A Triazatruxene‐Based Glycocluster as a Fluorescent Sensor for Concanavalin A

A new triazatruxene‐based fluorescent glycocluster has been designed, synthesized, and fully characterized by NMR spectroscopy and mass spectrometry. Furthermore, its specific and selective binding properties with concanavalin A (Con A) have been investigated by fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and turbidity assay. The obtained results showed that the multivalent mannose‐modified triazatruxene exhibited specific binding with Con A, but no binding to peanut agglutinin (PNA) lectin or bovine serum albumin (BSA), corresponding to a two‐orders‐of‐magnitude higher affinity than that of monovalent mannose ligands. Most interestingly, a fluorescence enhancement of the triazatruxene‐based glycocluster was observed upon binding with Con A because of hydrophobic interactions involving sites close to the triazatruxene moiety. Furthermore, the inhibitory ability of the triazatruxene‐based glycocluster against ORN178‐ induced haemagglutination has been investigated by haemagglutination inhibition assay. The results indicated selective binding with ORN178.     

Synthesis of Glutamic Acid-based Cluster Galactosides and Their Binding Affinities with Liver Cells

Structurally well defined di-, tri- and tetra-valent cluster galactosides were synthesized in a convenient way.Oligo-glutamic acids were assembled as scaffolds. The presence of amine groups in these three ligands is expected to couple with drugs or genes for delivery. The binding affinities of these cluster galactoses to liver cells were determined by in vitro binding studies. Among them, the tetravalent cluster galactose (19) showed the highest affinity to liver cell. It is therefore a promising targeting device for the specific delivery of drugs or genes to parenchymal liver cells.     

Structure‐Based Optimization of the Terminal Tripeptide in Glycopeptide Dendrimer Inhibitors of Pseudomonas aeruginosa Biofilms Targeting LecA

The galactopeptide dendrimer GalAG2 ((β‐Gal‐OC₆H₄CO‐Lys‐Pro‐Leu)₄(Lys‐Phe‐Lys‐Ile)₂Lys‐His‐Ile‐NH₂) binds strongly to the Pseudomonas aeruginosa (PA) lectin LecA, and it inhibits PA biofilms, as well as disperses already established ones. By starting with the crystal structure of the terminal tripeptide moiety GalA‐KPL in complex with LecA, a computational mutagenesis study was carried out on the galactotripeptide to optimize the peptide–lectin interactions. 25 mutants were experimentally evaluated by a hemagglutination inhibition assay, 17 by isothermal titration calorimetry, and 3 by X‐ray crystallography. Two of these tripeptides, GalA‐KPY (dissociation constant (K_D)=2.7 μM ) and GalA‐KRL (K_D=2.7 μM ), are among the most potent monovalent LecA ligands reported to date. Dendrimers based on these tripeptide ligands showed improved PA biofilm inhibition and dispersal compared to those of GalAG2 , particularly G2KPY ((β‐Gal‐OC₆H₄CO‐Lys‐Pro‐Tyr)₄(Lys‐Phe‐Lys‐Ile)₂Lys‐His‐Ile‐NH₂). The possibility to retain and even improve the biofilm inhibition in several analogues of GalAG2 suggests that it should be possible to fine‐tune this dendrimer towards therapeutic use by adjusting the pharmacokinetic parameters in addition to the biofilm inhibition through amino acid substitutions.     

Synthesis and Immunological Evaluation of Self‐Assembling and Self‐Adjuvanting Tricomponent Glycopeptide Cancer‐Vaccine Candidates

Self‐adjuvanting tricomponent vaccines were prepared and assessed for their self‐assembly and immunological activity in mouse models. The vaccines each consisted of a peptide or glycopeptide antigen that corresponds to a complete copy of the variable‐number tandem repeat (VNTR) of the tumor‐associated mucin 1 (MUC1) glycoprotein, the universal T‐cell helper peptide epitope PADRE, and the immunoadjuvant Pam₃CysSer. The vaccines were shown to spontaneously self‐assemble in water to form isotropic particles varying in size from 17 to 25 nm and elicited robust humoral responses in murine models without the addition of an external adjuvant. The serum antibodies could recognize tumor‐associated MUC1 epitopes on the surface of MCF7 breast‐cancer cells and B16 melanoma cells, which overexpress this tumor‐associated glycoprotein.     

