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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.

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.

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.

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.

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.

International audience; The recently discovered enzyme Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt), which catalyses the phosphorylation of deoxythymidine monophosphate (dTMP) to give deoxythymidine diphosphate (dTDP), is indispensable for the growth and survival of M. tuberculosis as it plays an essential role in DNA synthesis. Inhibition of TMPKmt is an attractive avenue for the development of novel anti-tuberculosis agents. Based on the premise that sulfamide may be a suitable isostere of phosphate, deoxythymidine analogues comprising various substituted sulfamides at C5′ were modelled in silico into the active site of TMPKmt (PDB accession code: 1N5K) using induced-fit docking methods. A selection of modelled compounds was synthesized, and their activity as inhibitors of TMPKmt was evaluated. Three compounds showed competitive inhibition of TMPKmt in the micromolar range (10–50 μM). Compounds were tested in vitro for anti-mycobacterial activity against M. smegmatis: three compounds showed weak anti-mycobacterial activity (MIC 250 μg/mL).

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.

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.

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.

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 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.

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.

A series of potential UDP-sugar mimics were readily synthesised by copper(I) catalysed modified Huisgen cycloaddition of the corresponding alpha-propargyl glycosides with 5-azido uridine in aqueous solution. None of the compounds accessed displayed significant inhibitory activity at concentrations of up to 4.5mM in an assay against bovine milk beta-1,4-galactosyltransferase.

The total synthesis of the tetrasaccharide Glcα(1→2)Glcα(1→3)Glcα(1→3)ManαOMe, which corresponds to the terminal tetrasaccharide portion of the glucose terminated arm of the N-glycan tetradecasaccharide, was achieved by the use of differentially protected selenoglycosides and thioglycosides as glycosyl donors, all of which possessed non-participating protection of the 2-hydroxyl group. Favourable anomeric stereoselectivity was achieved for the glycosylation reactions by the use of ether as solvent, or co-solvent. Global deprotection by catalytic hydrogenation with palladium acetate in a mixture of ethanol and acetic acid yielded the target tetrasaccharide. © 2002 Published by Elsevier Science Ltd.

Dehydration of both γ- and δ- hexonolactones, either by intramolecular nucleophilic displacement of triflate leaving groups at C-2, or by Mitsunobu type displacement of the OH-6, provides access to bicyclic lactones which contain tetrahydropyran rings. Reduction or nucleophilic ring opening of these bicyclic lactones furnishes polyfunctionalised tetrahydropyrans in good yield.

Tetra-n-propyl ammonium perruthenate in the presence of morpholine-N-oxide induced the unanticipated transformation of the epimeric a-azido-1,4-lactones 4 into the bicyclic amine 3 and provided the key step in the synthesis of 5- epi hydantocidin 2 . Brief investigations into the acid catalysed equilibration of hydantocidin 1 and 5- epi hydantocidin 2 are described.

A five step synthesis of the pseudo C 2 symmetric (1S,2R,6R,7S)-1,2,6,7-tetrahydroxypyrrolizidine from 2,3:5,6-di- O -isopropylidene-D- glycero -D- talo -heptono-1,4-lactone is reported. The effects of structure of some polyhydroxylated pyrrolizidines and pyrrolidines on inhibition of mannosidases are compared.

An efficient aldol condensation of a 2-iodo-5-formyl-1,5-lactone provides a short route to precursors for both cis -β-cyclopentane amino acids and trehalostatin analogues with control of the stereochemistry at all 5 carbons of the cyclopentane ring.

A series of UDP-D-glucose derivatives and precursors that have been modified at C-3 were synthesised from D-glucose as potential chain terminators of beta-glucan biosynthesis. None of the UDP-derivatives or the precursors tested displayed significant anti-fungal activity in a series of germination assays on the dermatophyte Trichophyton rubrum.

Efficient but reversible aldol condensations of 2-azido-5-formyl-1,5-lactones provide short routes to precursors for α-cyclopentane amino acids, some aminopseudosugars and spirohydantoins of cyclopentanes with control of the stereochemistry at all 5 carbons bearing functional groups. The effects of some of the compounds on human liver-glycosidases are described.

