Visualizing association of lipidated signaling proteins in heterogeneous membranes−Partitioning into subdomains, lipid sorting, interfacial adsorption, and protein association

In a combined chemical biological and biophysical approach, we studied the partitioning of differently fluorescent-labeled palmitoyl and/or farnesyl lipidated peptides, which represent membrane recognition model systems, as well as the full lipidated N-Ras protein into various model membrane systems including canonical model raft mixtures. To this end, two-photon fluorescence microscopy on giant unilamellar vesicles, complemented by tapping-mode atomic force microscopy (AFM) measurements, was carried out. The measurements were performed over a wide temperature range, ranging from 30 to 80 °C to cover different lipid phase states (solid-ordered (gel), fluid/gel, liquid-ordered/liquid-disordered, all-fluid). The results provide direct evidence that partitioning of the lipidated peptides and N-Ras occurs preferentially into liquid-disordered lipid domains, which is also reflected in a faster kinetics of incorporation. The phase sequence of preferential binding of N-Ras to mixed-domain lipid vesicles is liquid-disordered > liquid-ordered ? solid-ordered. Intriguingly, we detect - using the better spatial resolution of AFM - also a large proportion of the lipidated protein located at the liquid-disordered/liquid-ordered phase boundary, thus leading to a favorable decrease in line tension that is associated with the rim of neighboring domains. In an all-liquid-ordered, cholesterol-rich phase, phase separation can be induced by an effective lipid sorting mechanism owing to the high affinity of the lipidated peptides and proteins to a fluid-like lipid environment. At low temperatures, where the overall acyl chain order parameter of the lipid bilayer has markedly increased, such an efficient lipid sorting mechanism is energetically too costly and self-association of the peptide into small clusters takes place. These data reveal the interesting ability of the lipidated peptides and proteins to induce formation of fluid microdomains at physiologically relevant high cholesterol concentrations. Furthermore, our results reveal self-association of the N-Ras protein at the domain boundaries which may serve as an important vehicle for association processes and nanoclustering, which has also been observed in in vivo studies.     

Synthesis of spacer-containing chlamydial disaccharides as analogues of the alpha-Kdop-(2-->8)-alpha-Kdop-(2-->4)-alpha-Kdop trisaccharide epitope

On the basis of high-resolution crystal structures of the antigen binding fragment of the chlamydia-specific monoclonal antibody S25-2 in complex with the trisaccharide alpha-Kdop-(2-->8)-alpha-Kdop-(2-->4)-alpha-Kdop and part structures thereof, seven modified alpha-Kdop-(2-->8)-alpha-Kdop disaccharide derivatives were synthesized starting from the protected disaccharide allyl ketoside 1. Hydroboration and subsequent oxidation as well as ozonolysis, respectively, followed by Wittig-reaction for chain elongation were used to install a terminal carboxylic group on spacer entities of various chain lengths. Furthermore, addition of methyl 2-thioacetate to the allyl group furnished the corresponding thioether derivative. Standard deprotection gave the target disaccharides as simplified trisaccharide analogues, which will be used to probe the contribution of the proximal carboxylic group in the binding of chlamydia-specific di- and trisaccharide-reactive monoclonal antibodies.     

Peptide conjugates as tools for the study of biological signal transduction

Today, many biological phenomena are being investigated and understood in molecular detail, and organic chemistry is increasingly being directed towards biological phenomena. This review is intended to highlight this interplay of organic chemistry and biology, using biological signal transduction as an example. Lipo-, glyco-, phospho- and nucleoproteins play key roles in the processes whereby chemical signals are passed across cell membranes and further to the cell nucleus. For the study of the biological phenomena associated with these protein conjugates, structurally well-defined peptides containing the characteristic linkage region of the peptide backbone with the lipid, the carbohydrate or the phosphoric acid ester can provide valuable tools. The multi-functionality and pronounced acid- and base-lability of such compounds renders their synthesis a formidable challenge to conventional organic synthesis. However, the recent development of enzymatic protecting groups, provides one of the central techniques which, when coupled with classic chemical synthesis, can provide access to these complex and sensitive biologically relevant peptide conjugates under particularly mild conditions and with high selectivity.     

