On-bead synthesis and binding assay of chemoselectively template-assembled multivalent neoglycopeptides

The investigation of recognition events between carbohydrates and proteins, especially the control of how spatial factors and binding avidity are correlated in, remains a great interest for glycomics. Therefore, the development of efficient methods for the rapid evaluation of new ligands such as multivalent glycoconjugates is essential for diverse diagnostic or therapeutic applications. In this paper we describe the synthesis of chemoselectively-assembled multivalent neoglycopeptides and the subsequent recognition assay on a solid support. Aminooxylated carbohydrates (betaLac-ONH(2) 4, alphaGalNAc-ONH(2) 9 and alphaMan-ONH(2) 13) have been prepared as carbohydrate-based recognition elements and assembled as clusters onto a cyclopeptidic scaffold by an oxime-based strategy in solid phase. Further binding tests between lectins and beads of resin derivatized with neoglycopeptides displaying clustered lactoses, N-acetylgalactoses and mannoses (18-20) have shown specific recognition and enhanced affinity through multivalent interactions, suggesting that the local density of carbohydrate-based ligands at the bead surface is crucial to improve the interaction of proteins of weak binding affinity. This solid phase strategy involving both molecular assembly and biological screening provides a rapid and efficient tool for various applications in glycomics.     

Tracking Surface Glycans on Live Cancer Cells with Single‐Molecule Sensitivity

Using a combination of metabolically labeled glycans, a bioorthogonal copper(I)‐catalyzed azide–alkyne cycloaddition, and the controlled bleaching of fluorescent probes conjugated to azide‐ or alkyne‐tagged glycans, a sufficiently low spatial density of dye‐labeled glycans was achieved, enabling dynamic single‐molecule tracking and super‐resolution imaging of N‐linked sialic acids and O‐linked N‐acetyl galactosamine (GalNAc) on the membrane of live cells. Analysis of the trajectories of these dye‐labeled glycans in mammary cancer cells revealed constrained diffusion of both N‐ and O‐linked glycans, which was interpreted as reflecting the mobility of the glycan rather than to be caused by transient immobilization owing to spatial inhomogeneities on the plasma membrane. Stochastic optical reconstruction microscopy (STORM) imaging revealed the structure of dynamic membrane nanotubes.     

Carbohydrate microarrays by microcontact "click" chemistry

Carbohydrate microarrays can be prepared by microcontact printing of carbohydrate alkyne conjugates on azide self-assembled monolayers (SAMs). The carbohydrates are immobilized by a "click" reaction in the contact area between the stamp and the substrate. The immobilized carbohydrates retain their characteristic selectivity toward lectins.     

Mechanistic Insight into Nanomolar Binding of Multivalent Neoglycopeptides to Wheat Germ Agglutinin

Multivalent carbohydrate–protein interactions are frequently involved in essential biological recognition processes. Accordingly, multivalency is often also exploited for the design of high‐affinity lectin ligands aimed at the inhibition of such processes. In a previous study (D. Schwefel et al., J. Am. Chem. Soc. 2010 , 132, 8704–8719) we identified a tetravalent cyclopeptide‐based ligand with nanomolar affinity to the model lectin wheat germ agglutinin (WGA). To unravel the structural features of this ligand required for high‐affinity binding to WGA, we synthesized a series of cyclic and linear neoglycopeptides that differ in their conformational freedom as well as the number of GlcNAc residues. Combined evidence from isothermal titration calorimetry (ITC), enzyme‐linked lectin assays (ELLA), and dynamic light scattering (DLS) revealed different binding modes of tetra‐ and divalent ligands and that conformational preorganization of the ligands by cyclization is not a prerequisite for achieving high binding affinities. The high affinities of the tetravalent ligands rather stem from their ability to form crosslinks between several WGA molecules. The results illustrate that binding affinities and mechanisms are strongly dependent on the used multivalent system which offers opportunities to tune and control binding processes.     

A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands

We describe a general synthetic strategy for developing high‐affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full‐length protein to identify the best binder. We describe development of epitope‐targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies.     

