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Emerging evidence suggests that host glycans influence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we reveal that the receptor-binding domain (RBD) of the spike (S) protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (Sia), with preference for monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind to the RBD. The monomeric affinities (Kd = 100–200 μM) of gangliosides for the RBD are similar to another negatively charged glycan ligand of the RBD proposed as a viral co-receptor, heparan sulfate (HS) dp2–dp6 oligosaccharides. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to angiotensin-converting enzyme 2 (ACE2)-expressing cells is decreased following depletion of cell surface Sia levels using three approaches: sialyltransferase (ST) inhibition, genetic knockout of Sia biosynthesis, or neuraminidase treatment. These effects on RBD binding and both pseudotyped and authentic SARS-CoV-2 viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2. Mass spectrometric profiling of a glycan library reveals that sialylated glycans, especially sialic acid-containing gangliosides, interact with the RBD of the SARS-CoV-2 spike protein and are involved in ACE2-dependent viral infection.

The central dogma of biology does not allow for the study of glycans using DNA sequencing. We report a liquid glycan array (LiGA) platform comprising a library of DNA 'barcoded' M13 virions that display 30-1,500 copies of glycans per phage. A LiGA is synthesized by acylation of the phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido-modified glycans. Pulldown of the LiGA with lectins followed by deep sequencing of the barcodes in the bound phage decodes the optimal structure and density of the recognized glycans. The LiGA is target agnostic and can measure the glycan-binding profile of lectins, such as CD22, on cells in vitro and immune cells in a live mouse. From a mixture of multivalent glycan probes, LiGAs identify the glycoconjugates with optimal avidity necessary for binding to lectins on living cells in vitro and in vivo.

The biosynthesis of lipopolysaccharide (LPS), a key immunomodulatory molecule produced by gram-negative bacteria, has been a topic of long-term interest. To date, the chemical probes used as tools to study LPS biosynthetic pathways have consisted primarily of small fragments of the larger structure (e.g., the O-chain repeating unit). While such compounds have helped to provide significant insight into many aspects of LPS assembly, understanding other aspects will require larger, more complex probes. For example, the molecular interactions between polymeric LPS biosynthetic intermediates and the proteins that transfer them across the inner and outer membrane remain largely unknown. We describe the synthesis of two lipid-linked polysaccharides, containing 11 and 27 monosaccharide residues, that are related to LPS O-chain biosynthesis in Escherichia coli O9a. This work has led not only to multi-milligram quantities of two biosynthetic probes, but also provided insights into challenges that must be overcome in the chemical synthesis of structurally-defined polysaccharides., The synthesis of lipid-linked polysaccharides containing 11 and 27 monosaccharides via a ‘frame-shift’ strategy is described. The work provides biosynthetic probes and highlights challenges in synthesizing structurally-defined polymeric glycans.

Avocado consumption is associated with numerous health benefits. Avocadyne is a terminally unsaturated, 17-carbon long acetogenin found almost exclusively within avocados with noted anti-leukemia and anti-viral properties. In this study, specific structural features such as the terminal triple bond, odd number of carbons, and stereochemistry are shown to be critical to its ability to suppress mitochondrial fatty acid oxidation and impart selective activity in vitro and in vivo. Together, this is the first study to conduct a structure-activity analysis on avocadyne and outline the chemical moieties critical to fatty acid oxidation suppression.

The arabinogalactan of Corynebacterianeae is a critical heteropolysaccharide that tethers outer membrane mycolic acids to peptidoglycan thus forming the characteristic cell wall core of these prokaryotes. An essential α-(1→5)-arabinosyltransferase, AftA, is responsible for the transfer of the first arabinofuranosyl (Araf) unit of the arabinan domain to the galactan backbone of arabinogalactan, but the number and precise position at which Araf residue(s) is/are added in mycobacteria remain ill-defined. Using membrane preparations from Mycobacterium smegmatis overexpressing aftA, farnesyl-phospho-arabinose as an Araf donor, and a series of synthetic galactan acceptors of various lengths, we here show that a single priming arabinosyl residue substitutes the C-5 position of a precisely positioned internal 6-linked galactofuranosyl residue of the galactan acceptors, irrespective of their length. This unexpected result suggests that, like the structurally related mycobacterial lipoarabinomannans, the arabinogalactan of mycobacteria may in fact harbor a single arabinan chain.

Chloroviruses produce a capsid protein containing N-linked glycans differing in structure from those found in all other organisms. These species feature a core "hyper-branched" fucose residue in which every hydroxyl group is glycosylated. We describe the synthesis of a nonasaccharide from Paramecium bursaria chlorella virus 1, one of most complex chlorovirus N-glycans reported, using a "counterclockwise" strategy involving the sequential addition of trisaccharide, disaccharide, and monosaccharide motifs to a trisaccharide containing the core fucose residue.

We report a siloxane-protected donor (7) for the highly stereoselective formation of β-(2,3-cis)-xylulofuranosyl bonds. Glycosylation reactions with 7 gave >80% yields, and only β-xylulofuranosides were isolated in all cases. The utility of 7 for the synthesis of complex glycans was shown by its successful application to the preparation of the repeating unit from the lipopolysaccharide O-antigen of Yersinia enterocolitica serovars O:5/O:5,27. This structure is a pentasaccharide with two β-xylulofuranose residues; using 7, both were introduced simultaneously with excellent stereocontrol.