Influence of the Lipid Anchor Motif of N-Ras on the Interaction with Lipid Membranes: A Surface Plasmon Resonance Study

Ras GTPases play a crucial role in signal transduction cascades involved in cell differentiation and proliferation, and membrane binding is essential for their proper function. To determine the influence of the nature of the lipid anchor motif and the difference between the active (GTP) and inactive (GDP) forms of N-Ras on partitioning and localization in the lipid membrane, five different N-Ras constructs with different lipid anchors and nucleotide loading (Far/Far (GDP), HD/Far (GDP), HD/HD (GDP), Far (GDP), and HD/Far (GppNHp)) were synthesized. Using the surface plasmon resonance technique, we were able to follow the insertion and dissociation process of the lipidated proteins into and out of model membranes consisting of pure liquid-ordered (l_o) or liquid-disordered (l_d) phase and a heterogeneous two-phase mixture, i.e., a raft mixture with l_o + l_d phase coexistence. In addition, we examined the influence of negatively charged headgroups and stored curvature elastic stress on the binding properties of the lipidated N-Ras proteins. In most cases, significant differences were found for the various anchor motifs. In general, N-Ras proteins insert preferentially into a fluidlike, rather than a rigid, ordered lipid bilayer environment. Electrostatic interactions with lipid headgroups or stored curvature elastic stress of the membrane seem to have no drastic effect on the binding and dissociation processes of the lipidated proteins. The monofarnesylated N-Ras exhibits generally the highest association rate and fastest dissociation process in fluidlike membranes. Double lipidation, especially including farnesylation, of the protein leads to drastically reduced initial binding rates but strong final association. The change in the nucleotide loading of the natural N-Ras HD/Far induces a slightly different binding and dissociation kinetics, as well as stability of association, and seems to influence the tendency to segregate laterally in the membrane plane. The GDP-bound inactive form of N-Ras with an HD/Far anchor shows stronger membrane association, which might be due to a more pronounced tendency to self-assemble in the membrane matrix than is seen with the active GTP-bound form.     

Synthesis of Lewis X trisaccharide analogues in which glucose and rhamnose replace N-acetylglucosamine and fucose,respectively

Two analogues of the Le(x) trisaccharide, alpha-L-Fucp-(1-->3)-[beta-D-Galp-(1-->4)]-D-Glcp were synthesized as allyl glycosides. In these derivatives either only the N-acetylglucosamine is replaced by glucose or both the N-acetylglucosamine and the fucosyl residue are replaced by glucose and rhamnose, respectively. Our synthetic scheme used armed beta-thiophenyl fuco- and rhamnoside glycosyl donors that were prepared anomerically pure from the corresponding alpha-glycosyl bromides. The protecting groups were chosen to allow access to the fully deprotected trisaccharides without reduction of the allyl glycosidic group. These analogues will be used as soluble antigens in binding experiments with anti-Le(x) antibodies and can also be conjugated to a carrier protein and used as immunogens. In the course of this synthetic work, we also describe the use of reversed-phase HPLC to purify key protected trisaccharide intermediates prior to their deprotection.     

Simultaneous Post-cysteine(S-Acm) Group Removal Quenching of Iodine and Isolation of Peptide by One Step Ether Precipitation

The S-acetamidomethyl (Acm) protecting group is widely used in the chemical synthesis of peptides that contain one or more disulfide bonds. Treatment of peptides containing S-Acm protecting group with iodine results in simultaneous removal of the sulfhydryl protecting group and disulfide formation. However, the excess iodine needs to be quenched or adsorbed as quickly as possible after completion of the disulfide bond formation in order to minimize side reactions that are often associated with the iodination step. We report a simple method for simultaneous post-cysteine (Acm) group removal quenching of iodination and isolation. Use of large volumes of diethyl ether for direct precipitation action of the oxidized peptide from the 90 or 95% aqueous acetic acid solution affords nearly quantitative recovery of largely iodine-free peptide ready for direct purification. It was successfully applied to the synthesis of various peptides including human insulin-like peptide 3 analogues. Although recovery yields were comparable to the traditionally used ascorbic acid quenching method, this new approach offers significant advantages such as more simple utility, minimal side reactions, and greater cost effectiveness.     

Identification and quantification of DNA repair proteins by liquid chromatography/isotope-dilution tandem mass spectrometry using their fully 15N-labeled analogues as internal standards

Oxidatively induced DNA damage is implicated in disease, unless it is repaired by DNA repair. Defects in DNA repair capacity may be a risk factor for various disease processes. Thus, DNA repair proteins may be used as early detection and therapeutic biomarkers in cancer and other diseases. For this purpose, the measurement of the expression level of these proteins in vivo will be necessary. We applied liquid chromatography/isotope-dilution tandem mass spectrometry (LC-MS/MS) for the identification and quantification of DNA repair proteins human 8-hydroxyguanine-DNA glycosylase (hOGG1) and Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), which are involved in base-excision repair of oxidatively induced DNA damage. We overproduced and purified (15)N-labeled analogues of these proteins to be used as suitable internal standards to ensure the accuracy of quantification. Unlabeled and (15)N-labeled proteins were digested with trypsin and analyzed by LC-MS/MS. Numerous tryptic peptides of both proteins were identified on the basis of their full-scan mass spectra. These peptides matched the theoretical peptide fragments expected from trypsin digestion and provided statistically significant protein scores that would unequivocally identify these proteins. We also recorded the product ion spectra of the tryptic peptides and defined the characteristic product ions. Mixtures of the analyte proteins and their (15)N-labeled analogues were analyzed by selected-reaction monitoring on the basis of product ions. The results obtained suggest that the methodology developed would be highly suitable for the positive identification and accurate quantification of DNA repair proteins in vivo as potential biomarkers for cancer and other diseases.     

