Glycosidase Inhibitors Research Articles

Synthesis of Halogenated-4-Nitrophenyl 2-deoxy-2-halogeno-pyranosides viaN-Halogenosuccinimide Activated Glucal

Reaction of 3,4,6-tri- O -acetyl-D-glucal with silver 4-nitrophenolate in the presence of N -iodosuccinimide and N -bromosuccinimide produced (2,6-dihalo-4-nitro)phenyl 2-halo-2-deoxy-α-D-glycopyranosides. Although bromination and iodination of the 4-nitrophenyl group could not be avoided, the resulting (2,6-dihalo-4-nitro)phenylated compounds can be used as substrates or covalent glycosidase inhibitors after deprotection. The stereoselectivity and regioselectivity of the halogenation reactions are described.

ToP-DNJ, a Selective Inhibitor of Endoplasmic Reticulum α-Glucosidase II Exhibiting Antiflaviviral Activity.

Iminosugars have therapeutic potential against a range of diseases, due to their efficacy as glycosidase inhibitors. A major challenge in the development of iminosugar drugs lies in making a compound that is selective for the glycosidase associated with a given disease. We report the synthesis of ToP-DNJ, an antiviral iminosugar–tocopherol conjugate. Tocopherol was incorporated into the design of the iminosugar in order to direct the drug to the liver and immune cells, specific tissues of interest for antiviral therapy. ToP-DNJ inhibits ER α-glucosidase II at low micromolar concentrations and selectively accumulates in the liver in vivo. In cellular assays, the drug showed efficacy exclusively in immune cells of the myeloid lineage. Taken together, these data demonstrate that inclusion of a native metabolite into an iminosugar provides selectivity with respect to target enzyme, target cell, and target tissue.

N-Butyl-l-deoxynojirimycin (l-NBDNJ): Synthesis of an Allosteric Enhancer of α-Glucosidase Activity for the Treatment of Pompe Disease.

The highly stereocontrolled de novo synthesis of l-NBDNJ (the unnatural enantiomer of the iminosugar drug Miglustat) and a preliminary evaluation of its chaperoning potential are herein reported. l-NBDNJ is able to enhance lysosomal α-glucosidase levels in Pompe disease fibroblasts, either when administered singularly or when coincubated with the recombinant human α-glucosidase. In addition, differently from its d-enantiomer, l-NBDNJ does not act as a glycosidase inhibitor.

Towards Tuneable Retaining Glycosidase-Inhibiting Peptides by Mimicry of a Plant Flavonol Warhead.

Retaining glycosidases are an important class of enzymes involved in glycan degradation. To study better the role of specific enzymes in deglycosylation processes, and thereby the importance of particular glycosylation patterns, a set of potent inhibitors, each specific to a particular glycosidase, would be an invaluable toolkit. Towards this goal, we detail here a more in-depth study of a prototypical macrocyclic peptide inhibitor of the model retaining glycosidase human pancreatic α-amylase (HPA). Notably, incorporation of l-DOPA into this peptide affords an inhibitor of HPA with potency that is tenfold higher (Ki =480 pm) than that of the previously found consensus sequence. This represents a first successful step in converting a recently discovered natural-product-derived motif, already specific for the catalytic side-chain arrangement conserved in the active sites of retaining glycosidases, into a tuneable retaining glycosidase inhibition warhead.

Covalent Probes for Carbohydrate-Active Enzymes: From Glycosidases to Glycosyltransferases.

Covalent probes for glycosidases and glycosyltransferases are of great interest as tool compounds for chemical biology. For glycosidases, a sizable number of such probes have been developed from covalent glycosidase inhibitors. We review selected recent examples and highlight different design strategies, including probes based on photoaffinity labels and mechanism-based inhibitors, as well as their applications in biology and for activity-based protein profiling. In contrast to glycosidases, only a limited number of covalent probes have been reported to date for glycosyltransferases. We describe a new class of covalent probes for the retaining α-1,4-galactosyltransferase LgtC from Neisseria meningitidis. On the basis of these probes, we have developed an operationally simple two-step protocol for the fluorescent labeling of recombinant LgtC both in purified form and in cell lysates. In principle, our approach is also applicable to other bacterial glycosyltransferases. Among other applications, our protocol may therefore be particularly useful for imaging of the differential expression of these enzymes in different bacterial species and strains.

