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Un-protected 2-acetamido sugars are stereoselectively converted into 1,2-trans glycosides and (1→6)-linked disaccharides without any protecting groups. Reaction proceeds via intermediate oxazolines which react with acceptors under acid catalysis.

β-Cyclodextrin (β-CD) and derivatives are approved therapeutics in30 clinical settings. β-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, β-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 β-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 β-CD, indicating that M6P-glycan conjugation may improve the therapeutic effectiveness of β-CD for the treatment of disorders involving hydrophobic metabolite accumulation in the lysosome.

Reducing sugars may be directly converted into the corresponding para-nitrophenyl (pNP) glycosides using 2-chloro-1,3-dimethylimidazolinium chloride (DMC), para-nitrophenol, and a suitable base in aqueous solution. The reaction is stereoselective for sugars with either a hydroxyl or an acetamido group at position 2, yielding the 1,2-trans pNP glycosides. A judicious choice of base allows extension to di- and oligosaccharide substrates, including a complex N-glycan oligosaccharide isolated from natural sources, without the requirement of any protecting group manipulations.

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

The majority of the world's best-selling biotherapeutics are glycoproteins. However their production using cellular expression systems invariably produces inseparable mixtures of materials which differ in their attached carbohydrates. As in many cases correct carbohydrate structure is vital for in vivo efficacy, the development of methods for the production of glycoproteins in homogeneous form has become a significant scientific objective. Here a brief overview of recent progress in the production of homogeneous glycoproteins, including monoclonal antibodies, will be discussed, centring on the use of endo-β-N-acetylglucosaminidase (ENGase) enzymes for protein glycoengineering.

2-Chloro-1,3-dimethylimidazolinium chloride (DMC, herein also referred to as Shoda's reagent) and its derivatives are useful for numerous synthetic transformations in which the anomeric centre of unprotected reducing sugars is selectively activated in aqueous solution. As such unprotected sugars can undergo anomeric substitution with a range of added nucleophiles, providing highly efficient routes to a range of glycosides and glycoconjugates without the need for traditional protecting group manipulations. This mini-review summarizes the development of DMC and some of its derivatives/analogues, and highlights recent applications for protecting group-free synthesis.

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.

M. Carmen Galan, Sabine Flitsch and Antony Fairbanks introduce the Organic & Biomolecular Chemistry themed collection on glycosylation: new methodologies and applications.

Reducing sugars may be directly converted into the corresponding

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.

The synthesis of analogues of natural enzyme substrates can be used to help deduce enzymatic mechanisms. N-Acetylmannosamine-6-phosphate 2-epimerase is an enzyme in the bacterial sialic acid catabolic pathway. To investigate whether the mechanism of this enzyme involves a re-protonation mechanism by the same neighbouring lysine that performed the deprotonation or a unique substrate-assisted proton displacement mechanism involving the substrate C5 hydroxyl, the syntheses of two analogues of the natural substrate, N-acetylmannosamine-6-phosphate, are described. In these novel analogues, the C5 hydroxyl has been replaced with a proton and a methyl ether respectively. As recently reported, Staphylococcus aureus N-acetylmannosamine-6-phosphate 2-epimerase was co-crystallized with these two compounds. The 5-deoxy variant bound to the enzyme active site in a different orientation to the natural substrate, while the 5-methoxy variant did not bind, adding to the evidence that this enzyme uses a substrate-assisted proton displacement mechanism. This mechanistic information may help in the design of potential antibacterial drug candidates.

