[Show abstract][Hide abstract]ABSTRACT: β-glucocerebrosidase deficiency leads to Gaucher disease and is a potential marker of Parkinson’s disease. We have have identified N-octyl conduritol aziridine as a potent and specific covalent inactivator of GBA1 in living cells. This compound is a promising lead towards a positron emission tomography probe intended to image GBA1 activity.
Full-text available · Article · Jun 2015 · Chemical Communications
[Show abstract][Hide abstract]ABSTRACT: A surface modification which renders a carbon electrode sensitive to β-glucosidase enzyme activity is reported. 2-Nitro-4-(4-diazophenoxymethyl)-phenyl β-D-glucopyranoside was prepared in four steps from the known starting material 2-nitro-4-benzyloxy 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside. A film was formed by the electrochemical deposition of the aromatic diazonium salt 2-nitro-4-(4-diazoophenoxymethyl)-phenyl β-D-glucopyranoside onto a glassy carbon electrode surface, as judged by the change in the oxidation potential for the test analyte ascorbic acid in the cyclic voltammogram (CV). This carbohydrate derivative incorporated a self-immolative linker to physically distance the sugar from the electrode surface and make it more accessible to the active site of the enzyme. A +0.341 V change in the oxidation potential for ascorbic acid was observed in the CV for a modified electrode following treatment with the retaining β-glucosidase from Agrobacterium sp. (Abg). Deposition of either 2-nitro-4-diazophenyl-β-D-glucopyranoside or 4-diazophenyl-β-D-glucopyranoside, both of which lacked the self-immolative linker, produced electrode surfaces that were relatively insensitive to Abg, demonstrating the importance of the distance from the electrode surface provided by the linker. This work represents an important step towards the development of a sensor that could be used to detect glycosidase activity.
Article · Nov 2014 · International journal of electrochemical science
[Show abstract][Hide abstract]ABSTRACT: 2-Deoxy-2-fluoroglycosides bearing dibenzyl phosphate and phosphonate aglycones were synthesised and tested as covalent inactivators of several retaining α- and β-glycosidases. β-d-Gluco-, -manno- and -galacto-configured benzyl-benzylphosphonate derivatives efficiently inactivated β-gluco-, β-manno- and β-galactosidases, while α-gluco- and α-manno-configured phosphate and phosphonate derivatives served instead as slow substrates.
Article · Mar 2014 · Organic & Biomolecular Chemistry
[Show abstract][Hide abstract]ABSTRACT: Chaperoning an enzyme: Fluorosugar glycosidase inactivators with tunable phosphorus-based leaving groups react quickly with the catalytic nucleophile in β glucocerebrosidase (blue circle; Bn=benzyl). In Western blot analysis, Gaucher patient cells treated with these inactivators show increased intracellular levels of mutant enzyme, presumably because of increased transit from the endoplasmic reticulum (pale blue) to the lysosome (pale pink).
Article · Oct 2011 · Angewandte Chemie International Edition
[Show abstract][Hide abstract]ABSTRACT: The title compound, which differs from the powerful O-GlcNAcase (OGA) inhibitor GlcNAc-thiazoline only at the chalcogen atom (Se for S), is a much weaker inhibitor in a direct OGA assay. In human cells, however, the selenazoline shows comparable ability to induce hyper-O-GlcNAc-ylation, and the two show similar reduction of insulin-stimulated translocation of glucose transporter 4 in differentiated 3T3 adipocytes.
