Engineering a therapeutic lectin by uncoupling mitogenicity from antiviral activity.

A key effector route of the Sugar Code involves lectins that exert crucial regulatory controls by targeting distinct cellular glycans. We demonstrate that a single amino-acid substitution in a banana lectin, replacing histidine 84 with a threonine, significantly reduces its mitogenicity, while preserving its broad-spectrum antiviral potency. X-ray crystallography, NMR spectroscopy, and glycocluster assays reveal that loss of mitogenicity is strongly correlated with loss of pi-pi stacking between aromatic amino acids H84 and Y83, which removes a wall separating two carbohydrate binding sites, thus diminishing multivalent interactions. On the other hand, monovalent interactions and antiviral activity are preserved by retaining other wild-type conformational features and possibly through unique contacts involving the T84 side chain. Through such fine-tuning, target selection and downstream effects of a lectin can be modulated so as to knock down one activity, while preserving another, thus providing tools for therapeutics and for understanding the Sugar Code.

A single sulfatase is required to access colonic mucin by a gut bacterium.

Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates these microorganisms from the intestinal epithelium1. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate degradation of the complex O-glycans found in mucins. In the distal colon, these glycans are heavily sulfated, but specific sulfatases that are active on colonic mucins have not been identified. Here we show that sulfatases are essential to the utilization of distal colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases produced by this species, showing that they are collectively active on all known sulfate linkages in O-glycans. Crystal structures of three enzymes provide mechanistic insight into the molecular basis of substrate specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also has a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by a prominent group of gut bacteria, an important process for both normal microbial gut colonization2 and diseases such as inflammatory bowel disease3.

The structure of a C. neoformans polysaccharide motif recognized by protective antibodies: A combined NMR and MD study.

Cryptococcus neoformans is a fungal pathogen responsible for cryptococcosis and cryptococcal meningitis. The C. neoformans' capsular polysaccharide and its shed exopolysaccharide function both as key virulence factors and to protect the fungal cell from phagocytosis. Currently, a glycoconjugate of these polysaccharides is being explored as a vaccine to protect against C. neoformans infection. In this study, NOE and J-coupling values from NMR experiments were consistent with a converged structure of the synthetic decasaccharide, GXM10-Ac3, calculated from MD simulations. GXM10-Ac3 was designed as an extension of glucuronoxylomannan (GXM) polysaccharide motif (M2) which is common in the clinically predominant serotype A strains and is recognized by protective forms of GXM-specific monoclonal antibodies. The M2 motif is a hexasaccharide with a three-residue α-mannan backbone, modified by ß-(1→2)-xyloses (Xyl) on the first two mannoses (Man) and a ß-(1→2)-glucuronic acid (GlcA) on the third Man. Combined NMR and MD analyses reveal that GXM10-Ac3 adopts an extended structure, with Xyl/GlcA branches alternating sides along the α-mannan backbone. O-acetyl esters also alternate sides and are grouped in pairs. MD analysis of a twelve M2-repeating unit polymer supports the notion that the GXM10-Ac3 structure is uniformly represented throughout the polysaccharide. This derived GXM model displays high flexibility while maintaining a structural identity, yielding insights to further explore intermolecular interactions between polysaccharides, interactions with anti-GXM mAbs, and the cryptococcal polysaccharide architecture.

A glycan FRET assay for detection and characterization of catalytic antibodies to the Cryptococcus neoformans capsule.

Classic antibody functions include opsonization, complement activation, and enhancement of cellular antimicrobial function. Antibodies can also have catalytic activity, although the contribution of catalysis to their biological functions has been more difficult to establish. With the ubiquity of catalytic antibodies against glycans virtually unknown, we sought to advance this knowledge. The use of a glycan microarray allowed epitope mapping of several monoclonal antibodies (mAbs) against the capsule of Cryptococcus neoformans From this, we designed and synthesized two glycan-based FRET probes, which we used to discover antibodies with innate glycosidase activity and analyze their enzyme kinetics, including mAb 2H1, the most efficient identified to date. The validity of the FRET assay was confirmed by demonstrating that the mAbs mediate glycosidase activity on intact cryptococcal capsules, as observed by a reduction in capsule diameter. Furthermore, the mAb 18B7, a glycosidase hydrolase, resulted in the appearance of reducing ends in the capsule as labeled by a hydroxylamine-armed fluorescent (HAAF) probe. Finally, we demonstrate that exposing C. neoformans cells to catalytic antibodies results in changes in complement deposition and increased phagocytosis by macrophages, suggesting that the antiphagocytic properties of the capsule have been impaired. Our results raise questions over the ubiquity of antibodies with catalytic activity against glycans and establish the utility of glycan-based FRET and HAAF probes as tools for investigating this activity.