Background Lipid hydroperoxides decompose to reactive aldehydes, such as acrolein. Measurement of oxidative stress markers in the clinic could improve risk stratification for patients. Methods To aid the development of diagnostic oxidative stress markers, we defined the acrolein modifications of haemoglobin using mass spectrometry. Results Acrolein modifications have little effect on the secondary structure of haemoglobin. They do not disrupt the quaternary structure, but instead promote crosslinked octamers. For acrolein modified haemoglobin the response to O2 binding is altered such that cooperativity is lost. Mass spectrometry experiments at a 1:1 acrolein:haemoglobin molar ratio demonstrate that the α-chain quickly forms an aza-Michael adduct (+56 Da), which then forms a more stable adduct, Ne-(3-methylpyridinium)lysine (MP-lysine, +76 Da) over 7 days. The β-chain remains relatively unchanged over the duration of the 7 days and the aza-Michael adduct is dominant. At 2:1 and 5:1 molar ratios the α-chain was consistently modified at K7, H20, H50, and the β-chain at C93 and H97 with the aza-Michael adduct. Beyond 5 h, an MP-adduct (+76 Da) was located predominantly at K7 of the α-chain, while an FDP-adduct (+94 Da) was observed at K95 of the β-chain. Conclusions We have generated qualitative evidence identifying the acrolein target sites on haemoglobin, a potential oxidative stress marker that is easily measured in circulation. General significance We provide data for the community to develop targeted mass spectrometric or immunometric assays for acrolein modified haemoglobin to further validate the potential of haemoglobin as an oxidative stress marker in patients .

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 β-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.

The endo-β-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.

High yielding selective acetylation of only the anomeric hydroxyl of unprotected sugars is possible in aqueous solution using 2-chloro-1,3-dimethylimidazolinium chloride (DMC), thioacetic acid, and a suitable base. The reaction, which may be performed on a multi-gram scale, is stereoselective for sugars that possess a hydroxyl group at position-2, exclusively yielding the 1,2-trans products. The use of an iterative reagent addition procedure allows the use of sodium carbonate as the base, avoiding the formation of triethylammonium salts, which may hamper product purification. The glycosyl acetate products may be used as donor substrates for glycosidase-catalysed synthesis. The crude aqueous acetylation reaction mixture may also be used for this purpose.

Enzymatic degradation of locust bean gum provides a Manβ(1 → 4)Man disaccharide, which may be converted into the core Manβ(1 → 4)GlcNAc disaccharide unit of all N-glycans via conversion to a 2-iodo-glycosyl azide, and Lafont rearrangement. The Manβ(1 → 4)GlcNAc disaccharide may be used as a key intermediate for elaboration into more complex N-glycan structures providing a route to N-glycan oxazolines as donor substrates for ENGase enzymes that is considerably shorter than those reported previously.

Antony J. Fairbanks is a Professor in the Department of Chemistry at the University of Canterbury in New Zealand. The research of his group focuses on the broad areas of organic synthesis, particularly applied to carbohydrates. He currently serves as an active Editorial Board member for ChemistryOpen.

N-Azidoacetyl-D-glucosamine (GlcNAz) is a particularly useful tool in chemical biology as the azide is a metabolically stable yet accessible handle within biological systems. Herein, we report a practical synthesis of FmocAsn(N-Ac3GlcNAz)OH, a building block for solid phase peptide synthesis (SPPS). Protecting group manipulations are minimised by taking advantage of the inherent chemoselectivity of phosphine-mediated azide reduction, and the resulting glycosyl amine is employed directly in the opening of Fmoc protected aspartic anhydride. We show potential application of the building block by establishing it as a substrate for enzymatic glycan extension using sugar oxazolines of varying size and biological significance with several endo-β-N-acetylglucosaminidases (ENGases). The added steric bulk resulting from incorporation of the azide is shown to have no or a minor impact on the yield of enzymatic glycan extension.

A series of glycosyl donors possessing a (thiophen-2-yl)methyl ether protecting group at position 2 were synthesised and the effect of the protecting group pattern of other hydroxyls on the stereochemical outcome of glycosylation was investigated. Studies revealed optimal α-selectivity for glycosylation using a fully armed tri-benzylated donor, whilst other protecting group patterns were significantly less effective. Low-temperature NMR studies of both fully armed and fully disarmed donors revealed the intermediacy of cyclised sulfonium ion intermediates. Reaction conditions were developed which allowed removal of the (thiophen-2-yl)methyl ether protecting group either selectively, or together with benzyl ethers.

