Serine versus Threonine Glycosylation with α‐O‐GalNAc: Unexpected Selectivity in Their Molecular Recognition with Lectins

The molecular recognition of several glycopeptides bearing Tn antigen (α‐O‐GalNAc‐Ser or α‐O‐GalNAc‐Thr) in their structure by three lectins with affinity for this determinant has been analysed. The work yields remarkable results in terms of epitope recognition, showing that the underlying amino acid of Tn (serine or threonine) plays a key role in the molecular recognition. In fact, while Soybean agglutinin and Vicia villosa agglutinin lectins prefer Tn‐threonine, Helix pomatia agglutinin shows a higher affinity for the glycopeptides carrying Tn‐serine. The different conformational behaviour of the two Tn biological entities, the residues of the studied glycopeptides in the close proximity to the Tn antigen and the topology of the binding site of the lectins are at the origin of these differences.     

The Plasticity of the Carbohydrate Recognition Domain Dictates the Exquisite Mechanism of Binding of Human Macrophage Galactose-Type Lectin

The human macrophage galactose-type lectin (MGL), expressed on macrophages and dendritic cells (DCs), modulates distinct immune cell responses by recognizing N-acetylgalactosamine (GalNAc) containing structures present on pathogens, self-glycoproteins, and tumor cells. Herein, NMR spectroscopy and molecular dynamics (MD) simulations were used to investigate the structural preferences of MGL against different GalNAc-containing structures derived from the blood group A antigen, the Forssman antigen, and the GM2 glycolipid. NMR spectroscopic analysis of the MGL carbohydrate recognition domain (MGL-CRD, C181-H316) in the absence and presence of methyl α-GalNAc (α-MeGalNAc), a simple monosaccharide, shows that the MGL-CRD is highly dynamic and its structure is strongly altered upon ligand binding. This plasticity of the MGL-CRD structure explains the ability of MGL to accommodate different GalNAc-containing molecules. However, key differences are observed in the recognition process depending on whether the GalNAc is part of the blood group A antigen, the Forssman antigen, or GM2-derived structures. These results are in accordance with molecular dynamics simulations that suggest the existence of a distinct MGL binding mechanism depending on the context of GalNAc moiety presentation. These results afford new perspectives for the rational design of GalNAc modifications that fine tune MGL immune responses in distinct biological contexts, especially in malignancy.     

Deciphering the Non‐Equivalence of Serine and Threonine O‐Glycosylation Points: Implications for Molecular Recognition of the Tn Antigen by an anti‐MUC1 Antibody

The structural features of MUC1‐like glycopeptides bearing the Tn antigen (α‐O‐GalNAc‐Ser/Thr) in complex with an anti MUC‐1 antibody are reported at atomic resolution. For the α‐O‐GalNAc‐Ser derivative, the glycosidic linkage adopts a high‐energy conformation, barely populated in the free state. This unusual structure (also observed in an α‐S‐GalNAc‐Cys mimic) is stabilized by hydrogen bonds between the peptidic fragment and the sugar. The selection of a particular peptide structure by the antibody is thus propagated to the carbohydrate through carbohydrate/peptide contacts, which force a change in the orientation of the sugar moiety. This seems to be unfeasible in the α‐O‐GalNAc‐Thr glycopeptide owing to the more limited flexibility of the side chain imposed by the methyl group. Our data demonstrate the non‐equivalence of Ser and Thr O‐glycosylation points in molecular recognition processes. These features provide insight into the occurrence in nature of the APDTRP epitope for anti‐MUC1 antibodies.     

Monitoring Glycan–Protein Interactions by NMR Spectroscopic Analysis: A Simple Chemical Tag That Mimics Natural CH–π Interactions

Detection of molecular recognition processes requires robust, specific, and easily implementable sensing methods, especially for screening applications. Here, we propose the difluoroacetamide moiety (an acetamide bioisoster) as a novel tag for detecting by NMR analysis those glycan–protein interactions that involve N‐acetylated sugars. Although difluoroacetamide has been used previously as a substituent in medicinal chemistry, here we employ it as a specific sensor to monitor interactions between GlcNAc‐containing glycans and a model lectin (wheat germ agglutinin). In contrast to the widely employed trifluoroacetamide group, the difluoroacetamide tag contains geminal ¹H and ¹⁹F atoms that allow both ¹H and ¹⁹F NMR methods for easy and robust detection of molecular recognition processes involving GlcNAc‐ (or GalNAc‐) moieties over a range of binding affinities. The CHF₂CONH‐ moiety behaves in a manner that is very similar to that of the natural acetamide fragment in the involved aromatic‐sugar interactions, providing analogous binding energy and conformations, whereas the perfluorinated CF₃CONH‐ analogue differs more significantly.     