International audience; Despite the impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus Mycobacterium tuberculosis adapts its cell wall structure and /composition in response to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes in its structure, but no reports exist supportings this assumption. Herein, using [MS2] recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we identified found that the mycobacterial protein GlfH1 (Rv3096), which protein exhibits an exo-β-D-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal β-(1,5) and β-(1,6)-Galf linkages. The characterization of this galactosidase represents the a first step towards understanding the remodeling of mycobacterial AG.

Lipopolysaccharide O-antigen is an attractive candidate for immunotherapeutic strategies targeting antibiotic-resistant Klebsiella pneumoniae. Several K. pneumoniae O-serotypes are based on a shared O2a-antigen backbone repeating unit: (→ 3)-α-Galp-(1 → 3)-β-Galf-(1 →). O2a antigen is synthesized on undecaprenol diphosphate in a pathway involving the O2a polymerase, WbbM, before its export by an ATP-binding cassette transporter. This dual domain polymerase possesses a C-terminal galactopyranosyltransferase resembling known GT8 family enzymes, and an N-terminal DUF4422 domain identified here as a galactofuranosyltransferase defining a previously unrecognized family (GT111). Functional assignment of DUF4422 explains how galactofuranose is incorporated into various polysaccharides of importance in vaccine production and the food industry. In the 2.1-A resolution structure, three WbbM protomers associate to form a flattened triangular prism connected to a central stalk that orients the active sites toward the membrane. The biochemical, structural and topological properties of WbbM offer broader insight into the mechanisms of assembly of bacterial cell-surface glycans. Structural characterization of WbbM, an enzyme involved in O2a-antigen biosynthesis in Klebsiella pneumoniae, reveals two unique active sites with galactopyranosyl- or galactofuranosyl-transferase activities for oligosaccharide polymerization.

Glycolipids are prominent components of bacterial membranes that play critical roles not only in maintaining the structural integrity of the cell but also in modulating host-pathogen interactions. PatA is an essential acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. We demonstrate by electron spin resonance spectroscopy and surface plasmon resonance that PatA is an integral membrane acyltransferase tightly anchored to anionic lipid bilayers, using a two-helix structural motif and electrostatic interactions. PatA dictates the acyl chain composition of the glycolipid by using an acyl chain selectivity “ruler.” We established this by a combination of structural biology, enzymatic activity, and binding measurements on chemically synthesized nonhydrolyzable acyl–coenzyme A (CoA) derivatives. We propose an interfacial catalytic mechanism that allows PatA to acylate hydrophobic PIMs anchored in the inner membrane of mycobacteria, through the use of water-soluble acyl-CoA donors., This work was supported by the MINECO/FEDER EU contracts BFU2016-77427-C2-2-R, BFU2017-92223-EXP, PID2019-105649RB-I00, Severo Ochoa Excellence Accreditation SEV-2016-0644; the Basque Government contract KK-2019/00076; NIH R01AI149297 (to M.E.G.); the MINECO/FEDER EU contract BFU-2015-68981-P and the Basque Government (IT1264-19) (to F.-X.C.); MINECO/FEDER EU contract RTI2018-099592-B-C21 (to F.C.); the Canadian Glycomics Network Grant SD-1 (to T.L.L.). European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 844905 (to B.T.); the Basque Government (to I.A.); CONACyT fellowship no. 291276 (to E.D.P.-P.).

The outer surfaces of mycobacteria, including the organism that causes tuberculosis, are decorated with an array of immunomodulatory glycans. Among these are lipooligosaccharides (LOSs), a class of molecules for which the function remains poorly understood. We describe the chemical synthesis of the glycan portion of a tridecasaccharide LOS from the opportunistic pathogen Mycobacterium kansasii. The target contains a number of unusual structural motifs that complicate its assembly and is the most complex mycobacterial LOS glycan to be synthesized to date when considering size and number of unique monosaccharides and glycosidic linkages. These studies not only provide a roadmap for the preparation of additional members of this family of glycans, but also provides a valuable probe for use in structure-activity relationship investigations.

Lipopolysaccharide is an important immunomodulatory and structural component found in the outer membrane of Gram-negative bacteria. The biosynthesis of this glycoconjugate proceeds by a highly conserved pathway and, as such, is an attractive target for antibiotic action. We highlight here recent work focused on understanding this pathway with an emphasis on new insights related to chain length control and transport across the inner and outer cell membranes.

The de novo asymmetric synthesis of all possible stereoisomers of two polyketide natural products, avocadyne, avocadene, and the saturated variant avocadane, is described. The stereodivergent synthesis of the 12 congeners is accomplished in 4-6 steps from an achiral acylpyruvate derivative, which, in turn, is prepared in five steps from commercially available materials. The approach uses, sequentially, a Noyori asymmetric reduction, a diastereoselective chelate- or directed reduction of a β-hydroxyketone, and an ester reduction to a primary alcohol.