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.

The use of a bi-functional linker, containing an alkyne and an alkene, allows the protecting group free conjugation of reducing sugars to thiols via a double click process. Firstly the linker is attached to the sugar via one-pot glycosyl azide formation and Cu-catalysed azide-alkyne cycloaddition. Photochemical thiol-ene click reaction then allows conjugation to a range of thiols, including cysteine residues of peptides.

Antony J. Fairbanks is a Professor in the Department of Chemistry at the University of Canterbury in New Zealand. The research of his group focuses on the broad areas of organic synthesis, particularly applied to carbohydrates. He currently serves as an active Editorial Board member for ChemistryOpen.

A series of arabinose glycosyl sulfamides with varying alkyl chain types and lengths were synthesised as mimics of decaprenolphosphoarabinose (DPA), and as potential inhibitors of mycobacterial cell wall biosynthesis. Unprecedented conversion of the desired furanose to the thermodynamically more stable pyranose form occurred during final de-protection. Biological testing against Mycobacterium smegmatis revealed low to moderate anti-mycobacterial activity with marked dependence on alkyl chain length, which in the case of mono-substituted sulfamides was maximal for a C-10 chain.

A flavinium catalyst, in conjunction with hydrogen peroxide as stoichiometric oxidant, allowed the aqueous conversion of non-protected thioglycosides into the corresponding glycosyl sulfoxides. These glycosyl sulfoxides displayed only very weak inhibitory activity against corresponding glycosidases.

Glycosylation of a fully armed donor bearing a 2-O-(trimethoxybenzenethiol) ethyl ether protecting group is completely α-selective with a range of carbohydrate alcohol acceptors. Low-temperature NMR studies confirm the intermediacy of cyclic sulfonium ion intermediates arising from six-membered β-sulfonium ring neighboring group participation. Selective protecting group removal is achieved in high yield in a single operation by S-methylation and base-induced β-elimination.

Gold nanoparticles decorated with full‐length sialic acid terminated complex bi‐antennary N‐glycans, synthesized with glycans isolated from egg yolk, were used as a sensor for the detection of both recombinant hemagglutinin (HA) and whole influenza A virus particles of the H1N1 subtype. Nanoparticle aggregation was induced by interaction between the sialic acid termini of the glycans attached to gold and the multivalent sialic acid binding sites of HA. Both dynamic light scattering (DLS) and UV/Vis spectroscopy demonstrated the efficiency of the sensor, which could detect viral HA at nanomolar concentrations and revealed a linear relationship between the extent of nanoparticle aggregation and the concentration of HA. UV/Vis studies also showed that these nanoparticles can selectively detect an influenza A virus strain that preferentially binds sialic acid terminated glycans with α(2→6) linkages over a strain that prefers glycans with terminal α(2→3)‐linked sialic acids.

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.

Convergent chemo-enzymatic synthesis of mannosylated glycopeptides enhances uptake by human antigen presenting cells whilst preserving the immunogenicity of peptide epitopes., The combination of solid phase peptide synthesis and endo-β-N-acetylglucosaminidase (ENGase) catalysed glycosylation is a powerful convergent synthetic method allowing access to glycopeptides bearing full-length N-glycan structures. Mannose-terminated N-glycan oligosaccharides, produced by either total or semi-synthesis, were converted into oxazoline donor substrates. A peptide from the human cytomegalovirus (CMV) tegument protein pp65 that incorporates a well-characterised T cell epitope, containing N-acetylglucosamine at specific Asn residues, was accessed by solid phase peptide synthesis, and used as an acceptor substrate. High-yielding enzymatic glycosylation afforded glycopeptides bearing defined homogeneous high-mannose N-glycan structures. These high-mannose containing glycopeptides were tested for enhanced targeting to human antigen presenting cells (APCs), putatively mediated via the mannose receptor, and for processing by the APCs for presentation to human CD8+ T cells specific for a 9-mer epitope within the peptide. Binding assays showed increased binding of glycopeptides to APCs compared to the non-glycosylated control. Glycopeptides bearing high-mannose N-glycan structures at a single site outside the T cell epitope were processed and presented by the APCs to allow activation of a T cell clone. However, the addition of a second glycan within the T cell epitope resulted in ablation of T cell activation. We conclude that chemo-enzymatic synthesis of mannosylated glycopeptides enhances uptake by human APCs while preserving the immunogenicity of peptide epitopes within the glycopeptides, provided those epitopes are not themselves glycosylated.

