Lysosomal Targeting of ß-Cyclodextrin.

ß-Cyclodextrin (ß-CD) and derivatives are approved therapeutics in >30 clinical settings. ß-CDs have also shown promise as therapeutics for treatment of some lysosomal storage disorders, such as Niemann-Pick disease type C, and other disease states which involve metabolite accumulation in the lysosome. In these cases, ß-CD activity relies on transport to the lysosome, wherein it can bind hydrophobic substrate and effect extraction. The post-translational attachment of N-glycans terminated in mannose-6-phosphate (M6P) residues is the predominant method by which lysosomal enzymes are targeted to the lysosome. In this work we covalently attach a synthetic biantennary bis-M6P-terminated N-glycan to ß-CD and study the effect of the added glycans in a mammalian cell line. The formation of a host guest complex with a Cy5 fluorophore allows study of both cellular internalisation and transport to the lysosome by fluorescence microscopy. Results indicate that the rates of both internalisation and lysosomal transport are increased by the attachment of M6P-glycans to ß-CD, indicating that M6P-glycan conjugation may improve the therapeutic effectiveness of ß-CD for the treatment of disorders involving hydrophobic metabolite accumulation in the lysosome.

N-acetylmannosamine-6-phosphate 2-epimerase uses a novel substrate-assisted mechanism to catalyze amino sugar epimerization.

There are five known general catalytic mechanisms used by enzymes to catalyze carbohydrate epimerization. The amino sugar epimerase N-acetylmannosamine-6-phosphate 2-epimerase (NanE) has been proposed to use a deprotonation-reprotonation mechanism, with an essential catalytic lysine required for both steps. However, the structural determinants of this mechanism are not clearly established. We characterized NanE from Staphylococcus aureus using a new coupled assay to monitor NanE catalysis in real time and found that it has kinetic constants comparable with other species. The crystal structure of NanE from Staphylococcus aureus, which comprises a triosephosphate isomerase barrel fold with an unusual dimeric architecture, was solved with both natural and modified substrates. Using these substrate-bound structures, we identified the following active-site residues lining the cleft at the C-terminal end of the ß-strands: Gln11, Arg40, Lys63, Asp124, Glu180, and Arg208, which were individually substituted and assessed in relation to the mechanism. From this, we re-evaluated the central role of Glu180 in this mechanism alongside the catalytic lysine. We observed that the substrate is bound in a conformation that ideally positions the C5 hydroxyl group to be activated by Glu180 and donate a proton to the C2 carbon. Taken together, we propose that NanE uses a novel substrate-assisted proton displacement mechanism to invert the C2 stereocenter of N-acetylmannosamine-6-phosphate. Our data and mechanistic interpretation may be useful in the development of inhibitors of this enzyme or in enzyme engineering to produce biocatalysts capable of changing the stereochemistry of molecules that are not amenable to synthetic methods.

Scope of the DMC mediated glycosylation of unprotected sugars with phenols in aqueous solution.

Activation of reducing sugars in aqueous solution using 2-chloro-1,3-dimethylimidazolinium chloride (DMC) and triethylamine in the presence of para-nitrophenol allows direct stereoselective conversion to the corresponding 1,2-trans para-nitrophenyl glycosides without the need for any protecting groups. The reaction is applicable to sulfated and phosphorylated sugars, but not to ketoses or uronic acids or their derivatives. When applied to other phenols the product yield was found to depend on the pKa of the added phenol, and the process was less widely applicable to 2-acetamido sugars. For 2-acetamido substrates an alternative procedure in which the glycosyl oxazoline was pre-formed, the reaction mixture freeze-dried, and the crude product then reacted with an added phenol in a polar aprotic solvent system with microwave irradiation proved to be a useful simplification.

The ENGases: versatile biocatalysts for the production of homogeneous N-linked glycopeptides and glycoproteins.