Synthesis of the 3'-O-sulfated TF antigen with a TEG-N3 linker for glycodendrimersomes preparation to study lectin binding.

The synthesis of gram quantities of the TF antigen (ß-ᴅ-Gal-(1→3)-α-ᴅ-GalNAc) and its 3'-sulfated analogue with a TEG-N3 spacer attached is described. The synthesis of the TF antigen comprises seven steps, from a known N-Troc-protected galactosamine donor, with an overall yield of 31%. Both the spacer (85%) and the galactose moiety (79%) were introduced using thioglycoside donors in NIS/AgOTf-promoted glycosylation reactions. The 3'-sulfate was finally introduced through tin activation in benzene/DMF followed by treatment with a sulfur trioxide-trimethylamine complex in a 66% yield.

A novel thiol-saccharide mucolytic for the treatment of muco-obstructive lung diseases.

BACKGROUND: Mucin disulfide cross-links mediate pathologic mucus formation in muco-obstructive lung diseases. MUC-031, a novel thiol-modified carbohydrate compound, cleaves disulfides to cause mucolysis. The aim of this study was to determine the mucolytic and therapeutic effects of MUC-031 in sputum from patients with cystic fibrosis (CF) and mice with muco-obstructive lung disease (ßENaC-Tg mice). METHODS: We compared the mucolytic efficacy of MUC-031 and existing mucolytics (N-acetylcysteine (NAC) and recombinant human deoxyribonuclease I (rhDNase)) using rheology to measure the elastic modulus (G') of CF sputum, and we tested effects of MUC-031 on airway mucus plugging, inflammation and survival in ßENaC-Tg mice to determine its mucolytic efficacy in vivo. RESULTS: In CF sputum, compared to the effects of rhDNase and NAC, MUC-031 caused a larger decrease in sputum G', was faster in decreasing sputum G' by 50% and caused mucolysis of a larger proportion of sputum samples within 15 min of drug addition. Compared to vehicle control, three treatments with MUC-031 in 1 day in adult ßENaC-Tg mice decreased airway mucus content (16.8±3.2 versus 7.5±1.2 nL·mm-2, p<0.01) and bronchoalveolar lavage cells (73 833±6930 versus 47 679±7736 cells·mL-1, p<0.05). Twice-daily treatment with MUC-031 for 2 weeks also caused decreases in these outcomes in adult and neonatal ßENaC-Tg mice and reduced mortality from 37% in vehicle-treated ßENaC-Tg neonates to 21% in those treated with MUC-031 (p<0.05). CONCLUSION: MUC-031 is a potent and fast-acting mucolytic that decreases airway mucus plugging, lessens airway inflammation and improves survival in ßENaC-Tg mice. These data provide rationale for human trials of MUC-031 in muco-obstructive lung diseases.

Synthesis of a B-Antigen Hexasaccharide, a B-Lewis b Heptasaccharide and Glycoconjugates Thereof to Investigate Binding Properties of Helicobacter pylori.

Infecting the stomach of almost 50 % of people, Helicobacter pylori is a causative agent of gastritis, peptic ulcers and stomach cancers. Interactions between bacterial membrane-bound lectin, Blood group Antigen Binding Adhesin (BabA), and human blood group antigens are key in the initiation of infection. Herein, the synthesis of a B-antigen hexasaccharide (B6) and a B-Lewis b heptasaccharide (BLeb7) and Bovine Serum Albumin glycoconjugates thereof is reported to assess the binding properties and preferences of BabA from different strains. From a previously reported trisaccharide acceptor a versatile key Lacto-N-tetraose tetrasaccharide intermediate was synthesized, which allowed us to explore various routes to the final targets, either via initial introduction of fucosyl residues followed by introduction of the B-determinant or vice versa. The first approach proved unsuccessful, whereas the second afforded the target structures in good yields. Protein conjugation using isothiocyanate methodology allowed us to reach high glycan loadings (up to 23 per protein) to mimic multivalent displays encountered in Nature. Protein glycoconjugate inhibition binding studies were performed with H. pylori strains displaying high or low affinity for Lewis b hexasaccharide structures showing that the binding to the high affinity strain was reduced due to the presence of the B-determinant in the Bleb7-conjugates and further reduced by the absence of the Lewis fucose residue in the B6-conjugate.