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.

Glycosylation of peptides and proteins has emerged as a promising strategy to improve the pharmacokinetic profile of peptide- and protein-based therapeutics. The synthesis of pure homogeneous N-linked glycopeptides and glycoproteins is a challenging task, and efficient routes to access them are in high demand. Endo-β-N-acetylglucosaminidise catalysed glycosylation of N-acetylglucosamine-tagged peptides, using activated oligosaccharide oxazolines as donors, has recently attracted attention due to the relative simplicity by which the process convergently affords glycoconjugates with complete control of stereo- and regioselectivity. Herein, a brief review of some examples of recent enzyme-mediated N-glycosylation used to synthesise glycopeptides with therapetic potential is provided.

N-Glycan oxazolines have found widespread use as activated donor substrates for endo-β-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 Manβ(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.

Advanced lipid peroxidation end-products (ALEs) accumulate with ageing and oxidative stress-related diseases. Despite their potential therapeutic value, there are no suitably protected ALE building blocks reported in the literature to enable their site-specific incorporation into synthetic peptides. The synthesis of an Fmoc-protected ALE building block, N∈-(3-methylpyridinium)lysine (MP-lysine) and its incorporation into collagen model peptides is reported.

Glycosyl thiols may be accessed from the corresponding reducing sugars in water without recourse to any sugar projecting groups by way of a DMC mediated reaction with thioacetic acid in the presence of base, and hydrolysis of the anomeric thioacetate. Glycosyl thiols produced by this method may be used to access glycoconjugates, such as glycopeptides by use of the thiol–ene click reaction.

The conversion of sugars into glycomimetics typically involves multiple protecting-group manipulations. The development of methodology allowing the direct aqueous conversion of free sugars into glycosides, and mimics of oligosaccharides and glycoconjugates in a high-yielding and stereoselective process is highly desirable. The combined use of 2-azido-1,3-dimethylimidazolinium hexafluorophosphate and the Cu-catalyzed Huisgen cycloaddition allowed the synthesis of a range of glycoconjugates in a one-step reaction directly from reducing sugars under aqueous conditions. The reaction, which is completely stereoselective, may be applied to the convergent synthesis of triazole-linked glycosides, oligosaccharides, and glycopeptides. The procedure provides a method for the one-pot aqueous ligation of oligosaccharides and peptides bearing alkyne side chains.

The development of new protecting groups that undergo neighbouring group participation (NGP) via six-membered ring intermediates to promote the formation of α-1,2-cis glycosidic linkages complements the established use of 5-ring NGP in terms of stereochemical outcome. A selection of glycosyl donors was synthesised that possessed novel 2-iodo- and 2-(phenylseleno)ethyl ether protecting groups in an attempt to promote highly α-selective glycosylation by 6-ring NGP. Although the fully armed donors produced α-glucosides as the predominant reaction products, low-temperature NMR studies did not show NGP by the observation of cyclised reaction intermediates. The corresponding disarmed glycosyl donors were unexpectedly less stereoselective. NMR spectroscopy revealed that the 2-iodoethyl ether did not participate in any of the glycosylation processes; however, the 2-(phenylseleno)ethyl ether did participate, and β-configured cyclic intermediates were observed. The fact that considerable amounts of β-glycoside product were formed in these latter cases indicated that the predominant reaction pathway to product did not occur through the observed cyclic species. Clearly, a fine balance exists during glycosylation reactions, and the reaction pathway to product depends on a variety of factors. Notably, the formation of cyclised intermediates by 6-ring NGP is not on its own sufficient to ensure high levels of α-stereoselectivity.