Conformational Selection in Glycomimetics: Human Galectin‐1 Only Recognizes syn‐Ψ‐Type Conformations of β‐1,3‐Linked Lactose and Its C‐Glycosyl Derivative

The human lectin galectin‐1 (hGal‐1) translates sugar signals, that is, β‐galactosides, into effects on the level of cells, for example, growth regulation, and has become a model for studying binding of biopharmaceutically relevant derivatives. Bound‐state conformations of Galβ‐C‐(1→3)‐Glcβ‐OMe ( 1 ) and its βGal‐(1→3)‐βGlc‐OMe disaccharide parent compound were studied by using NMR spectroscopy (transferred (TR)‐NOESY data), assisted by docking experiments and molecular dynamics (MD) simulations. The molecular recognition process involves a conformational selection event. Although free C‐glycoside access four distinct conformers in solution, hGal‐1 recognizes shape of a local minimum of compound 1 , the syn‐Φ/syn‐Ψ conformer, not the structure at global minimum. MD simulations were run to explain, in structural terms, the observed geometry of the complex.     

Synthesis and Conformational Analysis of Hybrid α/β‐Dipeptides Incorporating S‐Glycosyl‐β2,2‐Amino Acids

We synthesized and carried out the conformational analysis of several hybrid dipeptides consisting of an α‐amino acid attached to a quaternary glyco‐β‐amino acid. In particular, we combined a S‐glycosylated β^2,2‐amino acid and two different types of α‐amino acid, namely, aliphatic (alanine) and aromatic (phenylalanine and tryptophan) in the sequence of hybrid α/β‐dipeptides. The key step in the synthesis involved the ring‐opening reaction of a chiral cyclic sulfamidate, inserted in the peptidic sequence, with a sulfur‐containing nucleophile by using 1‐thio‐β‐D ‐glucopyranose derivatives. This reaction of glycosylation occurred with inversion of configuration at the quaternary center. The conformational behavior in aqueous solution of the peptide backbone and the glycosidic linkage for all synthesized hybrid glycopeptides was analyzed by using a protocol that combined NMR experiments and molecular dynamics with time‐averaged restraints (MD‐tar). Interestingly, the presence of the sulfur heteroatom at the quaternary center of the β‐amino acid induced θ torsional angles close to 180° (anti). Notably, this value changed to 60° (gauche) when the peptidic sequence displayed aromatic α‐amino acids due to the presence of CH–π interactions between the phenyl or indole ring and the methyl groups of the β‐amino acid unit.     

A Convergent Strategy for the Synthesis of Type‐1 Elongated Mucin Cores 1–3 and the Corresponding Glycopeptides

Mucins are a class of highly O‐glycosylated proteins found on the surface of cells in epithelial tissues. O‐Glycosylation is crucial for the functionality of mucins and changes therein can have severe consequences for an organism. With that in mind, the elucidation of interactions of carbohydrate binding proteins with mucins, whether in morbidly altered or unaltered conditions, continue to shed light on mechanisms involved in diseases like chronic inflammations and cancer. Despite the known importance of type‐1 and type‐2 elongated mucin cores 1–4 in glycobiology, the corresponding type‐1 structures are much less well studied. Here, the first chemical synthesis of extended mucin type‐1 O‐glycan core 1–3 amino acid structures based on a convergent approach is presented. By utilizing differentiation in acceptor reactivity, shared early stage Tn‐ and T‐acceptor intermediates were elongated with a common type‐1 [β‐D ‐Gal‐1,3‐β‐D ‐GlcNAc] disaccharide, which allows for straightforward preparation of diverse glycosylated amino acids carrying the type‐1 mucin core 1–3 saccharides. The obtained glycosylated 9‐fluorenylmethoxycarbonyl (Fmoc)‐protected amino acid building blocks were employed in synthesis of type‐1 mucin glycopeptides, which are useful in biological applications.     

