Self-organized glycoclusters along DNA: effect of the spatial arrangement of galactoside residues on cooperative lectin recognition

We describe herein the relationship between the spatial arrangement of self-organized galactose clusters and lectin recognition. beta-Galactose-modified deoxyuridine phosphoramidite was synthesized and applied to solid-phase synthesis to provide 18-, 20-, and 22-mers of site-specifically galactosylated oligodeoxynucleotides (Gal-ODNs). These Gal-ODNs were self-organized through hybridization with the corresponding 18-, 20-, and 22-mers of half-sliding complementary ODNs (hsc-ODNs) to give periodic galactoside clusters. The self-organization of ODNs was confirmed by size exclusion chromatography and gel electrophoresis. The binding of the Gal-clusters to the FITC-labeled RCA(120) lectin was analyzed by monitoring the change in fluorescence intensity. The assembly of 20-mer Gal-ODN with the 20-mer hsc-ODN was strongly and cooperatively recognized by the lectin. The 18-mer assembly was bound more weakly and less cooperatively, and the 22-mer assembly was minimally bound to the lectin. RCA(120) lectin recognized not only the density of galactoside residues, but also the spatial arrangement. The size of the Gal cluster was estimated from the association constant of Gal-ODN with hsc-ODN. The relationship between lectin-recognition and Gal-cluster size is also discussed.     

Saccharide-branched Cyclodextrins as Targeting Drug Carriers

A synthetic series of heptakis-galactose-branched cyclodextrins (termed CDs) having a longer spacer arm using two amino-caproic acids as an enlarging unit were prepared. Starting with heptakis-amino-β-CD or heptakis-amino-caproic-amide-β-CD, treated with galactosyl-glucono-amide-caproic acid, the new compounds heptakis (Gal-cap1)-CD (4) or heptakis (Gal-cap2)-CD (5) were obtained. The longer galactose spacer arm extremely favors the PNA association. The effect of branch length on K with PNA was enhanced up to 138-fold 3 as well as with DXR enhanced up to 81-fold. Hexakis (Gal-cap2)-CD (6) was prepared and the association constants with rat liver cells were observed to be 2.5 × 10¹⁰ M⁻¹. A multi-high mannose type oligosaccharide branched CD (7) showed a large association constant with DXR up to 1.1 × 10⁹ M⁻¹. The two-dimensional map for the association constants of newly synthesized oligosaccharide-branched CDs toward lectin or liver cells versus the association constants toward a drug (doxorubicin) suggested a method of finding a better targeting drug carrier. The structural effect of the oligosaccharide-CDs showed that the number and length of the branch were dominant factors in designing for enhanced dual recognition.     

Mannose‐based graft polyesters with tunable binding affinity to concanavalin A

Glycopolymers have been widely used to understand the interactions between carbohydrates and lectins, which facilitate the diagnosis and detection of disease and pathogens as well as the development of vaccines. While studies have been focused on the correlation of glycopolymer structure and their binding to lectins, graft‐type glycopolyesters are uncommon. Herein, we report the design and synthesis of mannose‐based graft polyesters by “grafting‐from” method and investigate their interactions with Concanavalin A (Con A). As confirmed by ¹H NMR spectroscopy and sulfuric acid‐UV method, graft polyesters with different lengths of mannose graft were successfully synthesized. Our results from turbidimetry binding assay showed that graft polyesters with longer mannose graft exhibit higher initial binding rate (k_i). Isothermal titration calorimetry measurements of these graft polyesters with Con A showed that polymers exhibit higher binding affinity (k_a) with the number of side chain mannose. This study provides understanding of the interaction between Con A and mannose‐based graft polyesters, which can be employed for the development of glycopolymeric therapeutics. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 3908–3917     

Comparison of RAFT‐derived poly(vinylpyrrolidone) verses poly(oligoethyleneglycol methacrylate) for the stabilization of glycosylated gold nanoparticles

Carbohydrates dictate many biological processes including infection by pathogens. Glycosylated polymers and nanomaterials which have increased affinity due to the cluster glycoside effect, are therefore useful tools to probe function, but also as prophylactic therapies or diagnostic tools. Here, the effect of polymer structure on the coating of gold nanoparticles is studied in the context of grafting density, buffer stability, and in a lectin binding assay. RAFT polymerization is used to generate poly(oligoethyleneglycol methacrylates) and poly(N‐vinylpyrrolidones) with a thiol end‐group for subsequent immobilization onto the gold. It is observed that poly(oligoethylene glycol methacrylates), despite being widely used particle coatings, lead to low grafting densities which in turn resulted in lower stability in biological buffers. A depression of the cloud point upon nanoparticle immobilization is also seen, which might compromise performance. In comparison poly(vinylpyrrolidones) resulted in stable particles with higher grafting densities due to the compact size of each monomer unit. The higher grafting density also enabled an increase in the number of carbohydrates which can be installed per nanoparticle at the chain ends, and gave increased binding in a lectin recognition assay. These results will guide the development of new nanoparticle biosensors with enhanced specificity, affinity, and stability. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 55, 1200–1208     

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.     