Bacterial capsular polysaccharides are important virulence factors. Capsular polysaccharides from several important Gram-negative pathogens share a conserved glycolipid terminus containing 3-deoxy-β-d- manno-oct-2-ulosonic acid (β-Kdo). The β-Kdo glycosyltransferases responsible for synthesis of this conserved glycolipid belong to a new family of glycosyltransferases that shares little homology with other such enzymes, thereby representing an attractive antivirulence target. Here, we report the development of a fluorescence polarization-based, high-throughput screening assay (FP-tag) for β-Kdo glycosyltransferases, and use it to identify a class of marine natural products as lead inhibitors. This "FP-tag" assay should be readily adaptable to high-throughput screens of other glycosyltransferases.

Emerging evidence suggests that host glycans influence infection by SARS-CoV-2. Here, we reveal that the receptor-binding domain (RBD) of the spike (S)-protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (SA), with preference for the oligosaccharide of monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind the RBD. The monomeric affinities (Kd = 100-200 μM) of gangliosides for the RBD are similar to heparan sulfate, another negatively charged glycan ligand of the RBD proposed as a viral co-receptor. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to ACE2-expressing cells is decreased upon depleting cell surface SA level using three approaches: sialyltransferase inhibition, genetic knock-out of SA biosynthesis, or neuraminidase treatment. These effects on RBD binding and pseudotyped viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.

Paramecium bursaria chlorella virus-1 (PBCV-1) is a large double-stranded DNA (dsDNA) virus that infects the unicellular green alga Chlorella variabilis NC64A. Unlike many other viruses, PBCV-1 encodes most, if not all, of the enzymes involved in the synthesis of the glycans attached to its major capsid protein. Importantly, these glycans differ from those reported from the three domains of life in terms of structure and asparagine location in the sequon of the protein. Previous data collected from 20 PBCV-1 spontaneous mutants (or antigenic variants) suggested that the a064r gene encodes a glycosyltransferase (GT) with three domains, each with a different function. Here, we demonstrate that: domain 1 is a ?-L-rhamnosyltransferase; domain 2 is an ?-L-rhamnosyltransferase resembling only bacterial proteins of unknown function, and domain 3 is a methyltransferase that methylates the C-2 hydroxyl group of the terminal ?-L-rhamnose (Rha) unit. We also establish that methylation of the C-3 hydroxyl group of the terminal ?-L-Rha is achieved by another virus-encoded protein A061L, which requires an O-2 methylated substrate. This study, thus, identifies two of the glycosyltransferase activities involved in the synthesis of the N-glycan of the viral major capsid protein in PBCV-1 and establishes that a single protein A064R possesses the three activities needed to synthetize the 2-OMe-?-L-Rha-(1->2)-?-L-Rha fragment. Remarkably, this fragment can be attached to any xylose unit.

Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) in the membranes of cells are implicated in a wide variety of normal and pathophysiological processes. Despite the critical biological roles these interactions play, the GSL ligands of most GBPs have not yet been identified. The limited availability of purified GSLs represents a significant challenge to the discovery and characterization of biologically relevant GBP-GSL interactions. The present work investigates the use of neoglycolipids (NGLs) as surrogates for GSLs for catch-and-release-electrospray ionization mass spectrometry (CaR-ESI-MS)-based screening, implemented with nanodiscs, for the discovery of GSL ligands. Three pairs of NGLs based on the blood group type A and B trisaccharides, with three different lipid head groups but all with "ring-closed" monosaccharide residue at the reducing end, were synthesized. The incorporation efficiencies (into nanodiscs) of the NGLs and their affinities for a fragment of family 51 carbohydrate-binding module (CBM) identified an amide-linked 1,3-di-O-hexadecyl-glycerol moiety as the optimal lipid structure. Binding measurements performed on cholera toxin B subunit homopentamer (CTB5) and nanodiscs containing an NGL consisting of the optimal lipid moiety and the GM1 ganglioside pentasaccharide yielded affinities similar, within a factor of 2, to those of native GM1. Finally, nanodiscs containing the optimal A and B trisaccharide NGLs, as well as the corresponding NGLs of lactose, A type 2 tetrasaccharide, and the GM1 and GD2 pentasaccharides were screened against the family 51 CBM, human galectin-7, and CTB5 to illustrate the potential of NGLs to accelerate the discovery of GSL ligands of GBPs.

Arabinosyltransferase B (EmbB) belongs to a family of membrane-bound glycosyltransferases that build the lipidated polysaccharides of the mycobacterial cell envelope, and are targets of anti-tuberculosis drug ethambutol. We present the 3.3 Å resolution single-particle cryo-electron microscopy structure of Mycobacterium smegmatis EmbB, providing insights on substrate binding and reaction mechanism. Mutations that confer ethambutol resistance map mostly around the putative active site, suggesting this to be the location of drug binding., The cryo-EM structure of Mycobacterium smegmatis arabinosyltransferase B EmbB involved in mycobacterial cell wall biosynthesis provides insights into the substrate binding and reaction mechanism. Mapping of the ethambutol resistance associated mutations onto the structure suggests the location of the drug binding site.