Mannose‐Binding Geometry of Pradimicin A

Pradimicins (PRMs) and benanomicins are the only family of non‐peptidic natural products with lectin‐like properties, that is, they recognize D ‐mannopyranoside (Man) in the presence of Ca²⁺ ions. Coupled with their unique Man binding ability, they exhibit antifungal and anti‐HIV activities through binding to Man‐containing glycans of pathogens. Notwithstanding the great potential of PRMs as the lectin mimics and therapeutic leads, their molecular basis of Man recognition has yet to be established. Their aggregate‐forming propensity has impeded conventional interaction analysis in solution, and the analytical difficulty is exacerbated by the existence of two Man binding sites in PRMs. In this work, we investigated the geometry of the primary Man binding of PRM‐A, an original member of PRMs, by the recently developed analytical strategy using the solid aggregate composed of the 1:1 complex of PRM‐A and Man. Evaluation of intermolecular distances by solid‐state NMR spectroscopy revealed that the C2–C4 region of Man is in close contact with the primary binding site of PRM‐A, while the C1 and C6 positions of Man are relatively distant. The binding geometry was further validated by co‐precipitation experiments using deoxy‐Man derivatives, leading to the proposal that PRM‐A binds not only to terminal Man residues at the non‐reducing end of glycans, but also to internal 6‐substituted Man residues. The present study provides new insights into the molecular basis of Man recognition and glycan specificity of PRM‐A.     

Highly Effective Recognition of Carbohydrates by Phenanthroline‐Based Receptors: α‐ versus β‐Anomer Binding Preference

¹H NMR spectroscopic titrations in competitive and non‐competitive media, as well as binding studies in two‐phase systems, such as phase transfer of sugars from aqueous into organic solvents and dissolution of solid carbohydrates in apolar media revealed both highly effective recognition of neutral carbohydrates and interesting binding preferences of an acyclic phenanthroline‐based receptor 1 . Compared to the previously described acyclic receptors, compound 1 displays significantly higher binding affinities, the rare capability to extract sugars from water into non‐polar organic solutions and α‐ versus β‐anomer binding preference in the recognition of glycosides, which differs from those observed for other receptor systems. X‐ray crystallographic investigations revealed the presence of water molecules in the binding pocket of 1 that are engaged in the formation of hydrogen‐bonding motifs similar to those suggested by molecular modelling for the sugar OH groups in the receptor–sugar complexes. The molecular modelling calculations, synthesis, crystal structure and binding properties of 1 are described and compared with those of the previously described receptors.     

Structural Insights into the Molecular Recognition Mechanism of the Cancer and Pathogenic Epitope,LacdiNAc by Immune-Related Lectins

Interactions of glycan-specific epitopes to human lectin receptors represent novel immune checkpoints for investigating cancer and infection diseases. By employing a multidisciplinary approach that combines isothermal titration calorimetry, NMR spectroscopy, molecular dynamics simulations, and X-ray crystallography, we investigated the molecular determinants that govern the recognition of the tumour and pathogenic glycobiomarker LacdiNAc (GalNAcβ1-4GlcNAc, LDN), including their comparison with the ubiquitous LacNAc epitope (Galβ1-4GlcNAc, LN), by two human immune-related lectins, galectin-3 (hGal-3) and the macrophage galactose C-type lectin (hMGL). A different mechanism of binding and interactions was observed for the hGal-3/LDN and hMGL/LDN complexes, which explains the remarkable difference in the binding specificity of LDN and LN by these two lectins. The new structural clues reported herein are fundamental for the chemical design of mimetics targeting hGal-3/hMGL recognition process.     