Magnetic Nanoparticles Coated with Thiomannosides or Iminosugars to Switch and Recycle Galactosidase Activity

Glycosidase effectors have rarely been reported despite their great potential interest in pharmaceutical sciences and industry. Magnetic nanoparticles were coated with thiomannosides (SMan@Fe3O4) or the broad spectrum glycosidase inhibitor deoxynojirimycin (DNJ@Fe3O4). The coated ligands were shown to exert a fully reverse effect on a model galactosidase (AgaB), with SMan@Fe3O4 or DNJ@Fe3O4 ligands acting as an enzyme inhibitor (Ki=3.7 μM) or a strong activator (250% higher AgaB velocity at 50 μM), respectively. This is striking considering that monovalent soluble SMan and DNJ analogues do not interact with AgaB at millimolar concentrations. The AgaB-DNJ@Fe3O4 enzyme-effector complex could be magnetically recycled and still showed a higher activity compared to free AgaB after four catalytic cycles. The “boost and recycle” procedure may provide interesting perspectives in glycosidase biocatalysis.

A quantitative analytical method for valienone and its application in the evaluation of valienone production by a breakthrough microbial process

AbstractValienone is a significant natural carbasugar member of the C7-cyclitol family as a valuable precursor for glycosidase inhibitor drugs. It is an intermediate of validamycin A biosynthesis pathway and exhibits minimal accumulation in the fermentation broth of the natural Streptomyces producer. A quantitative analytical method is crucial for the development of a breakthrough microbial process overcoming the consumption of the natural metabolic flux. The present study was designed to develop a pre-column derivatization high-performance liquid chromatography method for quantification of valienone and to help establish a straightforward fermentation process for valienone production by metabolically engineered Streptomyces hygroscopicus 5008. Valienone was derivatized by 2, 4-dinitrophenylhydrazine (DNPH) in 10 mmol·L−1 H3PO4 at 37 °C for 45 min and the derivatives were separated on Eclipse XDB-C18 (5 μm, 4.6 mm × 150 mm) column at 30 °C eluted with 50% acetonitrile for 18 min. The derivatives were detected by diode array detector at 380 nm and the configurations of the derivatives were determined by computational studies. The method was shown to be effective, sensitive, and reliable. Good linearity was found in the range of 5–2 000 μg·mL−1. The intra- and inter-day precisions were 1.1%–2.7% and 1.7%–2.2%, respectively. The absolute recovery of the spiked samples was 97.2%–102.6%. To date, this is the first reversed-phase high-performance liquid chromatography detection method for valienone in microbial culture medium. This method successfully helped evaluate the valienone production capability of the engineered Streptomyces hygroscopicus 5008 and could be promising for C7-cyclitol profiling of different engineered mutants combined with the metabonomics methods.

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 β-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.

Glycosidase Inhibition by Multivalent Presentation of Heparan Sulfate Saccharides on Bottlebrush Polymers.

We report herein the first-time exploration of the attachment of well-defined saccharide units onto a synthetic polymer backbone for the inhibition of a glycosidase. More specifically, glycopolymers endowed with heparan sulfate (HS) disaccharides were established to inhibit the glycosidase, heparanase, with an IC50 value in the low nanomolar range (1.05 ± 0.02 nm), a thousand-fold amplification over its monovalent counterpart. The monomeric moieties of these glycopolymers were designed in silico to manipulate the well-established glycotope of heparanase into an inhitope. Studies concluded that (1) the glycopolymers are hydrolytic stable toward heparanase, (2) longer polymer length provides greater inhibition, and (3) increased local saccharide density (monoantennary vs diantennary) is negligible due to hindered active site of heparanase. Furthermore, HS oligosaccharide and polysaccharide controls illustrate the enhanced potency of a multivalent scaffold. Overall, the results on these studies of the multivalent presentation of saccharides on bottlebrush polymers serve as the platform for the design of potent glycosidase inhibitors and have potential to be applied to other HS-degrading proteins.

