Molecular dynamics simulations of glycoclusters and glycodendrimers

Protein-carbohydrate recognition plays a crucial role in a wide range of biological processes, required both for normal physiological functions and the onset of disease. Nature uses multivalency in carbohydrate-protein interactions as a strategy to overcome the low affinity found for singular binding of an individual saccharide epitope to a single carbohydrate recognition domain of a lectin. To mimic the complex multi-branched oligosaccharides found in glycoconjugates, which form the structural basis of multivalent carbohydrate-protein interactions, so-called glycoclusters and glycodendrimers have been designed to serve as high-affinity ligands of the respective receptor proteins. To allow a rational design of glycodendrimer-type molecules with regard to the receptor structures involved in carbohydrate recognition, a deeper knowledge of the dynamics of such molecules is desirable. Most glycodendrimers have to be considered highly flexible molecules with their conformational preferences most difficult to elucidate by experimental methods. Longtime molecular dynamics (MD) simulations with inclusion of explicit solvent molecules are suited to explore the conformational space accessible to glycodendrimers. Here, a detailed geometric and conformational analysis of 15 glycodendrimers and glycoclusters has been accomplished, which differ with regard to their core moieties, spacer characteristics and the type of terminal carbohydrate units. It is shown that the accessible conformational space depends strongly on the structural features of the core and spacer moieties and even on the type of terminating sugars. The obtained knowledge about possible spatial distributions of the sugar epitopes exposed on the investigated hyperbranched neoglycoconjugates is detailed for all examples and forms important information for the interpretation and prediction of affinity data, which can be deduced from biological testing of these multivalent neoglycoconjugates.     

Glycodendrimers: novel glycotope isosteres unmasking sugar coding. case study with T-antigen markers from breast cancer MUC1 glycoprotein

Glycodendrimers are relatively novel synthetic biomacromolecules that are made of biologically relevant carbohydrate ligands constructed at the periphery of a wide range of highly functionalized and repetitive scaffolds having varied molecular weights and structures. They were aimed to fill the gap between glycopolymers, having generally dispersed higher molecular weight, and small glycoclusters, in the study of multivalent carbohydrate protein interactions. In a way, glycodendrimers, with their spheroidal or dendritic (wedge) type structures, were initially designed as bioisosteres of cell surface multiantennary glycans. Taken as a curiosity and elegant molecules at their beginning, they are now considered as potent inhibitors of microbial adhesins. They have also been shown to play some roles in signal transduction and in receptor cross-linking. This brief report will describe advances that have been made toward the syntheses of a range of glycodendrimers bearing the immunodominant T-antigen disaccharide [beta-D-Gal-(1-3)-alpha-D-GalNAc] found on malignant cells of carcinomas, particularly related to breast cancer. This antigen, usually cryptic on healthy tissues, is greatly increased on cancer cells as a result of aberrant glycosylation. It is considered to be an important cancer marker. The high incidence of these carcinomas to invade other tissues such as lymph nodes, lung, and liver by metastasis was one of the arguments raised to generate T-antigen dendrimers that might have the potential to block the receptor sites following surgery. The synthesis of the T-antigen disaccharide will be briefly described, followed by the elaboration of neoglycoproteins and glycopolymers used to raise monoclonal antibodies against the T-antigen and for screening purpose, respectively. Scaffolds made of poly(amidoamine) (PAMAM), poly(propylene imine), N,N'-bis(acrylamido)acetic acid, and finally hyperbranched L-lysine were used to construct relatively small glycodendrimers bearing T-antigen moieties. Few glycodendrimers were also linked to fluorescein and biotin probes to generate ligands that can be used to detect T-Ag receptor sites.     