Breaking the Limits in Analyzing Carbohydrate Recognition by NMR Spectroscopy: Resolving Branch‐Selective Interaction of a Tetra‐Antennary N‐Glycan with Lectins

The biological recognition of complex‐type N ‐glycans is part of many key physiological and pathological events. Despite their importance, the structural characterization of these events remains unsolved. The inherent flexibility of N ‐glycans hampers crystallization and the chemical equivalence of individual branches precludes their NMR characterization. By using a chemoenzymatically synthesized tetra‐antennary N ‐glycan conjugated to a lanthanide binding tag, the NMR signals under paramagnetic conditions discriminated all four N ‐acetyl lactosamine antennae with unprecedented resolution. The NMR data revealed the conformation of the N ‐glycan and permitted for the first time the direct identification of individual branches involved in the recognition by two N ‐acetyllactosamine‐binding lectins, Datura stramonium seed lectin (DSL) and Ricinus Communis agglutinin (RCA120).     

Glycopeptide Mimetics Recapitulate High‐Mannose‐Type Oligosaccharide Binding and Function

High‐mannose‐type glycans (HMTGs) decorating viral spike proteins are targets for virus neutralization. For carbohydrate‐binding proteins, multivalency is important for high avidity binding and potent inhibition. To define the chemical determinants controlling multivalent interactions we designed glycopeptide HMTG mimetics with systematically varied mannose valency and spacing. Using the potent antiviral lectin griffithsin (GRFT) as a model, we identified by NMR spectroscopy, SPR, analytical ultracentrifugation, and microcalorimetry glycopeptides that fully recapitulate the specificity and kinetics of binding to Man₉GlcNAc₂Asn and a synthetic nonamannoside. We find that mannose spacing and valency dictate whether glycopeptides engage GRFT in a face‐to‐face or an intermolecular binding mode. Surprisingly, although face‐to‐face interactions are of higher affinity, intermolecular interactions are longer lived. These findings yield key insights into mechanisms involved in glycan‐mediated viral inhibition.     

Microwave‐Assisted Solid‐Phase Synthesis of Antifreeze Glycopeptides

Microwave‐assisted solid‐phase synthesis allows for the rapid and large‐scale preparation and structure–activity characterization of tandem repeating glycopeptides, namely monodispersed synthetic antifreeze glycopeptides (syAFGPs, H‐[Ala‐Thr(Galβ1,3GalNAcα1→)‐Ala]_n‐OH, n=2–6). By employing novel AFGP analogues, we have demonstrated that of the monodispersed syAFGP_n (n=2–6, degree of polymerization, DP=2–6, M_w=1257–3690 Da), syAFGP₅ (DP=5, M_w=3082 Da) and syAFGP₆ (DP=6, M_w=3690 Da) exhibit the ability to form typical hexagonal bipyramidal ice crystals and satisfactory thermal hysteresis activity. Structural characterization by NMR and CD spectroscopy revealed that syAFGP₆ forms a typical poly‐L ‐proline type II helix‐like structure in aqueous solution whereas enzymatic modification by sialic acid of the residues at the C‐3 positions of the nonreducing Gal residues disturbs this conformation and eliminates the antifreeze activity.     

Synthesis and NMR elucidation of novel pentacycloundecane‐based peptides

The synthesis and NMR elucidation of two novel pentacycloundecane (PCU)‐based peptides are reported. The PCU cage amino acids were synthesised as racemates and the incorporation of the cage amino acid with (S)‐natural amino acids produced diastereomeric peptides. The diastereomeric ‘cage’ peptides were separated using preparative HPLC and the NMR elucidation of these PCU containing peptides are reported for the first time. The ¹H and ¹³C NMR spectra showed series of overlapping signals of the cage skeleton and that of the peptide, making it extremely difficult to resolve the structure using one‐dimensional NMR techniques only. The use of two‐dimensional NMR techniques proved to be a highly effective tool in overcoming this problem. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis and Biological Evaluation of the Forssman Antigen Pentasaccharide and Derivatives by a One‐Pot Glycosylation Procedure