IN VITRO ANTIOXIDANT CAPACITIES AND ANTIDIABETIC PROPERTIES OF PLUCHEA LEAVES AND GREEN TEA MIXTURES AT VARIOUS PROPORTIONS

Objective: This study was done to determine the effect of pluchea leaves and green tea mixtures at various proportions to antioxidant and antidiabetic properties.Methods: The research used a single factor randomized block design. Research factors were pluchea and green tea mixture proportions, including 100:0; 75:25; 50:50; 25:75; and 0:100% (w/w), respectively. Each of factors was repeated five times. The parameters observed in this study were phytochemical compounds, total phenols, total flavonoids, free radical DPPH scavenging activity, iron ion reducing power, alpha amylase and alpha glycosidase inhibition activities. The data were statistically α = 5%, if the data analysis showed a significant analyzed using analysis of variance (ANOVA) at effect then it was continued with duncan's multiple range test (DMRT).Results: The phytochemical compounds containing in pluchea leaves and green tea mixtures were alkaloids, flavonoids, phenolics, saponins, tannins, triterpenoids, sterols, and cardiac glycosides. The qualitative assay of phytochemical compounds from aqueous extract had a different pattern with total phenolic content (TPC) and total flavonoid content (TFC), especially at 75:25; 50:50 and 25:75 % (w/w) from pluchea leaves and green tea mixture proportions. The 50:50% (w/w) proportions of samples had the highest TPC and TFC, it was proposed that there was an interaction between bioactive compounds of pluchea leaves and green tea. TPC and TFC were correlated with free radical DPPH scavenging activity, iron reducing power and alpha glycosidase inhibition activity, but the alpha amylase inhibition activity had the different graph pattern with the TPC and TFC.Conclusion: The antioxidant capacity of pluchea leaves and green tea mixtures at various proportions had the graph pattern similar to the alpha glycosidase inhibitory activity and different with the alpha amylase inhibitory activity.

α-Geminal disubstituted pyrrolidine iminosugars and their C-4-fluoro analogues: Synthesis, glycosidase inhibition and molecular docking studies

A simple strategy for the synthesis of α-geminal disubstituted pyrrolidine iminosugars 3a-c and their C-4 fluorinated derivatives 4a-c has been described from d-glucose. The methodology involves the Corey-Link and Jocic-Reeve reaction with 3-oxo-α-d-glucofuranose and nucleophilic displacement reaction to get the furanose fused pyrrolidine ring skeleton with requisite CH2OH/CO2H functionalities at C-3. The fluorine substituent in target molecules was introduced by nucleophilic displacement of -OTf in 9a/9c with CsF. Appropriate manipulation of the anomeric carbon in the furanose fused pyrrolidine ring skeleton afforded α-geminal disubstituted pyrrolidine iminosugars 3a–c and C-4 fluoro derivatives 4a–c. The pyrrolidine iminosugars 3a and 3c were found to be potent inhibitors of α-galactosidase while, the fluoro derivatives 4a and 4b showed strong inhibition of β-glucosidase and β-galactosidase, respectively. These results were substantiated by in silico molecular docking studies.