Article · Oct 2010 · Bioorganic & medicinal chemistry
[Show abstract][Hide abstract]ABSTRACT: Direct enzyme replacement therapy (ERT) has been introduced as a means to treat a number of rare, complex genetic conditions associated with lysosomal dysfunction. Gaucher disease was the first for which this therapy was applied and remains the prototypical example. Although ERT using recombinant lysosomal enzymes has been shown to be effective in altering the clinical course of Gaucher disease, Fabry disease, Hurler syndrome, Hunter syndrome, Maroteaux-Lamy syndrome, and Pompe disease, the recalcitrance of certain disease manifestations underscores important unanswered questions related to dosing regimes, tissue half-life of the recombinant enzyme and the ability of intravenously administered enzyme to reach critical sites of known disease pathology. We have developed an innovative method for tagging acid beta-glucocerebrosidase (GCase), the recombinant enzyme formulated in Cerezyme(R) used to treat Gaucher disease, using an (18)F-labeled substrate analogue that becomes trapped within the active site of the enzyme. Using micro-PET we show that the tissue distribution of injected enzyme can be imaged in a murine model and that the PET data correlate with tissue (18)F counts. Further we show that PET imaging readily monitors pharmacokinetic changes effected by receptor blocking. The ability to (18)F-label GCase to monitor the enzyme distribution and tissue half-life in vivo by PET provides a powerful research tool with an immediate clinical application to Gaucher disease and a clear path for application to other ERTs.
Full-text available · Article · Jun 2010 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract]ABSTRACT: While covalent catalytic intermediates of retaining alpha-transglycosylases have been structurally characterized previously, no such information for a hydrolytic alpha-amylase has been obtained. This study presents a new "in situ" enzymatic elongation methodology that, for the first time, has allowed the isolation and structural characterization of a catalytically competent covalent glycosyl-enzyme intermediate with human pancreatic alpha-amylase. This has been achieved by the use of a 5-fluoro-beta-l-idosyl fluoride "warhead" in conjunction with either alpha-maltotriosyl fluoride or 4'-O-methyl-alpha-maltosyl fluoride as elongation agents. This generates an oligosaccharyl-5-fluoroglycosyl fluoride that then reacts with the free enzyme. The resultant covalent intermediates are extremely stable, with hydrolytic half-lives on the order of 240 h for the trisaccharide complex. In the presence of maltose, however, they undergo turnover via transglycosylation according to a half-life of less than 1 h. Structural studies of intermediate complexes unambiguously show the covalent attachment of a 5-fluoro-alpha-l-idosyl moiety in the chair conformation to the side chain of the catalytic nucleophile D197. The elongated portions of the intermediate complexes are found to bind in the high-affinity -2 and -3 binding subsites, forming extensive hydrogen-bonding interactions. Comparative structural analyses with the related noncovalent complex formed by acarbose highlight the structural rigidity of the enzyme surface during catalysis and the key role that substrate conformational flexibility must play in this process. Taken together, the structural data provide atomic details of several key catalytic steps. The scope of this elongation approach to probe the active sites and catalytic mechanisms of alpha-amylases is further demonstrated through preliminary experiments with porcine pancreatic alpha-amylase.
[Show abstract][Hide abstract]ABSTRACT: Four new difluorosugar fluorides, 2-deoxy-2,5-difluoro - l-idopyranosyl fluoride, 1,5-difluoro-d-glucopyranosyl fluoride, 1,5-difluoro-l-idopyranosyl fluoride, and 2-deoxy-1,2-difluoro-d-glucopyranosyl fluoride, were synthesized from known precursors by a radical bromination/fluoride displacement sequence, followed by deprotection. The compounds were tested as time-dependent inactivators of the -glucosidase from Agrobacterium sp. (Abg, EC 184.108.40.206) and, while they were shown to bind to the enzyme active site as reversible competitive inhibitors, the only time-dependent inactivation observed was traced to the presence of an extremely small amount (<0.1%) of a highly reactive contaminating impurity.
Article · Jan 2009 · Australian Journal of Chemistry
[Show abstract][Hide abstract]ABSTRACT: Glycoside hydrolases are important enzymes in a number of essential biological processes. Irreversible inhibitors of this
class of enzyme have attracted interest as probes of both structure and function. In this review we discuss some of the compounds
used to covalently modify glycosidases, their use in residue identification, structural and mechanistic investigations, and
finally their applications, both in vitro and in vivo, to complex biological systems.
Full-text available · Article · Sep 2008 · Glycobiology
[Show abstract][Hide abstract]ABSTRACT: Several C-1 homologated GlcNAc- and GalNAc-thiazolines, as well as a related GalNAc-thiazole, have been prepared. The compounds are analogues of GlcNAc-thiazoline, a potent transition-state-mimicking inhibitor of retaining beta-N-acetylglycosaminidases. Kinetic evaluation of these fused pyranose-heterocycles against the bacterial N-acetylhexosaminidase SpHex suggests active site steric restrictions around the substrate anomeric carbon.