Synthesis of novel, multivalent glycodendrimers as ligands for HIV-1 gp120

Multivalent neoglycoconjugates are valuable tools for studying carbohydrate-protein interactions. To study the interaction of HIV-1 gp120 with its reported alternate glycolipid receptors, galactosyl ceramide (GalCer) and sulfatide, galactose- and sulfated galactose-derivatized dendrimers were synthesized, analyzed as ligands for rgp120 by surface plasmon resonance, and tested for their ability to inhibit HIV-1 infection of CXCR4- and CCR5-expressing indicator cells. Four different series of glycodendrimers were made by amine coupling spacer-arm derivatized galactose residues, either sulfated or nonsulfated, to poly(propylenimine) dendrimers, generations 1-5. One series of glycodendrimers was prepared from the ceramide saccharide derivative of purified natural GalCer, and another was from chemically synthesized 3-(beta-D-galactopyranosylthio)propionic acid. Synthesis of 3-sulfogalactopyranosyl-derivatized dendrimers was accomplished using the novel compound, 3-(beta-D-3-sulfogalactopyranosylthio)propionic acid. The fourth series was made by random sulfation of the 3-(beta-D-galactopyranosylthio)propionic acid functionalized dendrimers. Structures of the carbohydrate moieties were confirmed by NMR, and the average molecular weights and polydispersities of the different glycodendrimers were determined using MALDI-TOF MS. Surface plasmon resonance studies found that rgp120 IIIB bound to the derivatized dendrimers tested with nanomolar affinity, and to dextran sulfate with picomolar affinity. In vitro studies of the effectiveness of these compounds at inhibiting infection of U373-MAGI-CCR5 cells by HIV-1 Ba-L indicated that the sulfated glycodendrimers were better inhibitors than the nonsulfated glycodendrimers, but not as effective as dextran sulfate.     

Peptide and glycopeptide dendrimers. Part II

Recent progress in peptide and glycopeptide chemistry make the preparation of peptide and glycopeptide dendrimers of acceptable purity, with designed structural and immunochemical properties reliable. New methodologies using unprotected peptide building blocks have been developed to further increase the possibilities of their design and improve their preparation and separation. The sophisticated design of peptide and glycopeptide dendrimers has led to their use as antigens and immunogens, for serodiagnosis and other biochemical uses including drug delivery. Dendrimers bearing peptide with predetermined secondary structures are useful tools in protein de novo design. This article covers synthesis and applications of multiple antigen peptides (MAPs), multiple antigen glycopeptides (MAGs), multiple antigen peptides based on sequential oligopeptide carriers (MAP‐SOCs), glycodendrimers and template‐assembled synthetic proteins (TASPs). In part II the preparation of MAPs, and the utility of glycodendrimers and TASPs are discussed. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Versatile strategy for the synthesis of biotin-labelled glycans,their immobilization to establish a bioactive surface and interaction studies with a lectin on a biochip

The emerging role of glycans as versatile biochemical signals in diverse aspects of cellular sociology calls for establishment of sensitive methods to monitor carbohydrate recognition by receptors such as lectins. Most of these techniques involve the immobilization of one of the binding partners on a surface, e.g. atomic force microscopy, glycan array and Surface Plasmon Resonance (SPR), hereby simulating cell surface presentation. Here, we report the synthesis of fluorescent glycoconjugates, with a functionalization strategy which avoids the frequently occurring ring opening at the reducing end for further immobilization on a surface or derivatization with biotin. In order to improve the versatility of these derivatized glycans for biological studies, a new approach for the synthesis of biotinylated and fluorescent glycans has also been realized. Finally, to illustrate their usefulness the neoglycoconjugates were immobilized on different surfaces, and the interaction analysis with a model lectin, the toxin from mistletoe, proved them to act as potent ligands, underscoring the merit of the presented synthetic approach. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Multimeric bivalent immunogens from recombinant tetanus toxin H<Subscript>C</Subscript> fragment,synthetic hexasaccharides,and a glycopeptide adjuvant

Using recombinant tetanus toxin H_C fragment (rTT-H_C) as carrier, we prepared multimeric bivalent immunogens featuring the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Ogawa, in combination with either the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Inaba, or a synthetic disaccharide tetrapeptide peptidoglycan fragment as adjuvant. The conjugation reaction was effected by squaric acid chemistry and monitored in virtually real time by SELDI-TOF MS. In this way, we could prepare well-defined immunogens with predictable carbohydrate–carrier ratio, whose molecular mass and the amount of each saccharide attached could be independently determined. The ability to prepare such neoglycoconjugates opens unprecedented possibilities for preparation of conjugate vaccines for bacterial diseases from synthetic carbohydrates.     

Enzymatic synthesis of oligosaccharides and neoglycoconjugates

Oligosaccharides involved in glycoconjugates play important roles in a number of biological events. To elucidate the biological functions of oligosaccharides, sufficient quantities of structurally defined oligosaccharides, are of limited availability by traditional purification methods, are required. Hence, chemical and enzymatic syntheses of oligosaccharides are becoming increasingly important in glycobiology and glycotechnology. In addition, oligosaccharides often occur as glycoconjugates attached to proteins or lipids. Hence, the development of simple and effective methods for synthesizing neoglycoconjugates such as neoglycoprotein and neoglycolipids is essential for an understanding of the biological function of these molecules. Here we review the most recent developments in the enzymatic synthesis of oligosaccharides and neoglycoconjugates.     