Carbohydrate chips for studying high-throughput carbohydrate-protein interactions

Carbohydrate-protein interactions play important biological roles in living organisms. For the most part, biophysical and biochemical methods have been used for studying these biomolecular interactions. Less attention has been given to the development of high-throughput methods to elucidate recognition events between carbohydrates and proteins. In the current effort to develop a novel high-throughput tool for monitoring carbohydrate-protein interactions, we prepared carbohydrate microarrays by immobilizing maleimide-linked carbohydrates on thiol-derivatized glass slides and carried out lectin binding experiments by using these microarrays. The results showed that carbohydrates with different structural features selectively bound to the corresponding lectins with relative binding affinities that correlated with those obtained from solution-based assays. In addition, binding affinities of lectins to carbohydrates were also quantitatively analyzed by determining IC(50) values of soluble carbohydrates with the carbohydrate microarrays. To fabricate carbohydrate chips that contained more diverse carbohydrate probes, solution-phase parallel and enzymatic glycosylations were performed. Three model disaccharides were in parallel synthesized in solution-phase and used as carbohydrate probes for the fabrication of carbohydrate chips. Three enzymatic glycosylations on glass slides were consecutively performed to generate carbohydrate microarrays that contained the complex oligosaccharide, sialyl Le(x). Overall, these works demonstrated that carbohydrate chips could be efficiently prepared by covalent immobilization of maleimide-linked carbohydrates on the thiol-coated glass slides and applied for the high-throughput analyses of carbohydrate-protein interactions.     

Preparation of Carbohydrate Arrays by Using Diels–Alder Reactions with Inverse Electron Demand

Carbohydrate microarrays are an emerging tool for the high‐throughput screening of carbohydrate–protein interactions that represent the basis of many biologically and medicinally relevant processes. The crucial step in the preparation of carbohydrate arrays is the attachment of carbohydrate probes to the surface. We examined the Diels–Alder reaction with inverse‐electron‐demand (DARinv) as an irreversible, chemoselective ligation reaction for that purpose. After having shown the efficiency of the DARinv in solution, we prepared a series of carbohydrate–dienophile conjugates that were printed onto tetrazine‐modified glass slides. Binding experiments with fluorescently labeled lectins proved successful and homogeneous immobilization was achieved by the DARinv. For immobilization of nonfunctionalized reducing oligosaccharides we developed a bifunctional chemoselective linker that enabled the attachment of a dienophile tag to the oligosaccharides through oxime ligation. The conjugates obtained were successfully immobilized on glass slides. The presented strategies for the immobilization of both synthetic carbohydrate derivatives and unprotected reducing oligosaccharides facilitate the preparation of high‐quality carbohydrate microarrays by means of the chemoselective DARinv. This concept can be readily adapted for the preparation of other biomolecule arrays.     

Chemical Synthesis Elucidates the Immunological Importance of a Pyruvate Modification in the Capsular Polysaccharide of Streptococcus pneumoniae Serotype 4

Carbohydrate modifications are believed to strongly affect the immunogenicity of glycans. Capsular polysaccharides (CPS) from bacterial pathogens are frequently equipped with a pyruvate that can be placed across the 4,6‐, 3,4‐, or 2,3‐positions. A trans‐2,3‐linked pyruvate is present on the CPS of the Gram‐positive bacterium Streptococcus pneumoniae serotype 4 (ST4), a pathogen responsible for pneumococcal infections. To assess the immunological importance of this modification within the CPS repeating unit, the first total synthesis of the glycan was carried out. Glycan microarrays containing a series of synthetic antigens demonstrated how antibodies raised against natural ST4 CPS specifically recognize the pyruvate within the context of the tetrasaccharide repeating unit. The pyruvate modification is a key motif for designing minimal synthetic carbohydrate vaccines for ST4.     

Glycopolymer Brushes for Specific Lectin Binding by Controlled Multivalent Presentation of N‐Acetyllactosamine Glycan Oligomers

A new multivalent glycopolymer platform for lectin recognition is introduced in this work by combining the controlled growth of glycopolymer brushes with highly specific glycosylation reactions. Glycopolymer brushes, synthetic polymers with pendant saccharides, are prepared by surface‐initiated atom transfer radical polymerization (SI‐ATRP) of 2‐O‐(N‐acetyl‐β‐d ‐glucosamine)ethyl methacrylate (GlcNAcEMA). Here, the fabrication of multivalent glycopolymers consisting of poly(GlcNAcEMA) is reported with additional biocatalytic elongation of the glycans directly on the silicon substrate by specific glycosylation using recombinant glycosyltransferases. The bioactivity of the surface‐grafted glycans is investigated by fluorescence‐linked lectin assay. Due to the multivalency of glycan ligands, the glycopolymer brushes show very selective, specific, and strong interactions with lectins. The multiarrays of the glycopolymer brushes have a large potential as a screening device to define optimal‐binding environments of specific lectins or as new simplified diagnostic tools for the detection of cancer‐related lectins in blood serum.