The Central Dogma of Biology does not allow for the study of glycans using DNA sequencing. We report a “Liquid Glycan Array” (LiGA) platform comprising a library of DNA ‘barcoded’ M13 virions that display 30-1500 copies of glycans per phage. A LiGA is synthesized by acylation of phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido-modified glycans. Pulldown of the LiGA with lectins followed by deep sequencing of the barcodes in the bound phage decodes the optimal structure and density of the recognized glycans. The LiGA is target agnostic and can measure the glycan-binding profile of lectins such as CD22 on cells in vitro and immune cells in a live mouse. From a mixture of multivalent glycan probes, LiGAs identifies the glycoconjugates with optimal avidity necessary for binding to lectins on living cells in vitro and in vivo; measurements that cannot be performed with canonical glass slide-based glycan arrays.DedicationThe paper is dedicated to Laura L. Kiessling on the occasion of her 60th birthday.

Reported is the first stereoselective method for β-xylulofuranosylation, which employs 3,4-O-xylylene-protected thioglycoside donors. For most acceptors, the best results were observed with a donor (8) that possesses both the xylylene group and a benzoate ester at O-1. To demonstrate its utility, the methodology was applied to the first synthesis of the pentasaccharide repeating unit from the lipopolysaccharide O-antigen of Yersinia enterocolitica serovars O:5/O:5,27, a structure containing two β-xylulofuranose residues.

The design and synthesis of multivalent ligands displaying complex oligosaccharides is necessary for the development of therapeutics, diagnostics, and research tools. Here, we report an efficient conjugation strategy to prepare complex glycoconjugates with 4 copies of 1 or 2 separate glycan epitopes, providing 4-8 carbohydrate residues on a tetravalent poly(ethylene glycol) scaffold. This strategy provides complex glycoconjugates that approach the size of glycoproteins (15-18 kDa) while remaining well-defined. The synthetic strategy makes use of three orthogonal functional groups, including a reactive N-hydroxysuccinimide (NHS)-ester moiety on the linker to install the first carbohydrate epitope via reaction with an amine. A masked amine functionality on the linker is revealed after the removal of a fluorenylmethyloxycarbonyl (Fmoc)-protecting group, allowing the attachment to the NHS-activated poly(ethylene glycol) (PEG) scaffold. An azide group in the linker was then used to incorporate the second carbohydrate epitope via catalyzed alkyne-azide cycloaddition. Using a known tetravalent PEG scaffold (PDI, 1.025), we prepared homofunctional glycoconjugates that display four copies of lactose and the A-type II or the B-type II human blood group antigens. Using our trifunctional linker, we expanded this strategy to produce heterofunctional conjugates with four copies of two separate glycan epitopes. These heterofunctional conjugates included Neu5Ac, 3'-sialyllactose, or 6'-sialyllactose as a second antigen. Using an alternative strategy, we generated heterofunctional conjugates with three copies of the glycan epitope and one fluorescent group (on average) using a sequential dual-amine coupling strategy. These conjugation strategies should be easily generalized for conjugation of other complex glycans. We demonstrate that the glycan epitopes of heterofunctional conjugates engage and cluster target B-cell receptors and CD22 receptors on B cells, supporting the application of these reagents for investigating cellular response to carbohydrate antigens of the ABO blood group system.

Accurate identification of tuberculosis (TB), caused by Mycobacterium tuberculosis, is important for global disease management. Point-of-care serological tests may improve TB diagnosis; however, specificities of available serodiagnostics are sub-optimal. We employed genetically encoded fragment-based discovery (GE-FBD) to select ligands for antibodies directed against the mycobacterial cell wall component lipoarabinomannan (LAM), a potent antigen. GE-FBD employed a phage displayed library of 108 heptapeptides, chemically modified with an arabinofuranosyl hexasaccharide fragment of LAM (Ara6), and the anti-LAM antibody CS-35 as a bait. The selection gave rise to glycopeptides with an enhanced affinity and selectivity for CS-35 but not for 906.4321 antibody, both of which bind to Ara6 with a comparable affinity. Multivalent assays incorporating the discovered ligands Ara6-ANSSFAP, Ara6-DAHATLR and Ara6-TTYVVNP exhibited up to 19-fold discrimination between CS-35 and 906.4321. The use of the Ara6 antigen alone failed to distinguish these antibodies. Thus, GE-FBD gives rise to ligands that differentiate monoclonal antibodies with enhanced specificity. This technology could facilitate the development of effective point-of-care serological tests for mycobacterial and other infections.

The convenient preparation of 1,4-dideoxy-1,4-imino-L-arabinitol derivatives carrying a propargyl group by way of the addition of TMS-propargyl Grignard reagent to a N-sulfinylglycosylamine is reported. This compound was coupled via a triazole-tether to various aromatic and heteroaromatic groups, thus providing structures that can be considered as simplified mimics of UDP-Galf, the substrate of GlfT2, a galactofuranosyltransferase present in mycobacteria. These new compounds were evaluated as inhibitors of this enzyme and found to have significant activity (IC50 values in the high M to low mM range) in spite of the absence of the diphospho linkage present in the parent sugar nucleotide.