Influence of mannan epitopes in glycoproteins--Concanavalin A interaction. Comparison of natural and synthetic glycosylated proteins

Two natural glycoproteins/glycoenzymes, invertase and glucoamylase, and two neoglycoconjugates, synthetized from Saccharomyces cerevisiae mannan, bovine serum albumin and penicillin G acylase were tested for interaction with lectin Concanavalin A (Con A). The interaction of natural and synthetic glycoproteins with Con A was studied using three different experimental methods: (i) quantitative precipitation in solution (ii) sorption to Con A immobilized on bead cellulose; and (iii) kinetic measurement of the interaction by surface plasmon resonance. Prepared neoglycoproteins were further characterized: saccharide content, molecular weight, polydispersion, kinetic and equilibrium association constants with Con A were determined. It can be concluded that the used conjugation method proved to be able to produce neoglycoproteins with similar properties like natural glycoproteins, i.e. enzymatic activity (protein part) and lectin binding activity (mannan part) were preserved and the neoglycoconjugates interact with Con A similarly as natural mannan-type glycoproteins.     

Cell type-dependent alterations of binding of synthetic blood group antigen-related oligosaccharides in lung cancer

Blood group antigen-related oligosaccharides have been implicated in growth regulation, cell mobility control and adhesion; we are therefore interested in the localization of receptors for these oligosaccharides in tumour cells. Labelled neoglycoconjugates that carry synthetic sugar structures are suitable tools to determine: whether such binding sites are present in human lung cancer; whether structural alterations of the glycoligand part will affect extent of binding; and whether cell type-associated alterations can be detected. Sections from 121 cases of lung cancer, representing small cell and non-small cell lung carcinoma, mesothelioma and metastases from extrapulmonary primary carcinomas were used to study the binding of nine synthetic AH- and Le-related oligosaccharides. Probes with fucose-1-3/4-N-acetylglucosamine-1-R, an A-like disaccharide and 3-sulfated galactose as ligand appear to bind less well to small cell than to non-small cell lung cancer cases, whereas Le^c-disaccharide distinguishes mesothelioma from metastatic carcinoma. The latter ligand, A-like disaccharide and H (type III)-like trisaccharide exhibit evident cell type-associated differences in extent of binding. Thus, tailor-made neoglycoconjugates constitute a promising class of histopathological tools that warrants further study.     

Peptide and glycopeptide dendrimers. Part I

Recent progress in peptide and glycopeptide chemistry make the preparation of peptide and glycopeptide dendrimers of acceptable purity, with designed structural and immunochemical properties reliable. New methodologies using unprotected peptide building blocks have been developed to further increase possibilities of their design and improve their preparation and separation. Sophisticated design of peptide and glycopeptide dendrimers has led to their use as antigens and immunogens, for serodiagnosis and other biochemical uses including drug delivery. Dendrimers bearing peptide with predetermined secondary structures are useful tools in protein de novo design. This article covers synthesis and applications of multiple antigen peptides (MAPs), multiple antigen glycopeptides (MAGs), multiple antigen peptides based on sequential oligopeptide carriers (MAP‐SOCs), glycodendrimers and template‐assembled synthetic proteins (TASPs). Part I deals with the development of various structural forms of MAPs as well as their application as antigens, immunogens, and for immunodiagnostic and biochemical purposes. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Neoglycoconjugates of mannan with bovine serum albumin and their interaction with lectin concanavalin A.

Neoglycoconjugates were prepared from mannan isolated from yeast Saccharomyces cerevisiae and activated by periodate oxidation to create aldehyde groups. Various degrees of oxidation introduced 11-28 aldehyde groups per mannan molecule and simultaneously resulted in a molar mass decrease from 46 to 44.5-31 kDa. The activated mannans were subsequently conjugated with bovine serum albumin forming neoglycoconjugates. Some parameters of these mannan-bovine serum albumin conjugates were characterized: saccharide content 25-30% w/w, molar mass within the range 169-246 kDa, and polydispersion (M(w)/M(n)) from 2.8 to 3.6. The interaction of these conjugates with lectin concanavalin A was studied using three different methods: (i) quantitative precipitation in solution; (ii) sorption to concanavalin A immobilized on bead cellulose; and (iii) kinetic measurement of the interaction by surface plasmon resonance. Quantitative precipitation assay showed only negligible differences in the precipitation course of original mannan and the corresponding mannan-bovine serum albumin conjugates. Both the sorption method (equilibrium method) and the surface plasmon resonance measurement (kinetic method) demonstrates that the values of dissociation constant K(D) of all synthetic neoglycoconjugates were within the range 10(-7) - 10(-8) mol x L(-1) (close to K(D) = 10(-8) mol x L(-1) determined by the sorption method for the original mannan). In conclusion, characterization of synthetic neoglycoconjugates confirmed that the method used for their preparation retained the ability of mannan moiety to interact with concanavalin A.     