Synthesis of fluoro- and seleno-containing D-lactose and D-galactose analogues.

Synthetic deoxy-fluoro-carbohydrate derivatives and seleno-sugars are useful tools in protein-carbohydrate interaction studies using nuclear magnetic resonance spectroscopy because of the presence of the 19F and 77Se reporter nuclei. Seven saccharides containing both these atoms have been synthesized, three monosaccharides, methyl 6-deoxy-6-fluoro-1-seleno-ß-D-galactopyranoside (1) and methyl 2-deoxy-2-fluoro-1-seleno-α/ß-D-galactopyranoside (2α and 2ß), and four disaccharides, methyl 4-O-(ß-D-galactopyranosyl)-2-deoxy-2-fluoro-1-seleno-ß-D-glucopyranoside (3), methyl 4-Se-(ß-D-galactopyranosyl)-2-deoxy-2-fluoro-4-seleno-ß-D-glucopyranoside (4), and methyl 4-Se-(2-deoxy-2-fluoro-α/ß-D-galactopyranosyl)-4-seleno-ß-D-glucopyranoside (5α and 5ß), the three latter compounds with an interglycosidic selenium atom. Selenoglycosides 1 and 3 were obtained from the corresponding bromo sugar by treatment with dimethyl selenide and a reducing agent, while compounds 2α/2ß, 4, and 5α/5ß were synthesized by the coupling of a D-galactosyl selenolate, obtained in situ from the corresponding isoselenouronium salt, with either methyl iodide or a 4-O-trifluoromethanesulfonyl D-galactosyl moiety. While benzyl ether protecting groups were found to be incompatible with the selenide linkage during deprotection, a change to acetyl esters afforded 4 in a 17% overall yield and over 9 steps from peracetylated D-galactosyl bromide. The synthesis of 5 was performed similarly, but the 2-fluoro substituent led to reduced stereoselectivity in the formation of the isoselenouronium salt (α/ß âˆ¼ 1 : 2.3). However, the ß-anomer of the uronium salt could be obtained almost pure (∼98%) by precipitation from the reaction mixture. The following displacement reaction occurred without anomerisation, affording, after deacetylation, pure 5ß.

Exploring antiviral and anti-inflammatory effects of thiol drugs in COVID-19.

The redox status of the cysteine-rich SARS-CoV-2 spike glycoprotein (SARS-2-S) is important for the binding of SARS-2-S to angiotensin-converting enzyme 2 (ACE2), suggesting that drugs with a functional thiol group ("thiol drugs") may cleave cystines to disrupt SARS-CoV-2 cell entry. In addition, neutrophil-induced oxidative stress is a mechanism of COVID-19 lung injury, and the antioxidant and anti-inflammatory properties of thiol drugs, especially cysteamine, may limit this injury. To first explore the antiviral effects of thiol drugs in COVID-19, we used an ACE-2 binding assay and cell entry assays utilizing reporter pseudoviruses and authentic SARS-CoV-2 viruses. We found that multiple thiol drugs inhibit SARS-2-S binding to ACE2 and virus infection. The most potent drugs were effective in the low millimolar range, and IC50 values followed the order of their cystine cleavage rates and lower thiol pKa values. To determine if thiol drugs have antiviral effects in vivo and to explore any anti-inflammatory effects of thiol drugs in COVID-19, we tested the effects of cysteamine delivered intraperitoneally to hamsters infected with SARS-CoV-2. Cysteamine did not decrease lung viral infection, but it significantly decreased lung neutrophilic inflammation and alveolar hemorrhage. We speculate that the concentration of cysteamine achieved in the lungs with intraperitoneal delivery was insufficient for antiviral effects but sufficient for anti-inflammatory effects. We conclude that thiol drugs decrease SARS-CoV-2 lung inflammation and injury, and we provide rationale for future studies to test if direct (aerosol) delivery of thiol drugs to the airways might also result in antiviral effects.

What is the Sugar Code?