1,6-Cyclophellitol Cyclosulfates: A New Class of Irreversible Glycosidase Inhibitor

The essential biological roles played by glycosidases, coupled to the diverse therapeutic benefits of pharmacologically targeting these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivalents of epoxides in organic synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors. Here we present the synthesis, conformational analysis, and application of novel 1,6-cyclophellitol cyclosulfates. We show that 1,6-epi-cyclophellitol cyclosulfate (α-cyclosulfate) is a rapidly reacting α-glucosidase inhibitor whose 4C1 chair conformation matches that adopted by α-glucosidase Michaelis complexes. The 1,6-cyclophellitol cyclosulfate (β-cyclosulfate) reacts more slowly, likely reflecting its conformational restrictions. Selective glycosidase inhibitors are invaluable as mechanistic probes and therapeutic agents, and we propose cyclophellitol cyclosulfates as a valuable new class of carbohydrate mimetics for application in these directions.

Synthesis and glycosidase inhibition of C-7 modified casuarine derivatives

A series of C-7 modified analogues of casuarine have been synthesized from sugar-derived nitrone and assayed against various glycosidases. Introduction of C-7 aminomethyl or amide group led to sharp decrease of the inhibitory activities.

Alkoxyallene-Based Stereodivergent Syntheses of (-)-Hyacinthacine B4 and of Putative Hyacinthacine C5 Epimers: Proposal of Hyacinthacine C5 Structure.

Hyacinthacines are members of the class of polyhydroxylated pyrrolizidines exhibiting outstanding biological activity as glycosidases inhibitors. Their structural complexity is embodied in the densely functionalized core, possessing a series of contiguous stereogenic centers. In this synthetic study we report a route to the more complex congeners of this class of alkaloids exploiting the diastereoselective addition of an axially chiral lithiated alkoxyallene to an enantiopure cyclic nitrone. Our stereodivergent approach enabled the installation of the targeted configuration at the ring A by minimal synthetic manipulations and at ring B by using stage dependent selective functionalizations. The versatility and robustness of this methodology were demonstrated by the syntheses of (-)-hyacinthacine B4 and of two epimers of (+)-hyacinthacine C5, allowing a suggestion of the likely structure of the isolated natural product.

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

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

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.

Metabolite profiling, antioxidant activity, and glycosidase inhibition property of the mesocarp tissue extracts of sugar date palm [Phoenix sylvestris (L.) Roxb.] fruits

ABSTRACTPhoenix sylvestris is an underutilized seasonal fruit in West Bengal, India. Methanol extract and extracts after alkaline hydrolysis of the mesocarp tissue of full-mature edible fruits of P. sylvestris were analyzed by GC-MS following a metabolomics approach. The fractions were tested for their antioxidant and inhibitory properties against the two key enzymes involved in diabetes, α-amylase, and α-glucosidase. Total 71 metabolites belonging to organic acids, amino acids, sugars, sugar alcohols, fatty acids, and phenols were identified in the methanol extract and in fractions after saponification. All the extracts and fractions showed high antioxidant, α-glucosidase, and α-amylase inhibitory activities. Sugars like raffinose (IC50 = 0.36 μM), sucrose (IC50 = 0.51 μM), trehalose (IC50 = 0.85 μM), and phenols like taxifolin (IC50 = 0.31 μM), benzoic acid (IC50 = 2.74 μM) inhibited only the enzyme α-amylase. Phenolic components which inhibited both the enzymes were caffeic acid (IC50 = 1.42 μM for α-amylase and IC50 = 1.8 μM for α-glucosidase), 3, 4-dihydroxy benzoic acid (IC50 = 0.23 μM for α-amylase and IC50 = 2.58 μM for α-glucosidase), and quinic acid (IC50 = 4.91 μM for α-amylase and IC50 = 4.95 μM for α-glucosidase). Ferulic acid (IC50 = 0.52 μM) and 4-hydroxycinnamic acid (IC50 = 0.23 μM) inhibited only α-glucosidase. This study suggested that the metabolites present in the fruit mesocarp tissue showed the potential antioxidant activity and properties to inhibit the enzymes α-amylase and α-glucosidase. Further in vivo study is to be carried out to prove the efficacy of the fruits.Abbreviations: FM: crude methanol extract; SI: ethyl acetate extract after alkaline hydrolysis step I; SII: ethyl acetate extract after alkaline hydrolysis step II