The endo-ß-N-acetylglucosaminidases (ENGases) are an enzyme class (EC 3.2.1.96) produced by a range of organisms, ranging from bacteria, through fungi, to higher order species, including humans, comprising two-sub families of glycosidases which all cleave the chitobiose core of N-linked glycans. Synthetic applications of these enzymes, i.e. to catalyse the reverse of their natural hydrolytic mode of action, allow the attachment of N-glycans to a wide variety of substrates which contain an N-acetylglucosamine (GlcNAc) residue to act as an 'acceptor' handle. The use of N-glycan oxazolines, high energy intermediates on the hydrolytic pathway, as activated donors allows their high yielding attachment to almost any amino acid, peptide or protein that contains a GlcNAc residue as an acceptor. The synthetic effectiveness of these biocatalysts has been significantly increased by the production of mutant glycosynthases; enzymes which can still catalyse synthetic processes using oxazolines as donors, but which do not hydrolyse the reaction products. ENGase biocatalysts are now finding burgeoning application for the production of biologically active glycopeptides and glycoproteins, including therapeutic monoclonal antibodies (mAbs) for which the oligosaccharides have been remodelled to optimise effector functions.

Synthetic and semi-synthetic approaches to unprotected N-glycan oxazolines.

N-Glycan oxazolines have found widespread use as activated donor substrates for endo-ß-N-acetylglucosaminidase (ENGase) enzymes, an important application that has correspondingly stimulated interest in their production, both by total synthesis and by semi-synthesis using oligosaccharides isolated from natural sources. Amongst the many synthetic approaches reported, the majority rely on the fabrication (either by total synthesis, or semi-synthesis from locust bean gum) of a key Manß(1-4)GlcNAc disaccharide, which can then be elaborated at the 3- and 6-positions of the mannose unit using standard glycosylation chemistry. Early approaches subsequently relied on the Lewis acid catalysed conversion of peracetylated N-glycan oligosaccharides produced in this manner into their corresponding oxazolines, followed by global deprotection. However, a key breakthrough in the field has been the development by Shoda of 2-chloro-1,3-dimethylimidazolinium chloride (DMC), and related reagents, which can direct convert an oligosaccharide with a 2-acetamido sugar at the reducing terminus directly into the corresponding oxazoline in water. Therefore, oxazoline formation can now be achieved in water as the final step of any synthetic sequence, obviating the need for any further protecting group manipulations, and simplifying synthetic strategies. As an alternative to total synthesis, significant quantities of several structurally complicated N-glycans can be isolated from natural sources, such as egg yolks and soy bean flour. Enzymatic transformations of these materials, in concert with DMC-mediated oxazoline formation as a final step, allow access to a selection of N-glycan oxazoline structures both in larger quantities and in a more expedient fashion than is achievable by total synthesis.

Recent applications of click chemistry for the functionalization of gold nanoparticles and their conversion to glyco-gold nanoparticles.

Glycoscience, despite its myriad of challenges, promises to unravel the causes of, potential new detection methods for, and novel therapeutic strategies against, many disease states. In the last two decades, glyco-gold nanoparticles have emerged as one of several potential new tools for glycoscientists. Glyco-gold nanoparticles consist of the unique structural combination of a gold nanoparticle core and an outer-shell comprising multivalent presentation of carbohydrates. The combination of the distinctive physicochemical properties of the gold core and the biological function/activity of the carbohydrates makes glyco-gold nanoparticles a valuable tool in glycoscience. In this review we present recent advances made in the use of one type of click chemistry, namely the azide-alkyne Huisgen cycloaddition, for the functionalization of gold nanoparticles and their conversion to glyco-gold nanoparticles.

Synthesis and anti-mycobacterial activity of glycosyl sulfamides of arabinofuranose.

A series of arabino N-glycosyl sulfamides, forced to adopt the furanose form by removal of the 5-hydroxyl group, were synthesised as putative isosteric mimics of decaprenolphosphoarabinose, the donor processed by arabinosyltransferases during mycobacterial cell wall assembly. Compounds showed moderate anti-mycobacterial activity, which was maximal for a C10 sulfamide side chain.

Direct aqueous synthesis of cyanomethyl thioglycosides from reducing sugars; ready access to reagents for protein glycosylation.