A code is defined by the nature of the symbols, which are used to generate information-storing combinations (e. g. oligo- and polymers). Like nucleic acids and proteins, oligo- and polysaccharides are ubiquitous, and they are a biochemical platform for establishing molecular messages. Of note, the letters of the sugar code system (third alphabet of life) excel in coding capacity by making an unsurpassed versatility for isomer (code word) formation possible by variability in anomery and linkage position of the glycosidic bond, ring size and branching. The enzymatic machinery for glycan biosynthesis (writers) realizes this enormous potential for building a large vocabulary. It includes possibilities for dynamic editing/erasing as known from nucleic acids and proteins. Matching the glycome diversity, a large panel of sugar receptors (lectins) has developed based on more than a dozen folds. Lectins 'read' the glycan-encoded information. Hydrogen/coordination bonding and ionic pairing together with stacking and C-H/π-interactions as well as modes of spatial glycan presentation underlie the selectivity and specificity of glycan-lectin recognition. Modular design of lectins together with glycan display and the nature of the cognate glycoconjugate account for the large number of post-binding events. They give an entry to the glycan vocabulary its functional, often context-dependent meaning(s), hereby building the dictionary of the sugar code.

Synthesis of a Lewis b hexasaccharide thioglycoside donor and its use towards an extended mucin core Tn heptasaccharide structure and a photoreactive biotinylated serine linked hexasaccharide.

Investigation into Heliobacter pylori binding to Lewis b (Leb) antigens through the blood group antigen binding adhesion protein (BabA) requires structurally well-defined tools. A Leb hexasaccharide thioglycoside donor was chemically prepared through a linear approach starting from D-lactose. This donor can be used to attach reducing end linkers providing a range of options for conjugation techniques or to further extend the oligosaccharide structure. To evaluate its efficiency as a donor, it was coupled to a 6-OH GalNAc acceptor, producing an extended Leb-containing Tn mucin core structure in 84% yield, and to L-serine in 72% yield. The latter compound was subsequently functionalized with a photolabile diazirine linker and biotin, creating a Leb hexasaccharide structure-function tool suitable for lectin tagging interaction studies. This donor opens a wide range of possibilities for conjugation of Leb structures to produce a variety of chemical biology tools to assist in the study of these interactions.

Design-functionality relationships for adhesion/growth-regulatory galectins.

Glycan-lectin recognition is assumed to elicit its broad range of (patho)physiological functions via a combination of specific contact formation with generation of complexes of distinct signal-triggering topology on biomembranes. Faced with the challenge to understand why evolution has led to three particular modes of modular architecture for adhesion/growth-regulatory galectins in vertebrates, here we introduce protein engineering to enable design switches. The impact of changes is measured in assays on cell growth and on bridging fully synthetic nanovesicles (glycodendrimersomes) with a chemically programmable surface. Using the example of homodimeric galectin-1 and monomeric galectin-3, the mutual design conversion caused qualitative differences, i.e., from bridging effector to antagonist/from antagonist to growth inhibitor and vice versa. In addition to attaining proof-of-principle evidence for the hypothesis that chimera-type galectin-3 design makes functional antagonism possible, we underscore the value of versatile surface programming with a derivative of the pan-galectin ligand lactose. Aggregation assays with N,N'-diacetyllactosamine establishing a parasite-like surface signature revealed marked selectivity among the family of galectins and bridging potency of homodimers. These findings provide fundamental insights into design-functionality relationships of galectins. Moreover, our strategy generates the tools to identify biofunctional lattice formation on biomembranes and galectin-reagents with therapeutic potential.

Exploring Cryptococcus neoformans capsule structure and assembly with a hydroxylamine-armed fluorescent probe.

Chemical biology is an emerging field that enables the study and manipulation of biological systems with probes whose reactivities provide structural insights. The opportunistic fungal pathogen Cryptococcus neoformans possesses a polysaccharide capsule that is a major virulence factor, but is challenging to study. We report here the synthesis of a hydroxylamine-armed fluorescent probe that reacts with reducing glycans and its application to study the architecture of the C. neoformans capsule under a variety of conditions. The probe signal localized intracellularly and at the cell wall-membrane interface, implying the presence of reducing-end glycans at this location where the capsule is attached to the cell body. In contrast, no fluorescence signal was detected in the capsule body. We observed vesicle-like structures containing the reducing-end probe, both intra- and extracellularly, consistent with the importance of vesicles in capsular assembly. Disrupting the capsule with DMSO, ultrasound, or mechanical shear stress resulted in capsule alterations that affected the binding of the probe, as reducing ends were exposed and cell membrane integrity was compromised. Unlike the polysaccharides in the assembled capsule, isolated exopolysaccharides contained reducing ends. The reactivity of the hydroxylamine-armed fluorescent probe suggests a model for capsule assembly whereby reducing ends localize to the cell wall surface, supporting previous findings suggesting that this is an initiation point for capsular assembly. We propose that chemical biology is a promising approach for studying the C. neoformans capsule and its associated polysaccharides to unravel their roles in fungal virulence.