Physico-chemical and biophysical study of the interaction of hexa- and heptaacyl lipid A from Erwinia carotovora with magainin 2-derived antimicrobial peptides

The neutralization of endotoxin structures such as the active 'endotoxic principle' lipid A by suitable compounds has been shown to be a key step in the treatment of infectious diseases, in particular in the case of Gram-negative bacteria which frequently may lead to the septic shock syndrome. An effective antimicrobial peptide, originally found in the skin of an African frog, is magainin 2. Here, the interaction of magainin 2-amide and a peptide derived thereof, M2V, with chemically defined and homogeneous hexaacyl and heptaacyl lipids A isolated from LPS of Erwinia carotovora, was investigated. By using Fourier-transform infrared spectroscopy, the gel to liquid crystalline phase transition of the acyl chains of lipid A and the conformation of their phosphate groups due to peptide binding was investigated. The former parameter was also determined by using differential scanning calorimetry. The electrophoretic mobility of lipid A aggregates under the influence of the peptides was studied to determine the Zeta potential, and small-angle X-ray scattering was applied for the elucidation of the types of aggregate structures in the absence and presence of the peptides. The lipid A-induced cytokine production in human mononuclear cells shows that the ability of the two peptides to inhibit a tumor necrosis factor-alpha production correlates with characteristic changes of the biophysical parameters. These are much stronger expressed for the peptide M2V than for magainin 2-amide, which apparently is connected with the higher number of positive as well as more hydrophobic amino acids, leading to a stronger amphiphilicity necessary to neutralize the amphiphilic lipid A aggregates.     

Physico-chemical and biophysical study of the interaction of hexa- and heptaacyl lipid A from Erwinia carotovora with magainin 2-derived antimicrobial peptides

The neutralization of endotoxin structures such as the active ‘endotoxic principle’ lipid A by suitable compounds has been shown to be a key step in the treatment of infectious diseases, in particular in the case of Gram-negative bacteria which frequently may lead to the septic shock syndrome. An effective antimicrobial peptide, originally found in the skin of an African frog, is magainin 2. Here, the interaction of magainin 2-amide and a peptide derived thereof, M2V, with chemically defined and homogeneous hexaacyl and heptaacyl lipids A isolated from LPS of Erwinia carotovora, was investigated. By using Fourier-transform infrared spectroscopy, the gel to liquid crystalline phase transition of the acyl chains of lipid A and the conformation of their phosphate groups due to peptide binding was investigated. The former parameter was also determined by using differential scanning calorimetry. The electrophoretic mobility of lipid A aggregates under the influence of the peptides was studied to determine the Zeta potential, and small-angle X-ray scattering was applied for the elucidation of the types of aggregate structures in the absence and presence of the peptides. The lipid A-induced cytokine production in human mononuclear cells shows that the ability of the two peptides to inhibit a tumor necrosis factor-α production correlates with characteristic changes of the biophysical parameters. These are much stronger expressed for the peptide M2V than for magainin 2-amide, which apparently is connected with the higher number of positive as well as more hydrophobic amino acids, leading to a stronger amphiphilicity necessary to neutralize the amphiphilic lipid A aggregates.     

Influence of the metal center and linker on the intracellular distribution and biological activity of organometal–peptide conjugates

Organometallic complexes conjugated to cell-penetrating peptides (CPPs) are promising systems for diagnostic imaging and therapeutic applications in human medicine. Recently, we reported on the synthesis of cymantrene(CpMn(CO)₃)–CPP conjugates with biological activity on different cancer cell lines. However, the precise mechanism of cytotoxicity remained elusive in these studies. To investigate the role of the metal center and the linker between the CpM(CO)₃ moiety and the peptide, a number of derivatives with manganese replaced by rhenium and the keto linker originally used substituted by a methylene group were prepared and fully characterized by ¹H NMR spectroscopy, infrared spectroscopy, electrospray ionization mass spectrometry, and elemental analysis as well as X-ray structure determination. The organometal–peptide conjugates as well as carboxyfluorescein-labeled derivatives thereof were prepared by solid-phase peptide synthesis, purified by high-performance liquid chromatography, and analyzed by mass spectrometry. Fluorescence microscopy studies of MCF-7 human breast cancer cells revealed an efficient cellular uptake and pronounced nuclear localization of the bioconjugates with the methylene linker compared with systems with the keto group. In addition, the latter also showed a higher cytotoxicity. In contrast, the variation of the metal center from manganese to rhenium had a negligible effect. The structure–activity relationships determined in the present work will aid in the further tuning of the biological activity of organometal–peptide conjugates.     