A Unified Strategy for the Synthesis of Mucin Cores 1–4 Saccharides and the Assembled Multivalent Glycopeptides

By displaying different O‐glycans in a multivalent mode, mucin and mucin‐like glycoproteins are involved in a plethora of protein binding events. The understanding of the roles of the glycans and the identification of potential glycan binding proteins are major challenges. To enable future binding studies of mucin glycan and glycopeptide probes, a method that gives flexible and efficient access to all common mucin core‐glycosylated amino acids was developed. Based on a convergent synthesis strategy starting from a shared early stage intermediate by differentiation in the glycoside acceptor reactivity, a common disaccharide building block allows for the creation of extended glycosylated amino acids carrying the mucin type‐2 cores 1–4 saccharides. Formation of a phenyl‐sulfenyl‐N‐Troc (Troc=trichloroethoxycarbonyl) byproduct during N‐iodosuccinimide‐promoted thioglycoside couplings was further characterized and a new methodology for the removal of the Troc group is described. The obtained glycosylated 9‐fluorenylmethoxycarbonyl (Fmoc)‐protected amino acid building blocks are incorporated into peptides for multivalent glycan display.     

Protecting‐Group‐Free One‐Pot Synthesis of Glycoconjugates Directly from Reducing Sugars

The conversion of sugars into glycomimetics typically involves multiple protecting‐group manipulations. The development of methodology allowing the direct aqueous conversion of free sugars into glycosides, and mimics of oligosaccharides and glycoconjugates in a high‐yielding and stereoselective process is highly desirable. The combined use of 2‐azido‐1,3‐dimethylimidazolinium hexafluorophosphate and the Cu‐catalyzed Huisgen cycloaddition allowed the synthesis of a range of glycoconjugates in a one‐step reaction directly from reducing sugars under aqueous conditions. The reaction, which is completely stereoselective, may be applied to the convergent synthesis of triazole‐linked glycosides, oligosaccharides, and glycopeptides. The procedure provides a method for the one‐pot aqueous ligation of oligosaccharides and peptides bearing alkyne side chains.     

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.     

Multivalency by Self‐Assembly: Binding of Concanavalin A to Metallosupramolecular Architectures Decorated with Multiple Carbohydrate Groups

Multiplication of functional units through self‐assembly is a powerful way to new properties and functions. In particular, self‐organization of components decorated with recognition groups leads to multivalent entities, amenable to strong and selective binding with multivalent targets, such as protein receptors. Here we describe an efficient, supramolecular, one‐pot valency multiplication process proceeding through self‐organization of monovalent components into well‐defined, grid‐shaped [2×2] tetranuclear complexes bearing eight sugar residues for multivalent interaction with the tetrameric lectin, concanavalin A (Con A). The grids are stable in water under physiological pH at a relatively high concentration, but dissociate readily at slightly more acidic pH or upon dilution below a certain threshold, in a type of on–off behavior. The carbohydrate‐decorated grids interact strongly and selectively with Con A forming triply supramolecular bio‐hybrid polymeric networks, which lead to a highly specific phase‐separation and quasi‐quantitative precipitation of Con A out of solution. Dramatic effects of valency number on agglutination properties were demonstrated by comparison of grids with divalent carbohydrates of covalent and non‐covalent (L ‐shaped, mononuclear zinc complex) scaffolds. The results presented here provide prototypical illustration of the power of multivalency generation by self‐assembly leading to defined arrays of functional groups and binding patterns.     

Masked Thiol Sugars: Chemical Behavior and Synthetic Applications of S‐Glycopyranosyl‐N‐monoalkyl Dithiocarbamates

The chemical behavior of S‐glycopyranosyl‐N‐monoalkyl dithiocarbamates (DTCs) as masked 1‐glycosyl thiols, easily prepared by the nucleophilic displacement of 1‐halo sugars with dithiocarbamate salts of primary amines, has been studied and synthetically exploited. This behavior relies on the abstraction of the proton of the carbamate functionality that allows controlled access to thiolate sugar intermediates. The basic character of the DTC salts used as reagents leads to thiolates that evolve in situ to symmetrical diglycosyldisulfides (DGDSs) when long reaction times are allowed. Alternatively, controlled unmasking of the thiolate function can be efficiently attained by treatment with an external base of isolated anomeric glycosyl DTCs, the formation of which is prevalent when using short reaction times. In this manner, a second methodology for the preparation of symmetrical DGDSs and a chemical protocol for the S‐glycosylation of any electrophilic substrate are established. The applications of this last strategy for the preparation of thioglycosyl vinyl sulfones, thiodisaccharides, and S‐linked homo‐ and heterodivalent neoglycoconjugates are described as a proof‐of‐concept of the great potential of the sugar DTCs in any chemical scenario in which the covalent attachment of a thiol sugar is required. The evaluation of the biological functionality of some divalent sulfurated sugar systems is also described.     