Synthesis of the trisaccharide repeating unit related to Klebsiella 012 serotype

Synthesis of the trisaccharide, allyl α-l-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-d-glucopyranosyl-(1→4)-α-l-rhamnopyranoside related to O-chain glycans isolated from the deaminated LPSs of Klebsiella pneumoniae serotype 012, was achieved through condensation of suitably synthesized disaccharide, allyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-β-d-glucopyranosyl-(1→4)-2,3-di-O-benzoyl-α-l-rhamnopyranoside and donor, ethyl 2,3,4-tri-O-acetyl-1-thio α-l-rhamnopyranoside starting from l-rhamnose and d-glucosamine hydrochloride. The trisaccharide can be utilized for the synthesis of neoglycoconjugates for use as a synthetic vaccine by coupling it with a suitable protein after deprotection. Various regio- and stereoselective protecting group strategies have been carefully considered, as protecting groups can influence the reactivity of the electrophile and nucleophile in glycosylation reactions on the basis of steric and electronic requirements.     

Dense shell glycodendrimers as potential nontoxic anti-amyloidogenic agents in Alzheimer's disease. Amyloid-dendrimer aggregates morphology and cell toxicity

Dendrimers have been proved to interact with amyloids, although most of dendrimers assayed in amyloidogenic systems are toxic to cells. The development of glycodendrimers, poly(propyleneimine) (PPI) dendrimers decorated with maltose (Mal), represents the possibility of using dendrimers with a low intrinsic toxicity. In the present paper we show that fourth (PPI-G4-Mal) and fifth (PPI-G5-Mal) generation glycodendrimers have the capacity to interfere with Alzheimer's amyloid peptide Aβ(1-40) fibrilization. The interaction is generation dependent: PPI-G5-Mal blocks amyloid fibril formation generating granular nonfibrillar amorphous aggregates, whereas PPI-G4-Mal generates clumped fibrils at low dendrimer-peptide ratios and amorphous aggregates at high ratios. Both PPI-G4-Mal and PPI-G5-Mal are nontoxic to PC12 and SH-SY5Y cells. PPI-G4-Mal reduces amyloid toxicity by clumping fibrils together, whereas amorphous aggregates are toxic to PC12 cells. The results show that glycodendrimers are promising nontoxic agents in the search for anti-amyloidogenic compounds. Fibril clumping may be an anti-amyloid toxicity strategy.     

Synthetic metalloporphyrins: a class of compounds of pharmacological interest

Studies of the regulation of heme oxygenase by synthetic metalloporphyrins reveal that within this group of compounds there exist both inducers and inhibitors of the synthesis of this enzyme or of its catalytic function. The ability of metalloporphyrins to alter heme catabolism is of considerable experimental and clinical interest since such alterations may have consequences for other aspects of heme homeostasis, including its synthesis and its function in the form of cytochrome(s) P-450. Examples of the metabolic effects – and their potential clinical and pharmacological consequences – produced by two synthetic metalloporphyrins, Sn-protoporphyrin and Co-protoporphyrin, are discussed.     

The use of nucleolytic enzymes (ribonucleases, polynucleotide phosphorylases and endonuclease from Serratia marcescens) for producing initial blocks of synthetic endoribonucleases]

The simplest variant of synthetic substrate-ribozyme complex has been proposed. The schemes of potential ribozyme "subunits" synthesis have been worked out: R1--GCUUGAAACAAA; R2--AAAAACUGAUGAAAGC. The macroscale synthesis of dinucleoside monophosphate ApU, GpC, CpU catalyzed by immobilized ribonucleases of different specificity and preparation of oligoadenylates by hydrolysis of poly-A in the presence of endonuclease Serratia marcescens, as well the synthesis of conservative sequences of potential ribozyme such as ApUpG, CpUpG, GpApU, ApApApG and others have been described.     

Solid-phase synthesis of multivalent glycoconjugates on a DNA synthesizer

Carbohydrate-oligonucleotide conjugates and glycodendrimers were synthesized utilizing a DNA synthesizer. The synthesis of multivalent glycoconjugates on solid-phase allows custom tailoring of their structure to the requirements of biological assays within hours, as opposed to traditional approaches that require weeks or months of work in the laboratory. Therefore it will become much easier to investigate carbohydrate-protein interactions and optimize for objectives such as the receptor-mediated targeting of antisense oligonucleotides.     