Attachment of human noroviruses to histo blood group antigens (HBGAs) is thought to be critical for the infection process. Therefore, we have determined binding epitopes of synthetic type 1 to 6 blood group A- and B-tetrasaccharides binding to GII.4 human Norovirus virus like particles (VLPs) using STD NMR experiments. So far, little information is available from crystal structure analysis studies on the interactions of the reducing-end sugars with the protruding domain (P-domain) of the viral coat protein VP1. Here, we show that the reducing-end sugars make notable contacts with the protein surface. The type of glycosidic linkage, and the identity of the sugar at the reducing end modulate HBGA recognition. Most strikingly, type 2 structures yield only very poor saturation transfer indicating impeded binding. This observation is in accordance with previous mass spectrometry based affinity measurements, and can be understood based on recent crystal structure data of a complex of highly homologous GII.4 P-dimers with H-type 2 trisaccharide where the N-acetyl group of the reducing N-acetyl glucosamine residue points towards a loop comprising amino acids Q390 to H395. We suggest that in our case, binding of type 2 A- and B-tetrasaccharides leads to steric conflicts with this loop. In order to identify factors determining L-Fuc recognition, we also synthesized GII.4 VLPs with point mutations D391A and H395A. Prior studies had suggested that these residues, located in a second shell around the L-Fuc binding site, assist L-Fuc binding. STD NMR experiments with L-Fuc and B-trisaccharide in the presence of wild type and mutant VLPs yield virtually identical binding epitopes suggesting that these two mutations do not significantly alter HBGA recognition. Our study emphasizes that recognition of α-(1→2)-linked L-Fuc residues is a conserved feature of GII.4 noroviruses. However, structural variation of the HBGA core structures clearly modulates molecular recognition depending on the genotype.

SUMMARYMycobacterium tuberculosiscauses tuberculosis, a disease that kills over one million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides – arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9 Å resolution structure ofM. abscessusAftD determined by single particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4 and 3.5 Å structures of a mutant with impaired ACP binding reveal a conformational change that suggests the ACP may regulate AftD function. Using a conditional knock-out constructed inM. smegmatis, mutagenesis experiments confirm the essentiality of the putative active site and the ACP binding for AftD function.

A limited range of different structures is observed in O-antigenic polysaccharides (OPSs) from Klebsiella pneumoniae lipopolysaccharides. Among these, several are based on modifications of a conserved core element of serotype O2a OPS, which has a disaccharide repeat structure [→3)-α-d-Galp-(1→3)-β-d-Galf-(1→]. Here, we describe the enzymatic pathways for a highly unusual modification strategy involving the attachment of a second glycan repeat-unit structure to the nonreducing terminus of O2a. This occurs by the addition of the O1 [→3)-α-d-Galp-(1→3)-β-d-Galp-(1→] or O2c [→3)-β-d-GlcpNAc-(1→5)-β-d-Galf-(1→] antigens. The organization of the enzyme activities performing these modifications differs, with the enzyme WbbY possessing two glycosyltransferase catalytic sites solely responsible for O1 antigen polymerization and forming a complex with the O2a glycosyltransferase WbbM. In contrast, O2c polymerization requires glycosyltransferases WbmV and WbmW, which interact with one another but apparently not with WbbM. Using defined synthetic acceptors and site-directed mutants to assign the activities of the WbbY catalytic sites, we found that the C-terminal WbbY domain is a UDP-Galp–dependent GT-A galactosyltransferase adding β-(1→3)–linked d-Galp, whereas the WbbY N terminus includes a GT-B enzyme adding α-(1→3)–linked d-Galp. These activities build the O1 antigen on a terminal Galp in the O2a domain. Using similar approaches, we identified WbmV as the UDP-GlcNAc transferase and noted that WbmW represents a UDP-Galf–dependent enzyme and that both are GT-A members. WbmVW polymerizes the O2c antigen on a terminal Galf. Our results provide mechanistic and conceptual insights into an important strategy for polysaccharide antigen diversification in bacteria.

Similar to other prokaryotes, mycobacteria decorate their major cell envelope glycans with minor covalent substituents whose biological significance remains largely unknown. We report on the discovery of a mycobacterial enzyme, named here SucT, that adds succinyl groups to the arabinan domains of both arabinogalactan (AG) and lipoarabinomannan (LAM). Disruption of the SucT-encoding gene in Mycobacterium smegmatis abolished AG and LAM succinylation and altered the hydrophobicity and rigidity of the cell envelope of the bacilli without significantly altering AG and LAM biosynthesis. The changes in the cell surface properties of the mutant were consistent with earlier reports of transposon mutants of the closely related species Mycobacterium marinum and Mycobacterium avium harboring insertions in the orthologous gene whose ability to microaggregate and form biofilms were altered. Our findings point to an important role of SucT-mediated AG and LAM succinylation in modulating the cell surface properties of mycobacteria.

An approach for the assembly of polyprenol pyrophosphate-based probes of O-polysaccharide biosynthesis in Klebsiella pneumoniae serotype O2a is described. This convergent route features high-yielding, diastereoselective glycosylations and the late-stage installation of the polyprenol pyrophosphate moiety. Although applied to the synthesis of a nonasaccharide bearing a farnesyl group (1), the modular nature of the route makes it amenable to the synthesis of additional derivatives containing either larger glycans or different lipid domains.

The synthesis of bradyrhizose, the monosaccharide component of the lipopolysaccharide O-antigen of the nitrogen-fixing bacteria Bradyrhizobium sp. BTAi1 and sp. ORS278, has been achieved in 25 steps in an overall yield of 6% using myo-inositol and ethyl propiolate as the starting materials. The route involved the late-stage resolution of a racemic intermediate to provide both enantiomers of this unusual bicyclic monosaccharide. Both the natural d-enantiomer, and the unnatural and heretofore unknown l-enantiomer, were converted to disaccharide derivatives containing different forms of the monosaccharide (d,d; l,l; d,l; l,d). Evaluation of the synthetic compounds for their ability to act as microbe-associated molecular patterns in plants, through induction of reactive oxygen species, was investigated. These experiments suggest that the immunologically silent nature of the natural glycans is due to specific structural features.