Unprotected carbohydrates can be directly converted into cyanomethyl thioglycosides in a completely stereoselective manner by reaction with 2-chloro-1,3-dimethylimidazolinium chloride (DMC) and mercaptoacetonitrile in aqueous solution in the presence of triethylamine. Reaction with methoxide provides 2-imino-2-methoxyethyl (IME) thioglycosides, which may be used directly for the glycosylation of surface protein lysine residues.

Chemoenzymatic Synthesis of a Phosphorylated Glycoprotein.

The majority of lysosomal enzymes are targeted to the lysosome by post-translational tagging with N-glycans terminating in mannose-6-phosphate (M6P) residues. Some current enzyme replacement therapies (ERTs) for lysosomal storage disorders are limited in their efficacy by the extent to which the recombinant enzymes bear the M6P-terminated glycans required for effective trafficking. Chemical synthesis was combined with endo-ß-N-acetylglucosaminidase (ENGase) catalysis to allow the convergent synthesis of glycosyl amino acids bearing M6P residues. This approach can be extended to the remodeling of proteins, as exemplified by RNase. The powerful synergy of chemical synthesis and ENGase-mediated biocatalysis enabled the first synthesis of a glycoprotein bearing M6P-terminated N-glycans in which the glycans are attached to the peptide backbone by entirely natural linkages.

A surface plasmon resonance assay for measurement of neuraminidase inhibition, sensitivity of wild-type influenza neuraminidase and its H274Y mutant to the antiviral drugs zanamivir and oseltamivir.

Antiviral resistance is currently monitored by a labelled enzymatic assay, which can give inconsistent results because of the short half-life of the labelled product, and variations in assay conditions. In this paper, we describe a competitive surface plasmon resonance (SPR) inhibition assay for measuring the sensitivities of wild-type neuraminidase (WT NA) and the H274Y (histidine 274 tyrosine) NA mutant to antiviral drugs. The two NA isoforms were expressed in High-five™ (Trichoplusia ni) insect cells. A spacer molecule (1,6-hexanediamine (HDA)) was conjugated to the 7-hydroxyl group of zanamivir, and the construct (HDA-zanamivir) was immobilized onto a SPR sensor chip to obtain a final immobilization response of 431 response units. The immobilized HDA-zanamivir comprised a bio-specific ligand for the WT and mutant proteins. The effects of the natural substrate (sialic acid) and two inhibitors (zanamivir and oseltamivir) on NA binding to the immobilized ligand were studied. The processed SPR data was analysed to determine 50% inhibitory concentrations (IC50-spr ), using a log dose-response curve fit. Although both NA isoforms had almost identical IC50-spr values for sialic acid (WT = 5.5 nM; H274Y mutant = 3.25 nM) and zanamivir (WT = 2.16 nM; H274Y mutant = 2.42 nM), there were significant differences between the IC50-spr values obtained for the WT (7.7 nM) and H274Y mutant (256 nM) NA in the presence of oseltamivir, indicating that oseltamivir has a reduced affinity for the H274Y mutant. The SPR inhibition assay strategy presented in this work could be applied for the rapid screening of newly emerging variants of NA for their sensitivity to antiviral drugs.

Development of a surface plasmon resonance assay to measure the binding affinity of wild-type influenza neuraminidase and its H274Y mutant to the antiviral drug zanamivir.

Influenza is one of the most common infections of the upper respiratory tract. Antiviral drugs that are currently used to treat influenza, such as oseltamivir and zanamivir, are neuraminidase (NA) inhibitors. However, the virus may develop resistance through single-point mutations of NA. Antiviral resistance is currently monitored by a labelled enzymatic assay, which can be inconsistent because of the short half-life of the labelled product and variations in the assay conditions. In this paper, we describe a label-free surface plasmon resonance (SPR) assay for measuring the binding affinity of NA-drug interactions. Wild-type (WT) NA and a histidine 274 tyrosine (H274Y) mutant were expressed in High Five™ (Trichoplusia ni) insect cells. A spacer molecule (1,6-hexanediamine) was site-specifically conjugated to the 7-hydroxyl group of zanamivir, which is not involved in binding to NA, and the construct was immobilized onto a SPR sensor Chip to obtain a final immobilization response of 431 response units. Binding responses obtained for WT and H274Y mutant NAs were fitted to a simple Langmuir 1:1 model with drift to obtain the association (ka ) and dissociation (kd ) rate constants. The ratio between the binding affinities for the two isoforms was comparable to literature values obtained using labelled enzyme assays. Significant potential exists for an extension of this approach to test for drug resistance of further NA mutants against zanamivir and other antiviral drugs, perhaps paving the way for a reliable SPR biosensor assay that may replace labelled enzymatic assays.