A glycoconjugate antigen based on the recognition motif of a broadly neutralizing human immunodeficiency virus antibody, 2G12, is immunogenic but elicits antibodies unable to bind to the self glycans of gp120

The glycan shield of human immunodeficiency virus type 1 (HIV-1) gp120 contributes to viral evasion from humoral immune responses. However, the shield is recognized by the HIV-1 broadly neutralizing antibody (Ab), 2G12, at a relatively conserved cluster of oligomannose glycans. The discovery of 2G12 raises the possibility that a carbohydrate immunogen may be developed that could elicit 2G12-like neutralizing Abs and contribute to an AIDS vaccine. We have previously dissected the fine specificity of 2G12 and reported that the synthetic tetramannoside (Man(4)) that corresponds to the D1 arm of Man(9)GlcNAc(2) inhibits 2G12 binding to gp120 as efficiently as Man(9)GlcNAc(2) itself, indicating the potential use of Man(4) as a building block for creating immunogens. Here, we describe the development of neoglycoconjugates displaying variable copy numbers of Man(4) on bovine serum albumin (BSA) molecules by conjugation to Lys residues. The increased valency enhances the apparent affinity of 2G12 for Man(4) up to a limit which is achieved at approximately 10 copies per BSA molecule, beyond which no further enhancement is observed. Immunization of rabbits with BSA-(Man(4))(14) elicits significant serum Ab titers to Man(4). However, these Abs are unable to bind gp120. Further analysis reveals that the elicited Abs bind a variety of unbranched and, to a lesser extent, branched Man(9) derivatives but not natural N-linked oligomannose containing the chitobiose core. These results suggest that Abs can be readily elicited against the D1 arm; however, potential differences in the presentation of Man(4) on neoglycoconjugates, compared to glycoproteins, poses challenges for eliciting anti-mannose Abs capable of cross-reacting with gp120 and HIV-1.     

Biological applications of dendrimers

In the past year, significant advances have been made in the synthesis and study of glycodendrimers and peptide dendrimers. Application of these dendrimers to the study of carbohydrate-protein and protein-protein interactions has facilitated the understanding of these processes. In addition, dendrimers show great promise as DNA- and drug-delivery systems.     

Design, synthesis and the effect of 1,2,3-triazole sialylmimetic neoglycoconjugates on Trypanosoma cruzi and its cell surface trans-sialidase

This work describes the synthesis of a series of sialylmimetic neoglycoconjugates represented by 1,4-disubstituted 1,2,3-triazole-sialic acid derivatives containing galactose modified at either C-1 or C-6 positions, glucose or gulose at C-3 position, and by the amino acid derivative 1,2,3-triazole fused threonine-3-O-galactose as potential TcTS inhibitors and anti-trypanosomal agents. This series was obtained by Cu(I)-catalysed azide-alkyne cycloaddition reaction ('click chemistry') between the azido-functionalized sugars 1-N(3)-Gal (commercial), 6-N(3)-Gal, 3-N(3)-Glc and 3-N(3)-Gul with the corresponding alkyne-based 2-propynyl-sialic acid, as well as by click chemistry reaction between the amino acid N(3)-ThrOBn with 3-O-propynyl-GalOMe. The 1,2,3-triazole linked sialic acid-6-O-galactose and the sialic acid-galactopyranoside showed high Trypanosoma cruzitrans-sialidase (TcTS) inhibitory activity at 1.0mM (approx. 90%), whilst only the former displayed relevant trypanocidal activity (IC(50) 260μM). These results highlight the 1,2,3-triazole linked sialic acid-6-O-galactose as a prototype for further design of new neoglycoconjugates against Chagas' disease.     

Synthesis of a New Template with a Built-in Adjuvant and Its Use in Constructing Peptide Vaccine Candidates Through Polyoxime Chemistry

Synthetic lipopeptides are showing promise as vaccine candidates, but until now it has been very difficult to prepare them in homogeneous form. We describe the synthesis and characterization of a new water-soluble, four-branched template with a built-in lipophilic adjuvant (Pam₃Cys). Through the use of oxime chemistry, we attached four copies of an unprotected influenza virus peptide and characterized the product (13kDa) by reversed-phase HPLC and electrospray ionization mass spectrometry. Several other such constructions were made using the new template and different peptides. We seem to have a general method for making synthetic lipopeptides in homogeneous form.