Article · Jun 2008 · Bioorganic & medicinal chemistry letters
[Show abstract][Hide abstract]ABSTRACT: The known glucaro-1,5-lactam 8, its diastereoisomers 9–11, and the tetrahydrotetrazolopyridine-5-carboxylates 12–14 were synthesised as potential inhibitors of β-D-glucuronidases and α-L-iduronidases. The known 2,3-di-O-benzyl-4,6-O-benzylidene-D-galactose (16) was transformed into the D-galactaro- and L-altraro-1,5-lactams 9 and 11via the galactono-1,5-lactam 21 in twelve steps and in an overall yield of 13 and 2%, respectively. A divergent strategy, starting from the known tartaric anhydride 41, led to the D-glucaro-1,5-lactam 8, D-galactaro-1,5-lactam 9, L-idaro-1,5-lactam 10, and L-altraro-1,5-lactam 11 in ten steps and in an overall yield of 4–20%. The anhydride 41 was transformed into the L-threuronate 46. Olefination of 46 to the (E)- or (Z)-alkene 47 or 48 followed by reagent- or substrate-controlled dihydroxylation, lactonisation, azidation, reduction, and deprotection led to the lactams 8–11. The tetrazoles 12–14 were prepared in an overall yield of 61–81% from the lactams 54, 28, and 67, respectively, by treatment with Tf2O and NaN3, followed by saponification, esterification, and hydrogenolysis. The lactams 8–11 and 40 and the tetrazoles 12–14 are medium-to-strong inhibitors of β-D-glucuronidase from bovine liver. Only the L-ido-configured lactam 10 (Ki = 94 μM) and the tetrazole 14 (Ki = 1.3 mM) inhibit human α-L-iduronidase.
[Show abstract][Hide abstract]ABSTRACT: [reaction: see text] An efficient 10-step preparation from 4-methoxypyridine of (2R,3R,4R)-2-acetamido-3,4-dihydroxypiperidine ("XylNAc-isofagomine") in optically active form is described. Key steps include an enantioselective reduction with catecholborane/(S)-2-methyl-CBS-oxazaborolidine, and a stereoselective pseudo-glycosylation of lithium azide by a cyclic sulfite ester. The title compound showed a Ki = 21 microM when evaluated against the N-acetyl-beta-hexosaminidase from Streptomyces plicatus.
Article · Oct 2005 · The Journal of Organic Chemistry
[Show abstract][Hide abstract]ABSTRACT: Genotype-phenotype correlations in genetic diseases for which missense mutations lead to disease remain a challenge. This is particularly true for diseases caused by alterations of proteins for which no three-dimensional structure is available. One such disease is Mucopolysaccharidosis type I, a disorder arising from a lack of activity of the lysosomal enzyme alpha-l-iduronidase (IDUA, EC 220.127.116.11). This deficiency compromises the degradation pathway of glycosaminoglycans such as heparan sulfate and dermatan sulfate, leading to substrate accumulation, which ultimately results in a multisystem disorder. Patients with IDUA deficiency have a wide spectrum of disease ranging from an early onset, rapidly progressive form leading to death in the first decade of life, to an attenuated disease which manifests in adolescence and leads to progressive joint and cardiac disease but is associated with a normal life span. Many patients fit into a disease phenotype intermediate to these extremes. While a number of point mutations have been described as leading to varying degrees of disease severity, a structural basis for these genotype-phenotype correlations has not been available owing to the lack of a three-dimensional structure for IDUA. A homology model for the IDUA enzyme was constructed based on the recently solved crystal structure of the beta-xylosidase from Thermoanaerobacterium saccharolyticum (XyTS, EC 18.104.22.168), both of which belong to the same sequence-related family (CAZY family 39). This model provides insights into why certain point mutations produce severely misfolded proteins and thus lead to severe disease, and why other mutations produce proteins with only minor structural perturbations and therefore the attenuated form of the disease.
Article · Jun 2005 · Molecular Genetics and Metabolism