In this study, we designed a method to prepare 2-deoxy-2-azido-α-d-galactopyranosidic bonds using 4,6-di-O-benzylidenyl-3-O-t-butyldiphenylsilyl protected 2-deoxy-2-azido-1-thio-d-galactopyranoside 5 as donors. The donor 5 gives a good to excellent α-selectivity in the glycosylation with secondary alcohols, which was found to be associated with the benzylidenyl on 4,6-di-O and TBDPS on 3-O of the donor 5. Compared with results of the donor 6 and 7, the 3-O-TBDPS could increase the activity of the thioglycoside, and the lone pairs on 4,6-di-O-benzylidenyl group enhanced the gg-cofnormation, which plays a role in improving the stereoselectivity. Finally, this method was demonstrated through the synthesis of a α-galactosamine -containing pentasaccharide 26.

Mycobacteria, including the human pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis, produce a complex cell wall structure made of carbohydrates and lipids. The major structural element of the mycobacterial cell wall is a glycoconjugate called the mycolic acid – arabinogalactan – peptidoglycan (mAGP) complex. Inhibition of mAGP biosynthesis is a proven strategy for developing anti-mycobacterial drugs, and thus, understanding the pathways and enzymes involved in the assembly of this molecule is of interest. In this paper, we describe the chemical synthesis of a panel of nine oligosaccharide fragments (4–12) of the galactan domain of the mAGP complex designed as biosynthetic probes. These structures, ranging in size from a hexasaccharide to a tetradecasaccharide, are potential substrates for two biosynthetic enzymes, GlfT2 and AftA, and represent the largest mycobacterial galactan fragments synthesized to date. The route developed was iterative and provided multimilligram quantities of the target molecules 4–12 in good overall yield.

Mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce a complex cell wall structure that is essential to survival. A key component of this structure is a glycoconjugate, the mycolyl-arabinogalactan-peptidoglycan complex, which has at its core a galactan domain composed of galactofuranose (Galf) residues linked to peptidoglycan. Because galactan biosynthesis is essential for mycobacterial viability, compounds that interfere with this process are potential therapeutic agents for treating mycobacterial diseases, including tuberculosis. Galactan biosynthesis in mycobacteria involves two glycosyltransferases, GlfT1 and GlfT2, which have been the subject of increasing interest in recent years. This Synopsis summarizes efforts to characterize the mechanism and specificity of GlfT2, which is responsible for introducing the majority of the Galf residues into mycobacterial galactan.

We report a detailed study of the structure and stability of carbohydrate-lipid interactions. Complexes of a methylmannose polysaccharide (MMP) derivative and fatty acids (FAs) served as model systems. The dependence of solution affinities and gas-phase dissociation activation energies (Ea ) on FA length indicates a dominant role of carbohydrate-lipid interactions in stabilizing (MMP+FA) complexes. Solution (1) H NMR results reveal weak interactions between MMP methyl groups and FA acyl chain; MD simulations suggest the complexes are disordered. The contribution of FA methylene groups to the Ea is similar to that of heats of transfer of n-alkanes from the gas phase to polar solvents, thus suggesting that MMP binds lipids through dipole-induced dipole interactions. The MD results point to hydrophobic interactions and H-bonds with the FA carboxyl group. Comparison of collision cross sections of deprotonated (MMP+FA) ions with MD structures suggests that the gaseous complexes are disordered.

A route for the synthesis of glycuronamides via the intermediacy of 6-S-tolyl-substituted glycosides and uronic acid thioesters, is reported. The route, which is compatible with a variety of carbohydrate residues and protecting groups, was used to synthesize the repeating unit of the E. coli K54 capsular polysaccharide.

Silica microparticles were functionalized with A and B blood group carbohydrate antigens (A type I, A type II, B type I, and B type II) to enable the detection and monitoring of ABO antigen-specific B cells. Microparticles were prepared via the Stöber synthesis, labeled with an Alexafluor fluorescent dye, and characterized via TEM and fluorescence microscopy. The silica microparticles were functionalized with (3-aminopropyl)trimethoxysilane (APTMS), followed by the use of an established fluorenylmethyloxycarbonyl (Fmoc)-protected PEG-based linker. The terminal Fmoc moiety of the PEG-based linker was then deprotected, yielding free amino groups, to which the A and B antigens were coupled. The carbohydrate antigens were synthesized with a p-nitrophenol ester to enable conjugation to the functionalized silica microparticles via an amide bond. The number of free amine groups available for coupling for a given mass of PEG-functionalized silica microparticles was quantified via reaction with Fmoc-glycine. The antigen-functionalized microparticles were then evaluated for their specificity in binding to A and B antigen-reactive B-cells via flow cytometry, and for blocking of naturally occurring antibodies in human serum. Selective binding of the functionalized microparticles to blood group-reactive B cells was observed by flow cytometry and fluorescence microscopy. The modular approach outlined here is applicable to the preparation of silica microparticles containing any carbohydrate antigen and alternative fluorophores or labels. This approach therefore comprises a novel, general platform for screening B cell populations for binding to carbohydrate antigens, including, in this case, the human A and B blood group antigens.