Application of Lectin Microarrays for Biomarker Discovery

Many proteins in living organisms are glycosylated. As their glycan patterns exhibit protein-, cell-, and tissue-specific heterogeneity, changes in the glycosylation levels could serve as useful indicators of various pathological and physiological states. Thus, the identification of glycoprotein biomarkers from specific changes in the glycan profiles of glycoproteins is a trending field. Lectin microarrays provide a new glycan analysis platform, which enables rapid and sensitive analysis of complex glycans without requiring the release of glycans from the protein. Recent developments in lectin microarray technology enable high-throughput analysis of glycans in complex biological samples. In this review, we will discuss the basic concepts and recent progress in lectin microarray technology, the application of lectin microarrays in biomarker discovery, and the challenges and future development of this technology. Given the tremendous technical advancements that have been made, lectin microarrays will become an indispensable tool for the discovery of glycoprotein biomarkers.     

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.     

The development of an integrated platform to identify breast cancer glycoproteome changes in human serum

Protein glycosylation represents one of the major post-translational modifications and can have significant effects on protein function. Moreover, changes in the carbohydrate structure are increasingly being recognized as an important modification associated with cancer etiology. In this report, we describe the development of a proteomics approach to identify breast cancer related changes in either concentration and/or the carbohydrate structures of glycoprotein(s) present in blood samples. Diseased and healthy serum samples were processed by an optimized sample preparation protocol using multiple lectin affinity chromatography (M-LAC) that partitions serum proteins based on glycan characteristics. Subsequently, three separate procedures, 1D SDS-PAGE, isoelectric focusing and an antibody microarray, were applied to identify potential candidate markers for future study. The combination of these three platforms is illustrated in this report with the analysis of control and cancer glycoproteomic fractions. Firstly, a molecular weight based separation of glycoproteins by 1D SDS-PAGE was performed, followed by protein, glycoprotein staining, lectin blotting and LC–MS analysis. To refine or confirm the list of interesting glycoproteins, isoelectric focusing (targeting sialic acid changes) and an antibody microarray (used to detect neutral glycan shifts) were selected as the orthogonal methods. As a result, several glycoproteins including alpha-1B-glycoprotein, complement C3, alpha-1-antitrypsin and transferrin were identified as potential candidates for further study.     

A Facile Latex Agglutination Lectin Assay (LALA) for Weakly Binding Ligands

To apply the latex agglutination lectin assay (LALA) to carbohydrate ligands, monosaccharide derivatives were incorporated onto latex beads by various methods, and the usefulness of the resulting beads was evaluated. The best outcome, which resulted in aggregation with lectin concentrations of 1 to 4 μg/mL, was obtained when latex beads coated with bovine serum albumin were treated with divinylsulfone, a linker agent, and then with 2-aminoethyl glycosides. Monosaccharides with an amino or anomeric hydroxyl group other than N-acetylglucosamine were applicable in this direct LALA. For example, mannose- and 5-thiomannose-coupled latex beads showed aggregation with minimum concanavalin (ConA) concentrations of 4 and 32 μg/mL, respectively. An inhibition assay was more versatile than the direct LALA, and the standardized inhibition activity (EC⁰ ₅₀) was determined for several compounds. Representative EC⁰ ₅₀ data for mannose, methyl mannoside, and p-nitrophenyl mannoside (1, 0.12, and 0.06 mM, respectively) are consistent with those reported with other methods. We obtained EC⁰ ₅₀ values for some synthetic compounds with slightly different binding abilities to ConA, demonstrating a semiquantitative character of this method. The inhibition LALA can be performed without instrumentation or tedious derivatization and is thus suitable for the rapid evaluation of monovalent ligands prior to assemblage into multivalent ligands.     

Linear Precision Glycomacromolecules with Varying Interligand Spacing and Linker Functionalities Binding to Concanavalin A and the Bacterial Lectin FimH

A series of precision glycomacromolecules is prepared following previously established solid phase synthesis allowing for controlled variations of interligand spacing and the overall number of carbohydrate ligands. In addition, now also different linkers are installed between the carbohydrate ligand and the macromolecular scaffold. The lectin binding behavior of these glycomacromolecules is then evaluated in isothermal titration calorimetry (ITC) and kinITC experiments using the lectin Concanavalin A (Con A) in its dimeric and tetrameric form. The results indicate that both sterical and statistical effects impact lectin binding of precision glycomacromolecules. Moreover, ITC results show that highest affinity toward Con A can be achieved with an ethyl phenyl linker, which parallels earlier findings with the bacterial lectin FimH. In this way, a first set of glycomacromolecule structures is selected for testing in a bacterial adhesion–inhibition study. Here, the findings point to a one‐sugar binding mode mainly affected by sterical restraints of the nonbinding parts of the respective glycomacromolecule.