Sialic acids are one of the most important carbohydrate classes in biology. Some bacterial pathogens can scavenge sialic acids from their surrounding environment and degrade them as a source of carbon, nitrogen and energy. This sequestration and subsequent catabolism of sialic acid require a cluster of genes known as the `Nan-Nag' cluster. The enzymes coded by these genes are important for pathogen colonization and persistence. Importantly, the Nan-Nag genes have proven to be essential forStaphylococcus aureusgrowth on sialic acids, suggesting that the pathway is a viable antibiotic drug target. The enzymeN-acetylmannosamine-6-phosphate 2-epimerase is involved in the catabolism of sialic acid; specifically, the enzyme convertsN-acetylmannosamine-6-phosphate intoN-acetylglucosamine-6-phosphate. The gene was cloned into an appropriate expression vector, and recombinant protein was expressed inEscherichia coliBL21 (DE3) cells and purifiedviaa three-step procedure. PurifiedN-acetylmannosamine-6-phosphate 2-epimerase was screened for crystallization. The best crystal diffracted to a resolution of beyond 1.84 Å in space groupP21212. Understanding the structural nature of this enzyme from methicillin-resistantS. aureuswill provide us with the insights necessary for the development of future antibiotics.

Endo-β-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.

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.

The ruthenium-catalyzed transfer hydrogenation of electron-rich amino-substituted acetophenones is reported. Variation of the reductant, ligands, base, and solvent allowed reaction optimization. A key discovery was the use of 1,4-butanediol as an irreversible reducing agent, which significantly improved the conversion. A range of amino- and amido-substituted aryl ketones were explored, and they all gave the corresponding alcohols in good yield, which demonstrates the wider applicability of this process.

The synthetic application of endohexosaminidase enzymes (e.g., Endo A, Endo M, Endo D) promises to allow ready access to a wide variety of defined homogeneous glycoproteins and glycopeptides. The use of N-glycan oligosaccharides that are activated at the reducing terminus as oxazolines allows their high-yielding attachment to almost any amino acid, peptide, or protein that contains a GlcNAc residue as an acceptor. A wide variety of oxazoline donors are readily available, either by total synthesis or by isolation of the corresponding oligosaccharide from natural sources and then conversion to the oxazoline in water. The synthetic potential of the enzymes is particularly augmented by the production of mutant glycosynthases, the use of which allows the synthesis of a wide variety of glycopeptides and glycoproteins bearing defined homogeneous N-glycan structures.

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 Å resolution 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.

Organic azides are readily reduced to the corresponding amines by treatment with sodium iodide in the presence of acidic ion exchange resin. The process, optimal when performed at 40 °C and 200 mbar pressure on a rotatory evaporator, is extremely efficient, clean, and tolerant of a variety of functional groups.

N-Acetylneuraminate lyase is the first committed enzyme in the degradation of sialic acid by bacterial pathogens. In this study, we analysed the kinetic parameters of N-acetylneuraminate lyase from methicillin-resistant Staphylococcus aureus (MRSA). We determined that the enzyme has a relatively high KM of 3.2 mM, suggesting that flux through the catabolic pathway is likely to be controlled by this enzyme. Our data indicate that sialic acid alditol, a known inhibitor of N-acetylneuraminate lyase enzymes, is a stronger inhibitor of MRSA N-acetylneuraminate lyase than of Clostridium perfringens N-acetylneuraminate lyase. Our analysis of the crystal structure of ligand-free and inhibitor-bound MRSA N-acetylneuraminate lyase suggests that subtle dynamic differences in solution and/or altered binding interactions within the active site may account for species-specific inhibition. This article is protected by copyright. All rights reserved.