Several important Gram-negative bacterial pathogens possess surface capsular layers composed of hypervariable long-chain polysaccharides linked via a conserved β-Kdo oligosaccharide to a phosphatidylglycerol residue. The pathway for synthesis of the terminal glycolipid was elucidated by determining structures of reaction intermediates. In Escherichia coli, KpsS transfers a single Kdo residue to phosphatidylglycerol; this primer is extended using a single enzyme (KpsC), possessing two CMP-Kdo-dependent glycosyltransferase catalytic centres with different linkage specificities. The structure of the N-terminal β-(2→4)-Kdo transferase from KpsC reveals two α/β domains, supplemented by several additional helices. The N-terminal Rossmann-like domain, typically responsible for acceptor binding, is severely reduced in size compared to canonical GT-B folds in glycosyltransferases. The similar structure of the C-terminal β-(2→7)-Kdo transferase indicates a past gene duplication event. Both Kdo transferases have a narrow active site tunnel, lined with key residues shared with GT99 β-Kdo transferases. This enzyme provides the prototype for the GT107 family. Canadian Institutes of Health Research, Natural Sciences and Engineering Research Council, Canadian Glycomics Network, Canada Research Chairs

Chlorella viruses produce N-linked glycoproteins with carbohydrate moieties that differ in structure from all other N-linked glycans. In addition, unlike most viruses, these organisms do not hijack the biosynthetic machinery of the host to make glycocoproteins; instead, they produce their own carbohydrate-processing enzymes. A better understanding of the function and assembly of these fascinating and structurally-unprecedented glycans requires access to probe molecules. This work describes the first synthesis of a chlorella virus N-linked glycan, a highly branched hexasaccharide that contains the pentasaccharide present in all of the >15 structures reported to date. The target molecule includes a glucosyl-asparagine linkage and a "hyperbranched" fucose residue in which all of the hydroxyl groups are glycosylated. Both convergent and linear approaches were investigated with the latter being successful in providing the target in 16 steps and 13 % overall yield.

The O-methyl phosphoramidate (MeOPN) motif is a non-stoichiometric modification of capsular polysaccharides (CPS) in ≈70 % of all Campylobacter jejuni strains. Infections by C. jejuni lead to food-borne illnesses and the CPS they produce are key virulence factors. The MeOPN phosphorus atom in these CPS is stereogenic and is found as a single stereoisomer. However, to date, the absolute stereochemistry at this atom has been undefined. We report the synthesis of the three repeating units found in C. jejuni 81-176 CPS; one of these possesses a MeOPN group. In the course of these studies we established that the stereochemistry of the phosphorus atom in this MeOPN group is R. These studies represent the first unequivocal proof of stereochemistry of this group in any C. jejuni CPS. The compounds produced are anticipated to be useful tools in investigations targeting the function and biosynthesis of this structurally-interesting modification, which so far has only been identified in campylobacter.

The stereocontrolled synthesis of 2-deoxy-d- arabino-hexopyranosides ("galactopyranosides") using 3,4- O-isopropylidene-6- O- tert-butyldiphenylsilyl-protected glycosyl donors is reported. 2-Deoxy-thioglycoside 3e gives excellent α-selectivity, while galactal 9 leads to, in a two-step protocol, 2-deoxy-β-glycosides in high stereoselectivity. The selectivity of both reagents is believed to arise from the combination of the isopropylidene acetal spanning O-3 and O-4 together with the sterically demanding silyl group on O-6. The utility of the method was demonstrated through the synthesis of a trisaccharide that contains both 2-deoxy α- and β-d-galactopyranosyl residues.

Mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce a complex cell wall that is critical for their survival. The largest structural component of the cell wall, the mycolyl-arabinogalactan-peptidoglycan complex, has at its core a galactan domain composed of d-galactofuranose residues. Mycobacterial galactan biosynthesis has been proposed to involve two glycosyltransferases, GlfT1 and GlfT2, which elongate polyprenol-pyrophosphate linked glycosyl acceptor substrates using UDP-galactofuranose as the donor substrate. We here report the first chemical synthesis of GlfT1 and GlfT2 acceptor substrates containing pyrophosphate and polyprenol moieties (compounds 3, 4, 22 and 23). The approach involves chemical synthesis of an oligosaccharide, subsequent phosphorylation at the reducing end and coupling to a polyprenol phosphate. These compounds were shown to be substrates for either GlfT1 (22 and 23) or GlfT2 (3 and 4) and all were substantially more active than the corresponding alkyl glycoside substrates reported previously. Mass spectrometric analysis of the products formed from the reaction of 3, 4, 22 and 23 with the respective cognate enzyme and UDP-galactofuranose provide additional evidence for the galactan biosynthetic model in which GlfT1 adds the first two galactofuranose residues with the remainder being installed via GlfT2. Overall, these results highlight the importance of the pyrophosphate motif in recognition of acceptor substrates by both enzymes and demonstrate a straightforward route for the preparation of such compounds. The work also provides additional support for the process by which this important glycan is biosynthesized using, for the first time, close structural analogs to the natural substrates.