Size-optimized galactose-capped gold nanoparticles for the colorimetric detection of heat-labile enterotoxin at nanomolar concentrations.

The development of a galactose-capped gold nanoparticle-based colorimetric sensor for the detection of the lectin heat-labile enterotoxin is reported. Heat-labile enterotoxin is one of the pathogenic agents responsible for the intestinal disease called 'traveller's diarrhoea'. By means of specific interaction between galactose moieties attached to the surface of gold nanoparticles and receptors on the B-subunit of heat-labile enterotoxin (LTB), the gold nanoparticles reported here act as an efficient colorimetric sensor, which can detect the toxin at nanomolar concentrations. The effect of gold nanoparticle size on the detection sensitivity was investigated in detail. Amongst the various sizes of gold nanoparticles studied (2, 7, 12, and 20 nm), the 12 nm sized gold nanoparticles were found to be the most efficient, with a minimum heat-labile enterotoxin detection concentration of 100 nM. The red to purple colour change of the gold nanoparticle solution occurred within two minutes, indicating rapid toxin sensing.

Unexpected furanose/pyranose equilibration of N-glycosyl sulfonamides, sulfamides and sulfamates.

De-protected arabino N-glycosyl sulfamides, sulfonamides and sulfamates were found to mutarotate and convert from the furanose to the thermodynamically more stable pyranose form in aqueous solution. The presence of a strongly electron withdrawing group in the alkyl chain stopped mutarotation and furanose/pyranose equilibration, allowing the isolation of the first unprotected furanose N-glycosyl sulfonamide.

Endo-ß-N-acetylglucosaminidase catalysed glycosylation: tolerance of enzymes to structural variation of the glycosyl amino acid acceptor.

Endo-ß-N-Acetylglucosaminidases (ENGases) are highly useful biocatalysts that can be used to synthetically access a wide variety of defined homogenous N-linked glycoconjugates in a convergent manner. The synthetic efficiency of a selection of family GH85 ENGases was investigated as the structure of the acceptor substrate was varied. Several different GlcNAc-asparagine acceptors were synthesised, and used in conjunction with penta- and decasaccharide oxazoline donors. Different enzymes showed different tolerances of modification of the GlcNAc acceptor. Whilst none tolerated modification of either the 4- or 6-hydroxyl, both Endo M and Endo D tolerated modification of OH-3. For Endo D the achievable synthetic efficiency was increased by a factor of three by the use a 3-O-benzyl protected acceptor. The presence of a fucose at position-3 was not tolerated by any of the enzymes assayed.

Convergent chemoenzymatic synthesis of a library of glycosylated analogues of pramlintide: structure-activity relationships for amylin receptor agonism.

Pramlintide (Symlin®), a synthetic analogue of the naturally occurring pancreatic hormone amylin, is currently used with insulin in adjunctive therapy for type 1 and type 2 diabetes mellitus. Herein we report a systematic study into the effect that N-glycosylation of pramlintide has on activation of amylin receptors. A highly efficient convergent synthetic route, involving a combination of solid phase peptide synthesis and enzymatic glycosylation, delivered a library of N-glycosylated variants of pramlintide bearing either GlcNAc, the core N-glycan pentasaccharide [Man3(GlcNAc)2] or a complex biantennary glycan [(NeuAcGalGlcNAcMan)2Man(GlcNAc)2] at each of its six asparagine residues. The majority of glycosylated versions of pramlintide were potent receptor agonists, suggesting that N-glycosylation may be used as a tool to optimise the pharmacokinetic properties of pramlintide and so deliver improved therapeutic agents for the treatment of diabetes and obesity.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of N-acetylneuraminate lyase from methicillin-resistant Staphylococcus aureus.