Key role of a structural water molecule for the specificity of 14F7-An antitumor antibody targeting the NeuGc GM3 ganglioside.

Tumor-associated glycolipids such as NeuGc GM3 are auspicious molecular targets in antineoplastic therapies and vaccine strategies. 14F7 is a monoclonal IgG1 with high clinical potential in cancer immunotherapy as it displays extraordinary specificity for NeuGc GM3, while it does not recognize the very similar, ubiquitous NeuAc GM3. Here we present the 2.3 Å crystal structure of the 14F7 antigen-binding domain (14F7 scFv) in complex with the NeuGc GM3 trisaccharide. Modeling analysis and previous mutagenesis data suggest that 14F7 may also bind to an alternative NeuGc GM3 conformation, not observed in the crystal structure. The most intriguing finding, however, was that a water molecule centrally placed in the complementarity-determining region directly mediates the specificity of 14F7 to NeuGc GM3. This has profound impact on the complexity of engineering in the binding site and provides an excellent example of the importance in understanding the water structure in antibody-antigen interactions.

A detailed picture of a protein-carbohydrate hydrogen-bonding network revealed by NMR and MD simulations.

Cyanovirin-N (CV-N) is a cyanobacterial lectin with antiviral activity towards HIV and several other viruses. Here, we identify mannoside hydroxyl protons that are hydrogen bonded to the protein backbone of the CV-N domain B binding site, using NMR spectroscopy. For the two carbohydrate ligands Manα(1→2)ManαOMe and Manα(1→2) Manα(1→6)ManαOMe five hydroxyl protons are involved in hydrogen-bonding networks. Comparison with previous crystallographic results revealed that four of these hydroxyl protons donate hydrogen bonds to protein backbone carbonyl oxygens in solution and in the crystal. Hydrogen bonds were not detected between the side chains of Glu41 and Arg76 with sugar hydroxyls, as previously proposed for CV-N binding of mannosides. Molecular dynamics simulations of the CV-N/Manα(1→2)Manα(1→6)ManαOMe complex confirmed the NMR-determined hydrogen-bonding network. Detailed characterization of CV-N/mannoside complexes provides a better understanding of lectin-carbohydrate interactions and opens up to the use of CV-N and similar lectins as antiviral agents.

Galectin-Glycan Interactions: Guidelines for Monitoring by 77 Se NMR Spectroscopy, and Solvent (H2 O/D2 O) Impact on Binding.

Functional pairing between cellular glycoconjugates and tissue lectins like galectins has wide (patho)physiological significance. Their study is facilitated by nonhydrolysable derivatives of the natural O-glycans, such as S- and Se-glycosides. The latter enable extensive analyses by specific 77 Se NMR spectroscopy, but still remain underexplored. By using the example of selenodigalactoside (SeDG) and the human galectin-1 and -3, we have evaluated diverse 77 Se NMR detection methods and propose selective 1 H,77 Se heteronuclear Hartmann-Hahn transfer for efficient use in competitive NMR screening against a selenoglycoside spy ligand. By fluorescence anisotropy, circular dichroism, and isothermal titration calorimetry (ITC), we show that the affinity and thermodynamics of SeDG binding by galectins are similar to thiodigalactoside (TDG) and N-acetyllactosamine (LacNAc), confirming that Se substitution has no major impact. ITC data in D2 O versus H2 O are similar for TDG and LacNAc binding by both galectins, but a solvent effect, indicating solvent rearrangement at the binding site, is hinted at for SeDG and clearly observed for LacNAc dimers with extended chain length.

Facile anomer-oriented syntheses of 4-methylumbelliferyl sialic acid glycosides.

As part of a program to find new sialidases and determine their enzymatic specificity and catalytic activity, a library of 4-methylumbelliferyl sialic acid glycosides derivatised at the C-5 position were prepared from N-acetylneuraminic acid. Both α- and ß-4-methylumbelliferyl sialic acid glycosides were prepared in high yields and stereoselectivity. α-Anomers were accessed via reagent control by utilising additive CH3CN and TBAI, whereas the ß-anomers were synthesised through a diastereoselective addition reaction of iodine and the aglycone to the corresponding glycal followed by reduction of the resulting 3-iodo compounds. Both anomer-oriented synthetic pathways allow for gram-scale stereoselective syntheses of the desired C-5 modified neuraminic acid derivatives for use as tools to quantify the enzymatic activity and substrate specificity of known sialidases, and potential detection and investigation of novel sialidases.