The synthesis and biological evaluation of the Forssman antigen pentasaccharide and derivatives thereof by using a one‐pot glycosylation and polymer‐assisted deprotection is described. The Forssman antigen pentasaccharide, composed of GalNAcα(1,3)GalNAcβ(1,3)Galα(1,4)Galβ(1,4)Glc, was recently identified as a ligand of the lectin SLL‐2 isolated from an octocoral Sinularia lochmodes. The chemo‐ and α‐selective glycosylation of a thiogalactoside with a hemiacetal donor by using a mixture of Tf₂O, TTBP and Ph₂SO, followed by activation of the remaining thioglycoside, provided the trisaccharide at the reducing end in a one‐pot procedure. The pentasaccharide was prepared by the α‐selective glycosylation of the N‐Troc‐protected (Troc=2,2,2‐trichloroethoxycarbonyl) thioglycoside with a 2‐azide‐1‐hydroxyl glycosyl donor, followed by glycosidation of the resulting disaccharide at the C3 hydroxyl group of the trisaccharide acceptor in a one‐pot process. We next applied the one‐pot glycosylation method to the synthesis of pentasaccharides in which the galactosamine units were partially and fully replaced by galactose units. Among the three possible pentasaccharides, Galα(1,3)GalNAc and Galα(1,3)Gal derivatives were successfully prepared by the established method. An assay of the binding of the synthetic oligosaccharides to a fluorescent‐labeled SLL‐2 revealed that the NHAc substituents and the length of the oligosaccharide chain were both important for the binding of the oligosaccharide to SLL‐2. The inhibition effect of the oligosaccharide relative to the morphological changes of Symbiodinium by SLL‐2, was comparable to their binding affinity to SLL‐2. In addition, we fortuitously found that the synthetic Forssman antigen pentasaccharide directly promotes a morphological change in Symbiodinium. These results strongly indicate that the Forssman antigen also functions as a chemical mediator of Symbiodinium.     

Chemoenzymatic synthesis of sialylated glycopeptides derived from mucins and T-cell stimulating peptides.

The Tn, T, sialyl-Tn, and 2,3-sialyl-T antigens are tumor-associated carbohydrate antigens expressed on mucins in epithelial cancers, such as those affecting the breast, ovary, stomach, and colon. Glycopeptides carrying these antigens are of interest for development of cancer vaccines and a short, chemoenzymatic strategy for their synthesis is reported. Building blocks corresponding to the Tn (GalNAc alpha-Ser/Thr) and T [Gal beta(1-->3)GalNAc alpha-Ser/Thr] antigens, which are relatively easy to obtain by chemical synthesis, were prepared and then used in the synthesis of glycopeptides on the solid phase. Introduction of sialic acid to give the sialyl-Tn [Neu5Ac alpha(2-->6)GalNAc alpha-Ser/Thr] and 2,3-sialyl-T [Neu5Ac alpha(2-->3)Gal beta(1-->3)GalNAc alpha-Ser/Thr] antigens is difficult when performed chemically at the building block level. Sialylation was therefore carried out with recombinant sialyltransferases in solution after cleavage of the Tn and T glycopeptides from the solid phase. In the same manner, the core 2 trisaccharide [Gal beta 1-->3(GlcNAc beta 1-->6)GalNAc] was incorporated in glycopeptides containing the T antigen by using a recombinant N-acetylglucosaminyltransferase. The outlined chemoenzymatic approach was applied to glycopeptides from the tandem repeat domain of the mucin MUC1, as well as to neoglycosylated derivatives of a T cell stimulating viral peptide.     

Substrate‐Guided Front‐Face Reaction Revealed by Combined Structural Snapshots and Metadynamics for the Polypeptide N‐Acetylgalactosaminyltransferase 2

The retaining glycosyltransferase GalNAc‐T2 is a member of a large family of human polypeptide GalNAc‐transferases that is responsible for the post‐translational modification of many cell‐surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum‐mechanics/molecular‐mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic‐electronic level of detail. Our study provides a detailed structural rationale for an ordered bi–bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front‐face S_Ni‐type reaction in which the substrate N‐acetyl sugar substituent coordinates efficient glycosyl transfer.     