     

Carbohydrate Chain of Ganglioside GM1 as a Ligand: Identification of the Binding Strategies of Three 15 mer Peptides and Their Divergence from the Binding Modes of Growth‐Regulatory Galectin‐1 and Cholera Toxin

The branched pentasaccharide chain of ganglioside GM₁ is a prominent cell surface ligand, for example, for cholera toxin or tumor growth‐regulatory homodimeric galectins. This activity profile via protein recognition prompted us to examine the binding properties of peptides with this specificity. Our study provides insights into the mechanism of molecular interaction of this thus far unexplored size limit of the protein part. We used three pentadecapeptides in a combined approach of mass spectrometry, NMR spectroscopy and molecular modelling to analyze the ligand binding in solution. Availability of charged and hydrophobic functionalities affected the intramolecular flexibility of the peptides differently. Backfolding led to restrictions in two cases; the flexibility was not reduced significantly by association of the ligand in its energetically privileged conformations. Major contributions to the interaction energy arise from the sialic acid moiety contacting Arg/Lys residues and the N‐terminal charge. Considerable involvement of stacking between the monovalent ligand and aromatic rings could not be detected. This carbohydrate binding strategy is similar to how an adenoviral fiber knob targets sialylated glycans. Rational manipulation for an affinity enhancement can now be directed to reduce the flexibility, exploit the potential for stacking and acquire the cross‐linking capacity of the natural lectins by peptide attachment to a suitable scaffold.     

Biotinylation of Peptides/Proteins Using Biocytin Hydrazide

Biocytin hydrazide is widely used to biotinylate the carbohydrate moieties of glycoproteins. In this study, however, biocytin hydrazide was found to be able to directly biotinylate peptides and proteins. This phenomenon may cause false identification of non‐glycopeptides/non‐glycoproteins as glycopeptides/glycoproteins. Here, we report a systematic investigation of the reaction of peptides/proteins with biocytin hydrazide. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry is used to analyze the biotinylation reaction between peptides/proteins and biocytin hydrazide. Peptides/proteins were reacted with biocytin hydrazide in diverse solvent systems with different biocytin hydrazide concentrations for up to 96 h at temperatures ranging from 4 °C to 65 °C. Singly biotinylated or multiply biotinylated peptides/proteins are observed. The efficiency of the biotinylation reaction increases with higher temperature, higher biocytin hydrazide concentration, or longer reaction time. The influence of buffer pH on the biotinylation reaction of peptides/proteins is less pronounced. The biotinylation efficiency is optimum at neutral pH. Data suggests that the peptides are biotinylated as efficiently as proteins. The observation that peptides/proteins condense only with biocytin hydrazide, 2‐iminobiotin hydrazide, adipic dihydrazide and phenyl hydrazine but not with biocytin HCl and 2‐iminobiotin, indicates that the biotinylation reaction of peptides/proteins occurs with the hydrazide moiety but not with biotin moiety of the biotinylated reagent. The postsource decay data of biotinylated P₁₄R indicates that biocytin hydrazide condenses with the guanidino group of arginine's side chain of P₁₄R, indicating that besides N‐terminal and lysine residue of peptides/proteins, arginine residue is capable of reacting with biocytin hydrazide.     

Promotion of sugar-lectin recognition through the multiple sugar presentation offered by regioselectively addressable functionalized templates (RAFT): a QCM-D and SPR study

The investigation of recognition events between carbohydrates and proteins, especially the understanding of how spatial factors and binding avidity are correlated, remains a great interest for glycobiology. In this context we have investigated by nanogravimetry (QCM-D) and surface plasmon resonance (SPR), the kinetics and thermodynamics of the interaction between concanavalin A (Con A) and various neoglycopeptide ligands of low molecular weight. Regioselectively addressable functionalized templates (RAFT) have been used as scaffolds for the design of multivalent neoglycopeptides bearing thiol or biotin functions for their anchoring on transducer surfaces. Although these multivalent neoglycopeptide ligands cannot span multiple binding sites within the same Con A protein, they have increased activities relative to their monovalent counterpart. Our results emphasize that the multivalent RAFT ligands function by clustering several lectins, which leads to enhanced affinities.     