Exploring functional pairing between surface glycoconjugates and human galectins using programmable glycodendrimersomes.

Precise translation of glycan-encoded information into cellular activity depends critically on highly specific functional pairing between glycans and their human lectin counter receptors. Sulfoglycolipids, such as sulfatides, are important glycolipid components of the biological membranes found in the nervous and immune systems. The optimal molecular and spatial design aspects of sulfated and nonsulfated glycans with high specificity for lectin-mediated bridging are unknown. To elucidate how different molecular and spatial aspects combine to ensure the high specificity of lectin-mediated bridging, a bottom-up toolbox is devised. To this end, negatively surface-charged glycodendrimersomes (GDSs), of different nanoscale dimensions, containing sulfo-lactose groups are self-assembled in buffer from a synthetic sulfatide mimic: Janus glycodendrimer (JGD) containing a 3'-O-sulfo-lactose headgroup. Also prepared for comparative analysis are GDSs with nonsulfated lactose, a common epitope of human membranes. These self-assembled GDSs are employed in aggregation assays with 15 galectins, comprising disease-related human galectins, and other natural and engineered variants from four families, having homodimeric, heterodimeric, and chimera architectures. There are pronounced differences in aggregation capacity between human homodimeric and heterodimeric galectins, and also with respect to their responsiveness to the charge of carbohydrate-derived ligand. Assays reveal strong differential impact of ligand surface charge and density, as well as lectin concentration and structure, on the extent of surface cross-linking. These findings demonstrate how synthetic JGD-headgroup tailoring teamed with protein engineering and network assays can help explain how molecular matchmaking operates in the cellular context of glycan and lectin complexity.

Fluorinated Carbohydrates as Lectin Ligands: Simultaneous Screening of a Monosaccharide Library and Chemical Mapping by 19F NMR Spectroscopy.

Molecular recognition of carbohydrates is a key step in essential biological processes. Carbohydrate receptors can distinguish monosaccharides even if they only differ in a single aspect of the orientation of the hydroxyl groups or harbor subtle chemical modifications. Hydroxyl-by-fluorine substitution has proven its merits for chemically mapping the importance of hydroxyl groups in carbohydrate-receptor interactions. 19F NMR spectroscopy could thus be adapted to allow contact mapping together with screening in compound mixtures. Using a library of fluorinated glucose (Glc), mannose (Man), and galactose (Gal) derived by systematically exchanging every hydroxyl group by a fluorine atom, we developed a strategy combining chemical mapping and 19F NMR T2 filtering-based screening. By testing this strategy on the proof-of-principle level with a library of 13 fluorinated monosaccharides to a set of three carbohydrate receptors of diverse origin, i.e. the human macrophage galactose-type lectin, a plant lectin, Pisum sativum agglutinin, and the bacterial Gal-/Glc-binding protein from Escherichia coli, it became possible to simultaneously define their monosaccharide selectivity and identify the essential hydroxyls for interaction.

Synthesis of type 1 Lewis b hexasaccharide antigen structures featuring flexible incorporation of l-[U-13C6]-fucose for NMR binding studies.

While 13C-labelled proteins are common tools in NMR studies, lack of access to 13C-labelled carbohydrate structures has restricted their use. l-Fucose is involved in a wide range of physiological and pathophysiological processes in mammalian organisms. Here, l-[U-13C6]-Fuc labelled type I Lewis b (Leb) structures have been synthesised for use in NMR binding studies with the Blood-group Antigen Binding Adhesin (BabA), a membrane-bound protein from the bacterium Helicobacter pylori. As part of this work, an efficient synthesis of a benzylated l-[U-13C6]-Fuc thioglycoside donor from l-[U-13C6]-Gal has been developed. The design and synthesis of an orthogonally protected tetrasaccharide precursor enabled controlled introduction of one or two 13C-labelled or non-labelled fucosyl residues prior to global deprotection. NMR analysis showed that it is straightforward to assign the anomeric centres as well as the H-5 positions to the individual fucosyl residues which are relevant for NMR binding studies.