TB diagnosis and treatment monitoring in resource limited regions rely heavily on serial sputum smear microscopy and bacterial culture. These microbiological methods are time-consuming, expensive and lack adequate sensitivity. The WHO states that improved TB diagnosis and treatment is imperative to achieve an end to the TB epidemic by 2030. Commercially available lipoarabinomannan (LAM) detection tools perform at low sensitivity that are highly dependent on the underlying immunological status of the patient; those with advanced HIV infection perform well. In this study, we have applied two novel strategies towards the sensitive diagnosis of TB infection based on LAM: Capture ELISA to detect LAM in paired urine and serum samples using murine and human monoclonal antibodies, essentially relying on LAM as an ‘immuno-marker’; and, secondly, detection of α- d -arabinofuranose and tuberculostearic acid (TBSA)- ‘chemical-markers’ unique to mycobacterial cell wall polysaccharides/lipoglycans by our recently developed gas chromatography/mass spectrometry (GC/MS) method. Blinded urine specimens, with microbiologically confirmed active pulmonary TB or non TB (HIV+/HIV–) were tested by the aforementioned assays. LAM in patient urine was detected in a concentration range of 3–28 ng/mL based on GC/MS detection of the two LAM-surrogates, d -arabinose and tuberculostearic acid (TBSA) correctly classifying TB status with sensitivity > 99% and specificity = 84%. The ELISA assay had high sensitivity (98%) and specificity (92%) and the results were in agreement with GC/MS analysis. Both tests performed well in their present form particularly for HIV-negative/TB-positive urine samples. Among the HIV+/TB+ samples, 52% were found to have >10 ng/mL urinary LAM. The detected amounts of LAM present in the urine samples also appears to be associated with the gradation of the sputum smear, linking elevated LAM levels with higher mycobacterial burden (odds ratio = 1.08–1.43; p = 0.002). In this small set, ELISA was also applied to parallel serum samples confirming that serum could be an additional reservoir for developing a LAM-based immunoassay for diagnosis of TB.

International audience; The convenient preparation of iminopentitol derivatives, based on a 1,4-dideoxy-1,4-imino-l-arabinitol scaffold carrying β-phosphono(difluoromethyl) or β-phosphonomethyl appendages, as Galf-1P mimics, is reported. The compounds were tested for their ability to inhibit GlfT2, a vital galactofuranosyltransferase involved in the cell wall biosynthesis of mycobacteria. Interestingly, the Galf-1P mimics lacking a fluorine atom (7 and 8) were very poor inhibitors, showing less than 20% inhibition of GlfT2, whereas compounds 2 and 3, which contains a difluoromethylenephosphonate moiety were more potent inhibitors. Compound 3 that is fully deprotected was the most potent showing a significant IC50 value (0.9 mm), despite the absence of the diphosphate linkage present in the parent sugar nucleotide. This study paves the way to the synthesis of more complex β-phosphonomethyl-imino-l-arabinitol derivatives as simplified mimics of UDP-α-d-Galf.

Mycobacteria produce a number of ‘extractable glycolipids’, which are present at the outer periphery of their cell wall structure. These molecules are believed to play important roles in mycobacteriahost interactions, although for many of these species the manner in which they mediate biological events is poorly understood. We summarize here the structures of one class of these molecules (lipooligosaccharides), highlight briefly their biological role, and discuss synthetic work done to access this complex and diverse group of bacterial glycolipids.

A convergent strategy was developed for the stereoselective synthesis of four unusual N-acylated monosaccharides (5-8), which are fragments of lipooligosaccharide IV (LOS-IV) from Mycobacterium marinum. A critical substrate-controlled asymmetric cyclization of an amino acid derived oxazolidine provided a key lactam intermediate 11, which was successfully converted to targets 5-7. The key step in the synthesis of 8 was a one-pot cascade oxidation-cyclization-oxidation reaction of a Boc-protected amino alcohol, prepared from 3-butynol, which led to the formation of lactam 15. The five-membered ring lactam intermediates in these synthetic routes were sensitive to elimination side reactions, but careful manipulation of the reaction sequence allowed for the stereoselective synthesis of the targets. This work represents the first synthesis of these unusual motifs, which have been shown to be essential to the bioactivity of LOS-IV.

The cell wall of pathogenic mycobacteria is abundant with virulence factors, among which phenolic glycolipids (PGLs) are prominent examples. Mycobacterium kansasii, an important opportunistic pathogen, produces seven PGLs and their effect on the release of important proinflammatory cytokines that mediate disease progression has not been investigated. We previously showed that proinflammatory cytokines are modulated by PGLs from M. tuberculosis, M. leprae and M. bovis. In this paper we describe the synthesis of a series of 17 analogs of M. kansasii PGLs containing a truncated aglycone. Subsequently, the effect of these compounds on the release of proinflammatory cytokines (TNF-α, IL-6, IL-1β, MCP-1) and nitric oxide (NO) was evaluated. These compounds exerted an immunoinhibitory effect on the release of the tested cytokines. The concentration-dependent inhibitory profile of the tested molecules was also found to be dependent on the methylation pattern of the molecule and was mediated via toll-like receptor (TLR)-2. This study led to the discovery of a glycolipid (18) that shows promising potent anti-inflammatory properties making it a potential candidate for further optimization of its anti-inflammatory profile.