An Apoptosis‐Inducing Peptidic Heptad That Efficiently Clusters Death Receptor 5

Multivalent ligands of death receptors hold particular promise as tumor cell‐specific therapeutic agents because they induce an apoptotic cascade in cancerous cells. Herein, we present a modular approach to generate death receptor 5 (DR5) binding constructs comprising multiple copies of DR5 targeting peptide (DR5TP) covalently bound to biomolecular scaffolds of peptidic nature. This strategy allows for efficient oligomerization of synthetic DR5TP‐derived peptides in different spatial orientations using a set of enzyme‐promoted conjugations or recombinant production. Heptameric constructs based on a short (60–75 residues) scaffold of a C‐terminal oligomerization domain of human C4b binding protein showed remarkable proapoptotic activity (EC₅₀=3 nm ) when DR5TP was ligated to its carboxy terminus. Our data support the notion that inter‐ligand distance, relative spatial orientation and copy number of receptor‐binding modules are key prerequisites for receptor activation and cell killing.     

Multi-mannosides based on a carbohydrate scaffold: synthesis, force field development, molecular dynamics studies, and binding affinities for lectin Con A

A short and efficient strategy for the synthesis of multi-valent mannosides based on a selectively functionalized carbohydrate scaffold was reported involving (i) direct regioselective azidation of unprotected commercial saccharides, (ii) acetylation, (iii) grafting of the mannosyl ligands by click chemistry, and (iv) deacetylation. New glycoclusters with a valency ranging from 1 to 4 and different spatial arrangements of the epitopes were obtained. Binding affinities of the new glycoclusters toward concanavalin A (Con A) lectin were investigated by an enzyme-linked lectin essay (ELLA). The synthetic multi-valent compounds exhibited a remarkable cluster effect with a relative potency per mannoside residue ranging from 8.1 to 9.1 depending on the structures. ELLA experiments were in agreement with the establishment of favorable interactions between triazole ring and Con A, increasing the binding affinity. A new force field topology database was developed in agreement with the GLYCAM 2004 force field. Molecular dynamics performed on representative glyco-conjugates revealed interesting structural features such as rigidity of the scaffold for a well-defined presentation of the ligands and highly flexible mannose counterparts. The new glycoconjugates reported may be promising tools as probes or effectors of biological processes involving lectins.     

Synthesis of Carbohydrate‐Functionalised Sequence‐Defined Oligo(amidoamine)s by Photochemical Thiol?Ene Coupling in a Continuous Flow Reactor

Poly/oligo(amidoamine)s (PAAs) have recently been recognised for their potential as well‐defined scaffolds for multiple carbohydrate presentation and as multivalent ligands. Herein, we report two complimentary strategies for the preparation of such sequence‐defined carbohydrate‐functionalised PAAs that use photochemical thiol? ene coupling (TEC) as an alternative to the established azide–alkyne cycloaddition (“click”) reaction. In the first approach, PAAs that contained multiple olefins were synthesised on a solid support from a new building block and subsequent conjugation with unprotected thio‐carbohydrates. Alternatively, a pre‐functionalised building block was prepared by using TEC and assembled on a solid support to provide a carbohydrate‐functionalised PAA. Both methods rely on the use of a continuous flow photoreactor for the TEC reactions. This system is highly efficient, owing to its short path length, and requires no additional radical initiator. Performing the reactions at 254 nm in Teflon AF‐2400 tubing provides a highly efficient TEC procedure for carbohydrate conjugation, as demonstrated in the reactions of O‐allyl glycosides with thiols. This method allowed the complete functionalisation of all of the reactive sites on the PAA backbone in a single step, thereby obtaining a defined homogeneous sequence. Furthermore, reaction at 366 nm in FEP tubing in the flow reactor enabled the large‐scale synthesis of an fluorenylmethyloxycarbonyl (Fmoc)‐protected glycosylated building block, which was shown to be suitable for solid‐phase synthesis and will also allow heterogeneous sequence control of different carbohydrates along the oligomeric backbone. These developments enable the synthesis of sequence‐defined carbohydrate‐functionalised PAAs with potential biological applications.     

Chemoenzymatic Assembly of Mammalian O‐Mannose Glycans

O‐Mannose glycans account up to 30 % of total O‐glycans in the brain. Previous synthesis and functional studies have only focused on the core M3 O‐mannose glycans of α‐dystroglycan, which are a causative factor for various muscular diseases. In this study, a highly efficient chemoenzymatic strategy was developed that enabled the first collective synthesis of 63 core M1 and core M2 O‐mannose glycans. This chemoenzymatic strategy features the gram‐scale chemical synthesis of five judiciously designed core structures, and the diversity‐oriented modification of the core structures with three enzyme modules to provide 58 complex O‐mannose glycans in a linear sequence that does not exceed four steps. The binding profiles of synthetic O‐mannose glycans with a panel of lectins, antibodies, and brain proteins were also explored by using a printed O‐mannose glycan array.     