SummaryThe complete degradation of N-linked glycans by the pathogenic bacterium Streptococcus pneumoniae is facilitated by the large multimodular cell wall-attached exo-β-D-N-acetylglucosaminidase StrH. Structural dissection of this virulence factor using X-ray crystallography showed it to have two structurally related glycoside hydrolase family 20 catalytic domains, which displayed the expected specificity for complex N-glycans terminating in N-acetylglucosamine but exhibited unexpected differences in their preferences for the substructures present in these glycans. The structures of the two catalytic domains in complex with unhydrolyzed substrates, including an N-glycan possessing a bisecting N-acetylglucosamine residue, revealed the specific architectural features in the active sites that confer their differential specificities. Inhibitors of StrH are demonstrated to be effective tools in modulating the interaction of StrH with components of the host, such as the innate immune system. Overall, new structural and functional insight into a carbohydrate-mediated component of the pneumococcus-host interaction is provided.

We report here the structural determination of the N-linked glycans in the 66-kDa glycoprotein, part of the unique sulfated complex cell wall polysaccharide of the red microalga Porphyridium sp. Structures were elucidated by a combination of normal phase/reverse phase HPLC, positive ion MALDI-TOF MS, negative ion electrospray ionization, and MS/MS. The sugar moieties of the glycoprotein consisted of at least four fractions of N-linked glycans, each composed of the same four monosaccharides, GlcNAc, Man, 6-O-MeMan, and Xyl, with compositions Man(8-9)Xyl(1-2)Me(3)GlcNAc(2). The present study is the first report of N-glycans with the terminal Xyl attached to the 6-mannose branch of the 6-antenna and to the 3-oxygen of the penultimate (core) GlcNAc. Another novel finding was that all four glycans contain three O-methylmannose residues in positions that have never been reported before. Although it is known that some lower organisms are able to methylate terminal monosaccharides in glycans, the present study on Porphyridium sp. is the first describing an organism that is able to methylate non-terminal mannose residues. This study will thus contribute to understanding of N-glycosylation in algae and might shed light on the evolutionary development from prokaryotes to multicellular organisms. It also may contribute to our understanding of the red algae polysaccharide formation. The additional importance of this research lies in its potential for biotechnological applications, especially in evaluating the use of microalgae as cell factories for the production of therapeutic proteins.

Glycoprotein remodelling represents a practical and potentially widely applicable method for the production of homogenous glycoconjugates bearing defined N -glycan structures, including therapeutic glycoproteins and monoclonal antibodies. Key to the remodelling process is the attachment of a defined oligosaccharide structure en bloc to GlcNAc residues at N -linked glycosylation sites. Endohexosaminidases (endo-β- N -acetyl-glucosaminidases, ENGases) are a class of enzyme that are capable of achieving this synthetic transformation with complete regio- and stereochemical control. In particular, the use of oxazolines as activated donor substrates for these enzymes greatly improves synthetic efficiencies as compared to transglycosylation using Asn-linked oligosaccharides. This article summarises recent work from within our laboratory focussing on the synthesis of a wide variety of N -glycan oxazolines and their use as substrates for endohexosaminidase-catalysed glycosylation. In particular, the problem of enzyme-catalysed competitive product hydrolysis may be countered, either by the use of structurally modified oxazoline donors, or by the production of mutant endohexosaminidase enzymes. The power of this methodology is exemplified by the production of a defined glycoform of ribonuclease B in a highly efficient fashion.

An investigation is undertaken into the propensity of glycosyloxazolines to undergo reductive cleavage by catalytic hydrogenation.Results indicate that the protecting groups on carbohydrate hydroxylgroups modulate the rate of glycosyl oxazoline reduction; electron-withdrawingester groups curtail reaction so that reductive cleavage of benzylethers and esters, or reduction of azide elsewhere in the moleculemay be readily achieved.