The enzyme N-acetylneuraminate lyase (EC 4.1.3.3) is involved in the metabolism of sialic acids. Specifically, the enzyme catalyzes the retro-aldol cleavage of N-acetylneuraminic acid to form N-acetyl-D-mannosamine and pyruvate. Sialic acids comprise a large family of nine-carbon amino sugars, all of which are derived from the parent compound N-acetylneuraminic acid. In recent years, N-acetylneuraminate lyase has received considerable attention from both mechanistic and structural viewpoints and has been recognized as a potential antimicrobial drug target. The N-acetylneuraminate lyase gene was cloned from methicillin-resistant Staphylococcus aureus genomic DNA, and recombinant protein was expressed and purified from Escherichia coli BL21 (DE3). The enzyme crystallized in a number of crystal forms, predominantly from PEG precipitants, with the best crystal diffracting to beyond 1.70 Šresolution in space group P21. Molecular replacement indicates the presence of eight monomers per asymmetric unit. Understanding the structural biology of N-acetylneuraminate lyase in pathogenic bacteria, such as methicillin-resistant S. aureus, will provide insights for the development of future antimicrobials.

Selective Anomeric Acetylation of Unprotected Sugars with Acetic Anhydride in Water.

Unprotected sugars are selectively acetylated simply by stirring in aqueous solution in the presence of acetic anhydride and a weak base such as sodium carbonate. The reaction is selective for acetylation of the anomeric hydroxyl group of mannose, 2-acetamido, and 2-deoxy sugars and can be performed on a large scale. Competitive intramolecular migration of the 1-O-acetate to the 2-hydroxyl group when these two substituents are cis causes over-reaction and the formation of product mixtures.

Protecting group free glycosylation: one-pot stereocontrolled access to 1,2-trans glycosides and (1→6)-linked disaccharides of 2-acetamido sugars.

Unprotected 2-acetamido sugars may be directly converted into their oxazolines using 2-chloro-1,3-dimethylimidazolinium chloride (DMC), and a suitable base, in aqueous solution. Freeze drying and acid catalysed reaction with an alcohol as solvent produces the corresponding 1,2-trans-glycosides in good yield. Alternatively, dissolution in an aprotic solvent system and acidic activation in the presence of an excess of an unprotected glycoside as a glycosyl acceptor, results in the stereoselective formation of the corresponding 1,2-trans linked disaccharides without any protecting group manipulations. Reactions using aryl glycosides as acceptors are completely regioselective, producing only the (1→6)-linked disaccharides.

Production and crystallization of processing α-glucosidase I: Pichia pastoris expression and a two-step purification toward structural determination.

Eukaryotic N-glycoprotein processing in the endoplasmic reticulum begins with the catalytic action of processing α-glucosidase I (αGlu). αGlu trims the terminal glucose from nascent glycoproteins in an inverting-mechanism glycoside hydrolysis reaction. αGlu has been studied in terms of kinetic parameters and potential key residues; however, the active site is unknown. A structural model would yield important insights into the reaction mechanism. A model would also be useful in developing specific therapeutics, as αGlu is a viable drug target against viruses with glycosylated envelope proteins. However, due to lack of a high-yielding overexpression and purification scheme, no eukaryotic structural model of αGlu has been determined. To address this issue, we overexpressed the Saccharomyces cerevisiae soluble αGlu, Cwht1p, in the host Pichia pastoris. It was purified in a simple two-step protocol, with a final yield of 4.2mg Cwht1p per liter of growth culture. To test catalytic activity, we developed a modified synthesis of a tetrasaccharide substrate, Glc(3)ManOMe. Cwht1p with Glc(3)ManOMe shows a K(m) of 1.26 mM. Cwht1p crystals were grown and subjected to X-ray irradiation, giving a complete diffraction dataset to 2.04 Å resolution. Work is ongoing to obtain phases so that we may further understand this fundamental member of the N-glycosylation pathway through the discovery of its molecular structure.