A chemoenzymatic approach enables the site‐specific conjugation of recombinant proteins

Many biotechniques including protein microarray, drug screening, biosensors rely on the immobilization of recombinant proteins on the solid supports. It is well known that random orientation of the immobilized proteins could impair their biologic functions. Thus, it is very important to develop new site‐specific immobilization approach. In this study, we presented a chemoenzymatic approach for site‐specific conjugation of recombinant proteins onto solid support. In this strategy, the affinity tag on recombinant protein was enzymatically cleaved to expose the N‐terminal serine, which was oxidized to carry an aldehyde group and was then covalently coupled to hydrazide resin through hydrazone ligation. As this approach takes advantage of the most frequently used TEV protease, it requires no further sequence design on recombinant protein. This method was validated by site specific coupling of a synthetic peptide and a recombinant protein onto solid supports. It was found that the site specific immobilized SH2 domain is functional and could be used to enrich tyrosine phosphorylated peptides.     

Automated Synthesis of Arabinoxylan‐Oligosaccharides Enables Characterization of Antibodies that Recognize Plant Cell Wall Glycans

Monoclonal antibodies that recognize plant cell wall glycans are used for high‐resolution imaging, providing important information about the structure and function of cell wall polysaccharides. To characterize the binding epitopes of these powerful molecular probes a library of eleven plant arabinoxylan oligosaccharides was produced by automated solid‐phase synthesis. Modular assembly of oligoarabinoxylans from few building blocks was enabled by adding (2‐naphthyl)methyl (Nap) to the toolbox of orthogonal protecting groups for solid‐phase synthesis. Conjugation‐ready oligosaccharides were obtained and the binding specificities of xylan‐directed antibodies were determined on microarrays.     

Synthetic Mucin‐Like Glycopeptides as Versatile Tools to Measure Effects of Glycan Structure/Density/Position on the Interaction with Adhesion/Growth‐Regulatory Galectins in Arrays

Functional pairing of cellular glycoconjugates with tissue lectins is a highly selective process, whose determinative factors have not yet been fully delineated. Glycan structure and modes of presentation, that is, its position and density, can contribute to binding, as different members of a lectin family can regulate degrees of responsiveness to these factors. Using a peptide repeat sequence motif of the glycoprotein mucin‐1, the principle of introducing synthetic (glyco)peptides with distinct variations in these three parameters to an array‐based screening of tissue lectins is illustrated. Interaction profiles of seven adhesion/growth‐regulatory galectins cover the range from intense signals with core 2 pentasaccharides and core 1 binding for galectins‐3 and ‐5 to a lack of binding for galectin‐1 and also the galectin‐related protein, which was included as a negative control. Remarkably, the two tandem‐repeat‐type galectins‐4 and ‐8 were distinguished by core 1 sialylation, as the two separated domains were. These results encourage further synthetic elaboration of the glycopeptide library and testing of the network of natural galectins and rationally engineered variants of the lectins.     

Bi- to tetravalent glycoclusters: synthesis, structure-activity profiles as lectin inhibitors and impact of combining both valency and headgroup tailoring on selectivity

The emerging functional versatility of cellular glycans makes research on the design of synthetic inhibitors a timely topic. In detail, the combination of ligand (or headgroup or contact site) structure with spatial parameters that depend on topological and geometrical factors underlies the physiological selectivity of glycan-protein (lectin) recognition. We herein tested a panel of bi-, tri- and tetravalent compounds against two plant agglutinins and adhesion/growth-regulatory lectins (galectins). In addition, we examined the impact of headgroup tailoring (converting lactose to 2'-fucosyllactose) in combination with valency increase in two assay types of increasing biorelevance (from solid-phase binding to cell binding). Compounds were prepared using copper-catalysed azide alkyne cycloaddition from peracetylated lactosyl or 2'-fucosyllactosyl azides. Significant inhibition was achieved for the plant toxin with a tetravalent compound. Different levels of sensitivity were noted for the three groups of the galectin family. The headgroup extension to 2'-fucosyllactose led to a selectivity gain, especially for the chimera-type galectin-3. Valency increase established discrimination against the homodimeric proteins, whereas the combination of valency with the headgroup extension led to discrimination against the tandem-repeat-type galectin-8 for chicken galectins but not human galectins-3 and -4. Thus, detailed structure-activity profiling of glycoclusters combined with suitably modifying the contact site for the targeted lectin will help minimize cross-reactivity among this class of closely related proteins.