Fullerene‐sp2‐Iminosugar Balls as Multimodal Ligands for Lectins and Glycosidases: A Mechanistic Hypothesis for the Inhibitory Multivalent Effect

Concerted functioning of lectins and carbohydrate‐processing enzymes, mainly glycosidases, is essential in maintaining life. It was commonly assumed that the mechanisms by which each class of protein recognizes their cognate sugar partners are intrinsically different: multivalency is a characteristic feature of carbohydrate–lectin interactions, whereas glycosidases bind to their substrates or substrate‐analogue inhibitors in monovalent form. Recent observations on the glycosidase inhibitory potential of multivalent glycomimetics have questioned this paradigm and led to postulate an inhibitory multivalent effect. Here the mechanisms at the origin of this phenomenon have been investigated. A D ‐gluco‐configured sp²‐iminosugar glycomimetic motif, namely 1‐amino‐5N,6O‐oxomethylydenenojirimycin (1N‐ONJ), behaving, simultaneously, as a ligand of peanut agglutinin (PNA) lectin and as an inhibitor of several glycosidases, has been identified. Both the 1N‐ONJ–lectin‐ and 1N‐ONJ–glycosidase‐recognition processes have been found to be sensitive to multivalency, which has been exploited in the design of a lectin–glycosidase competitive assay to explore the implication of catalytic and non‐glycone sites in enzyme binding. A set of isotropic dodecavalent C₆₀‐fullerene–sp²‐iminosugar balls incorporating matching or mismatching motifs towards several glycosidases (inhitopes) was synthesized for that purpose, thereby preventing differences in binding modes arising from orientational preferences. The data supports that: 1) multivalency allows modulating the affinity and selectivity of a given inhitope towards glycosidases; 2) multivalent presentation can switch on the inhibitory capacity for some inhitope–glycosidase pairs, and 3) interactions of the multivalent inhibitors with non‐glycone sites is critical for glycosidase recognition. The ensemble of results point to a shift in the binding mode on going from monovalent to multivalent systems: in the first case a typical ′′key–lock′′ model involving, essentially, the high‐affinity active site can be assumed, whereas in the second, a lectin‐like behavior implying low‐affinity non‐glycone sites probably operates. The differences in responsiveness to multivalency for different glycosidases can then be rationalized in terms of the structure and accessibility of the corresponding carbohydrate‐binding regions.     

Structure of the Complex between a Heparan Sulfate Octasaccharide and Mycobacterial Heparin‐Binding Hemagglutinin

Heparin‐binding hemagglutinin (HBHA) is a 199 amino acid virulence factor at the envelope of Mycobacterium tuberculosis that contributes to latent tuberculosis. The binding of HBHA to respiratory epithelial cells, which leads to extrapulmonary dissemination of the pathogen, is mediated by cell‐surface heparan sulfate (HS). We report the structural characterization of the HBHA/HS complex by NMR spectroscopy. To develop a model for the molecular recognition, the first chemically synthesized uniformly ¹³C‐ and ¹⁵N‐labeled HS octasaccharide and a uniformly ¹³C‐ and ¹⁵N‐labeled form of HBHA were prepared. Residues 180–195 at the C‐terminal region of HBHA show large chemical shift perturbation upon association with the octasaccharide. Molecular dynamics simulations conforming to the multidimensional NMR data revealed key electrostatic and even hydrophobic interactions between the binding partners that may aid in the development of agents targeting the binding event.     

a,a,a,b-ZnT(o-BocThr)APP对咪唑衍生物和氨基酸酯的分子识别研究

Molecular Recognition of α,α,α,β-ZnT（o-BocThr）APP （1） toward a series of imidazole derivatives and amino acid esters was investigated. Association constants were determined in chloroform by means of UV-Vis titration method. The association constants of 1 with imidazole derivatives are larger than those of 1 with amino acid esters. 1H NMR spectra were investigated to describe the binding mode of the recognition system, showing that all the protons of the guests were shifted to upfield. The circular dichroism spectra of 1-L-/D-ValOMe showed a split cotton effect in Soret region, while those of 1-L-/D-PheOMe showed no split cotton effect. Molecular modeling was performed to understand chiral recognition on a molecular level. Quantum chemical calculation was carried out based on the stable conformations of these recognition systems, which gave a reasonable explanation for the behavior of molecular recognition. The results indicated that the conformation of 1-D-ValOMe was more stable than that of 1-L-ValOMe.