A new pre-column derivatization for valienamine and beta-valienamine using o-phthalaldehyde to determine the epimeric purity by HPLC and application of this method to monitor enzymatic catalyzed synthesis of beta-valienamine

Valienamine and β-valienamine are representative C7 N aminocyclitols with significant glycosidase inhibition activity that have been developed as important precursors of drugs for diabetes and lysosomal storage diseases, respectively. The quantitative analysis of these chiral compounds is crucial for asymmetric in vitro biosynthetic processes for converting valienone into valienamine epimers using aminotransferase. Here, we developed an efficient and sensitive method for separation and quantitative analysis of chiral valienamine using reversed-phase high-performance liquid chromatography (HPLC) through o-phthalaldehyde (OPA) pre-column derivatization of the analytes. The epimers were derivatized by OPA in borate buffer (pH 9.0) at room temperature for 30 s, separated on an Eclipse XDB-C18 (5 μm, 4.6 × 150 mm) column, eluted with 22% acetonitrile at 30 °C for 18 min, and detected by a fluorescence detector using 445 nm emission and 340 nm excitation wavelengths. The average resolution of the epimers is 3.86, and the concentration linearity is in the range of 0.02–20 μg/ml. The method proved to be effective, sensitive, and reliable with good intra- and inter-day precision and accuracy, and successfully evaluated the enantiopreference and catalytic capability of the potential aminotransferases on an unnatural prochiral substrate, facilitating the design of an asymmetric biosynthetic route for optically pure valienamine and β-valienamine.

A Morita-Baylis-Hillman based route to C-5a-chain-extended 4-epi-isofagomine type glycosidase inhibitors

By Morita-Baylis-Hillman reaction of 2,3-O-isopropylidene-D-glyceraldehyde with α,β-unsaturated carbonyl as well as hetero analogous carbonyl compounds such as acrylonitrile, suitable precursors of isofagomine and of 4-epi-isofagomine are available. Elaboration of the structures by amine introduction, followed by intramolecular ring closure and subsequent hydroboration of the double bond provides 4-epi-isofagomine derivatives featuring chain extensions at C-5a which are determined by the structures of the carbonyl compounds employed. As an example, the synthesis of C-(5aR)- and C-(5aS)-5a-C-pentyl-4-epi-isofagomines, powerful inhibitors of β-galactosidases, is outlined. In line with reported data, the (C-5aR) epimer was found a highly potent experimental pharmacological chaperone for GM1-associated human lysosomal β-galactosidase mutant R201C.

Evaluation of 6‐Deoxy–amino–sugars as Potent Glycosidase Inhibitors. Importance of the CH2OH(6) Group for Enzyme‐Substrate Interaction

AbstractOptically active or racemic 6‐deoxy‐analogues of the nojirimycine d‐1 a in allose, glucose, fucose, talose and gulose series were evaluated as glycosidase inhibitors. These compounds were analysed either as their sulfite adducts or as the “true” amino‐sugars (the exact copies of aldoses with intracyclic oxygen replaced by nitrogen) or as their reduced 1‐deoxy‐form depending on the series. Some compounds are potent inhibitors of α‐ and β‐d‐glucosidases and α‐ and β‐d‐galactosidases. Racemic compounds in talose and gulose series were also micromolar inhibitors of α‐l‐fucosidase and β‐d‐galactosidase, while the talose analogues were active against β‐d‐mannosidase. Comparison of these inhibition data with those of amino‐hexoses possessing the CH2OH group permitted to evaluate the implication of the C(6)‐OH in enzyme‐inhibitor interactions, which seem to occur particularly with α‐d‐glucosidase (yeast) and α‐d‐galactosidase (green coffee beans). The cases of the β‐d‐glucosidase and β‐d‐galactosidase were discussed.