Structure‐Transport Relationship of Dendritic Core‐Shell Nanocarriers for Polar Dyes

TriPEGylated functionalized dendritic poly(ethylene imines) were synthesized and their transportation properties for rose Bengal (up to 50 guests per dendrimer) were evaluated. A critical aggregation concentration was observed, demonstrating the formation of aggregates among the dendrimers. The structure‐activity relationships show that their encapsulation capacities were dependent linearly on the density of the PEG shell, either as PEG length or degree of functionality, which confirmed that the PEG chains play a predominant role in the encapsulation process. It further gives some insights that the guest molecules appeared to be predominantly located in the PEG shell rather than in the core.

     

The Implications of (2S,4S)‐Hydroxyproline 4‐O‐Glycosylation for Prolyl Amide Isomerization

The conformations of peptides and proteins are often influenced by glycans O‐linked to serine (Ser) or threonine (Thr). (2S,4R)‐4‐Hydroxyproline (Hyp), together with L ‐proline (Pro), are interesting targets for O‐glycosylation because they have a unique influence on peptide and protein conformation. In previous work we found that glycosylation of Hyp does not affect the N‐terminal amide trans/cis ratios (K_trans/cis) or the rates of amide isomerization in model amides. The stereoisomer of Hyp—(2S,4S)‐4‐hydroxyproline (hyp)—is rarely found in nature, and has a different influence both on the conformation of the pyrrolidine ring and on K_trans/cis. Glycans attached to hyp would be expected to be projected from the opposite face of the prolyl side chain relative to Hyp; the impact this would have on K_trans/cis was unknown. Measurements of ³J coupling constants indicate that the glycan has little impact on the C^γ‐endo conformation produced by hyp. As a result, it was found that the D ‐galactose residue extending from a C^γ‐endo pucker affects both K_trans/cis and the rate of isomerization, which is not found to occur when it is projected from a C^γ‐exo pucker; this reflects the different environments delineated by the proline side chain. The enthalpic contributions to the stabilization of the trans amide isomer may be due to disruption of intramolecular interactions present in hyp; the change in enthalpy is balanced by a decrease in entropy incurred upon glycosylation. Because the different stereoisomers—Hyp and hyp—project the O‐linked carbohydrates in opposite spatial orientations, these glycosylated amino acids may be useful for understanding of how the projection of a glycan from the peptide or protein backbone exerts its influence.     

Sulfated and Non‐Sulfated Glycopeptide Recognition Domains of P‐Selectin Glycoprotein Ligand 1 and their Binding to P‐ and E‐Selectin

Total synthesis through block glycosylation and selective chemical O‐sulfation of tyrosine residues yielded the glycopeptide recognition domain A (X=SO₃^?) of the P‐selectin glycoprotein ligand 1, in which the terminal sialic acid of the complex hexasaccharide side chain was replaced by (S)‐cyclohexyl lactic acid. In binding assays the O‐sulfated structure A showed high affinity towards P‐selectin, the non‐sulfated towards E‐selectin.

     

Thiol‐ene and thiol‐yne‐based synthesis of glycodendrimers as nanomolar inhibitors of wheat germ agglutinin

Alkene and alkyne functional polyester‐based dendrimers of generation 1 to 4 have been prepared and reacted under free‐radical conditions with 2‐acetamido‐2‐deoxy‐1‐thio‐β‐D ‐glucose (GlcNAc‐SH). As the alkene‐dendrimers underwent the addition of one thiyl radical per ene group whereas each yne group of alkyne‐dendrimers was saturated by two thiyl radicals, a collection of glycodendrimers with glycan density ranging from six to ninety‐six GlcNAc per dendrimer was obtained. The recognition properties of the prepared glycodendrimers toward the wheat germ agglutinin (WGA) were evaluated by enzyme‐linked lectin assay (ELLA). The eight glycodendrimers were excellent ligands showing IC₅₀ values in the nanomolar range and relative potencies per sugar unit up to 2.27 e⁶ when compared to monosaccharidic GlcNAc used as monovalent reference. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 2422–2433