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-β-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.

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.

The use of a chiral Brønsted acid catalyst for the activation of trichloroacetimidate glycosyl donors has been demonstrated for the first time. In toluene the chirality of the acid catalyst is seen to influence the stereochemical outcome of the glycosylation processes, hinting that perhaps diastereocontrol of glycosylation processes may become achievable through the judicious use of chiral organic catalysts.

A series of beta-arabino glycosyl sulfones with varying alkyl chain lengths were synthesised in a stereoselective fashion as putative mimics of decaprenolphosphoarabinose (DPA), and as potential inhibitors of mycobacterial cell wall biosynthesis. Biological testing against Mycobacterium bovis BCG revealed low to moderate anti-mycobacterial activity with marked dependence on alkyl chain length, which was maximal for a C-12 chain.

As bacterial, viral and fungal drug resistance to currently administered treatments increase, the need for the development of new therapeutic strategies against infective agents becomes more urgent. Crucial to the survival of many pathogens are carbohydrate structures, which are either themselves structurally unique or specific to nonmammalian organisms. Indeed, inhibition or interference with correct biosynthesis of oligosaccharide materials represents an attractive and potentially highly selective strategy for the development of new classes of therapeutic agents. Over recent years substantial effort has been expended in the search for inhibitors of particular glycosyl transferase and glycosidase enzymes as a means of disrupting specific biosynthetic pathways. The design of such inhibitors can be problematic, since in many cases little is known about the precise enzymes involved. However, an alternative strategy, and one that does not rely on precise structural information about specific enzymes, in particular biochemical pathways, is to exploit structural analysis of the key targeted oligosaccharide structure. This strategy is to invoke chain termination of oligosaccharide biosynthesis. Chain-termination of oligonucleotide synthesis was originally developed by Sanger et al. as a means of sequencing DNA. Subsequently, chain termination strategies have found routine and widespread use in interference with oligonucleotide synthesis, and, moreover, have become the molecular basis of antiviral therapies in clinical use, perhaps most preeminently in the case of AZT. Although chain-termination processes have been implicated in the biological affects of some monosaccharide derivatives on mammalian glycoconjugate and glycosoaminoglycan biosynthesis, it is curious that a chain termination approach has not been widely promulgated as a strategy for the development of new classes of inhibitors of pathogenic oligosaccharide biosynthesis. Although there could be potential pitfalls in this approach, it is our considered opinion that this research route merits ACHTUNGTRENNUNGfurther investigation. For example, in cases in which multiplerepeat units of carbohydrate structures are essential for pathogenic survival (e.g. , in cell-wall formation), then the statistical chances of incorporations that lead to chain termination, would be more favourable. Moreover, there is now good evidence that glycosyl transferases do process activated donor substrates that are modified in a minimal way at a single hydroxyl group. Although certain of these compounds have been shown to act as enzyme inhibitors, many are good substrates that are readily processed. Indeed, as alluded to above, there is also literature precedent that chain-terminating modified carbohydrates have already been incorporated into mammalian glycoprotein oligosaccharides and glycosaminoglycans, with the net results of inhibition of their biosynthesis. As part of a program aimed at investigating the potential opportunities that chain termination of oligosaccharide biosynthesis offers to disease control, we became interested in the rational design of novel antifungal agents. Fungal infections represent a serious hazard to human and animal health, and—just as for bacterial infection—drug resistance to current therapies is increasing. The fungal cell wall consists of large sections of oligosaccharide materials including chitin, a polymer of bACHTUNGTRENNUNG(1–4)N-acetylglucosamine (GlcNAc), and also bglucan. The biosynthesis of both of these nonmammalian oligosaccharides could be potentially targeted by using a chain termination approach. The present study focuses on attempts to inhibit chitin biosynthesis. Chitin is assembled stepwise by the enzyme chitin synthase, which transfers single GlcNAc residues to a growing oligomeric chain; the donor substrate for the enzyme is UDP–GlcNAc. Potential chain terminators of this process are therefore GlcNAc residues in which the 4-hydroxyl has been modified. If such materials are processed by chitin synthase, then their transfer to the terminus of the growing chitin chain will result in a chain-termination step, since the required 4-hydroxyl at which subsequent units would be added will then be absent. Before embarking on the synthesis and testing of potential chain terminators two factors were considered. Firstly, the stability and polarity profile of potential drug compounds was borne in mind. Secondly, as it is a requirement that the potential chain terminator must be actively processed by the chitin synthase, thought was given as to what types of activated glycosyl donor should be synthesised and tested. The most obvious approach was that of synthesising and testing the modified UDP donors themselves, since these are the actual substrates processed by chitin synthase. Alternatively, a potential prodrug molecule could be synthesised, which might then be converted to the active UDP donor once inside the cell. Such a prodrug would have the added advantages of thermal stability and more appropriate polarity as compared to the UDP derivative. In line with previous literature precedent, it was reasoned that perhaps esters of GlcNAc derivatives could act as suitable prodrugs that might be able to penetrate into the cell without relying on carbohydrate-transport mechanisms. Therefore, peresters of GlcNAc derivatives in which the [a] Dr. R. Danac, Dr. T. Muller, Dr. A. J. Fairbanks Chemistry Research Laboratory, Oxford University Mansfield Road, Oxford, OX1 3TA (UK) Fax: (+44)1865-275674 E-mail : antony.fairbanks@chem.ox.ac.uk [b] L. Ball, Prof. S. J. Gurr Department of Plant Sciences, Oxford University South Parks Road, Oxford, OX1 3RB (UK) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

