars (2S,3R,4S)-2-((R)-1,2-dihydroxyethyl)-3-hydroxy-4-(hydroxymethyl)azetidine 2, (2S,3r,4R)-3-hydroxy-2,4-bis(hydroxymethyl)azetidine 3 and (2S,3R,4S)-3-hydroxy-4-(hydroxymethyl)-N-methylazetidine-2-carboxylic acid 4, starting from the d-glucose has been reported. The methodology involves preparation of the 3-amino-N-benzyloxycarbonyl-3-deoxy-6-O-tert-butyldimethylsillyl-1,2-O-isopropylidene-α-d-glucofuranose 9, which was converted to the C-5-OMs derivative 11. Intramolecular nucleophilic displacement of the C-5-OMs group with in situ generated 3-amino functionality provided the required key azetidine ring skeletons 10 with additional hydroxymethyl group. Removal of 1,2-acetonide protection, followed by reduction and hydrogenolysis afforded azetidine iminosugar 2. Alternatively, removal of 1,2-acetonide group and chopping of C1-anomeric carbon gave C2-aldehyde that on reduction or oxidation followed by hydrogenolysis gave 2,4-bis(hydroxymethyl) azetidine iminosugars 3 and N-methylazetidine-2-carboxylic acid 4 respectively. The glycosidase inhibitory activity of 2-4 iminosugars was screened against various glycosidase enzymes and compared with a standard miglitol. Amongst synthesized targets, the compound 2 was found to be more potent amyloglucosidase inhibitor than miglitol. These results were supported by molecular docking studies.