An efficient and completely stereocontrolled synthesis of the N-glycan Manβ(1–4)GlcNAc disaccharide is achieved by propargyl mediated intramolecular aglycon delivery (IAD). Isomerisation of the 2-O-progargyl group of a manno thioglycoside to an allene is followed by iodonium ion mediated mixed acetal formation with the 4-OH of a protected GlcNAc derivative, and subsequent intramolecular glycosylation with complete control of anomeric stereochemistry. Access to this key disaccharide intermediate allows completion of the total synthesis of the core N-glycan pentasaccharide.

Endo-β-N-acetylglucosaminidases (ENGases) are versatile biocatalysts that allow access to a wide variety of defined homogenous N-linked glycoconjugates in a convergent manner. A hybrid-type N-glycan was accessed by total synthesis, converted to an oxazoline, and used as a donor substrate with both wild type Endo M and an N175Q glycosynthase Endo M mutant allowing the convergent synthesis of a glycosylated amino acid bearing a hybrid N-glycan structure.

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.

Carbohydrate thionolactones may be efficiently synthesized from the corresponding 1-thio sugars via a two-step procedure involving formation of a glycosyl phenylthiosulfinate by treatment with either phenylsulfinyl chloride or 1-(phenylsulfinyl)piperidine (BSP), and subsequent thermal elimination in toluene.

Uronic acids, most efficiently synthesised from the corresponding alcohols by two step Dess-Martin and sodium chlorite mediated oxidation, may be used as coupling partners for esterification with an allo glycal as substrates for the tandem Tebbe/Claisen approach to the synthesis of 1-6 linked C-disaccharides. Whilst esters of glucuronic and mannuronic acids successfully undergo Tebbe methylenation, esters derived from galacturonic acids are unreactive under these conditions. Thermal Claisen rearrangement of vinyl ethers produced by methylenation yields α-C-disaccharides with complete control of anomeric stereochemistry.

The use of allyl protecting group mediated intramolecular aglycon delivery (IAD) as a strategy for intramolecular glycosylation has been extended to allow the stereoselective synthesis of α-glucofuranosides and β-rhamnopyranosides, in a totally stereoselective fashion. The efficiency of intramolecular glycosylation is dependent on the protecting group pattern of the glycosyl donor, and on the steric bulk of the glycosyl acceptor.

Site-selective glycosylation by Se-S-mediated ligation has led to the efficient formation of a wide variety of conjugates 1 without the need for a large excess of the carbohydrate reagent. By this convergent method it was possible to introduce a heptasaccharide glycan selectively, and to perform a multiple site-selective chemical glycosylation of protein. A chemically Cysglycosylated glycoprotein was elaborated enzymatically.