DHHC9 and GCP16 constitute a human protein fatty acyltransferase with specificity for H- and N-Ras

Covalent lipid modifications mediate the membrane attachment and biological activity of Ras proteins. All Ras isoforms are farnesylated and carboxyl-methylated at the terminal cysteine; H-Ras and N-Ras are further modified by palmitoylation. Yeast Ras is palmitoylated by the DHHC cysteine-rich domain-containing protein Erf2 in a complex with Erf4. Here we report that H- and N-Ras are palmitoylated by a human protein palmitoyltransferase encoded by the ZDHHC9 and GCP16 genes. DHHC9 is an integral membrane protein that contains a DHHC cysteine-rich domain. GCP16 encodes a Golgi-localized membrane protein that has limited sequence similarity to yeast Erf4. DHHC9 and GCP16 co-distribute in the Golgi apparatus, a location consistent with the site of mammalian Ras palmitoylation in vivo. Like yeast Erf2.Erf4, DHHC9 and GCP16 form a protein complex, and DHHC9 requires GCP16 for protein fatty acyltransferase activity and protein stability. Purified DHHC9.GCP16 exhibits substrate specificity, palmitoylating H- and N-Ras but not myristoylated G (alphai1) or GAP-43, proteins with N-terminal palmitoylation motifs. Hence, DHHC9.GCP16 displays the properties of a functional human ortholog of the yeast Ras palmitoyltransferase.     

Cellular palmitoylation and trafficking of lipidated peptides

Many important signaling proteins require the posttranslational addition of fatty acid chains for their proper subcellular localization and function. One such modification is the addition of palmitoyl moieties by enzymes known as palmitoyl acyltransferases (PATs). Substrates for PATs include C-terminally farnesylated proteins, such as H- and N-Ras, as well as N-terminally myristoylated proteins, such as many Src-related tyrosine kinases. The molecular and biochemical characterization of PATs has been hindered by difficulties in developing effective methods for the analysis of PAT activity. In this study, we describe the use of cell-permeable, fluorescently labeled lipidated peptides that mimic the PAT recognition domains of farnesylated and myristoylated proteins. These PAT substrate mimetics are accumulated by SKOV3 cells in a saturable and time-dependent manner. Although both peptides are rapidly palmitoylated, the SKOV3 cells have a greater capacity to palmitoylate the myristoylated peptide than the farnesylated peptide. Confocal microscopy indicated that the palmitoylated peptides colocalized with Golgi and plasma membrane markers, whereas the corresponding nonpalmitoylatable peptides accumulated in the Golgi but did not traffic to the plasma membrane. Overall, these studies indicate that the lipidated peptides provide useful cellular probes for quantitative and compartmentalization studies of protein palmitoylation in intact cells.     

Tandem Peptide Ligation for Synthetic and Natural Biologicals

We describe the concept and methods of peptide ligation and tandem peptide ligation for preparing synthetic and natural biologicals. Peptide ligation is a segment coupling method for free peptides or proteins through an amide bond without the use of a coupling reagent or a protecting group scheme. Because unprotected peptides or proteins prepared from either a chemical or biochemical source are being used as building blocks, the ligation removes the size limitation for peptide and protein synthesis. A key feature of the peptide ligation is that the coupling reaction is orthogonal, i.e. it is specific to a particular alpha-amino terminus (NT). This NT-amino acid-specific feature permits the development of a tandem peptide ligation method employing three unprotected peptide segments containing different NT-amino acids to form consecutively two amide bonds, an Xaa-SPro (thiaproline) and then an Xaa-Cys. This strategy was tested in peptides ranging from 28 to 70 amino acid residues, including analogues of somatostatins and two CC-chemokines MIP-1alpha and MIP-1beta. The thiaproline replacements in these peptides and proteins did not result in altered biological activity. By eliminating the protecting group scheme and coupling reagents, tandem ligation of multiple free peptide segments in aqueous solutions enhances the scope of protein synthesis and may provide a useful approach for preparing protein biologicals and synthetic vaccines.     

Parameters modulating the maximum insertion pressure of proteins and peptides in lipid monolayers

The lipid monolayer model membrane is useful for studying the parameters responsible for protein and peptide membrane binding. Different approaches have been used to determine the extent of protein and peptide binding to lipid monolayers. This review focuses on the use of the “maximum insertion pressure” (MIP) to estimate the extent of protein and peptide penetration in lipid monolayers. The MIP data obtained with different proteins and peptides have been reviewed and discussed which allowed to draw conclusions on the parameters modulating the monolayer binding of proteins and peptides. In particular, secondary structure components such as amphipathic α-helices of proteins and peptides as well as electrostatic interactions play important roles in monolayer binding. The MIPs have been compared to the estimated lateral pressure of biomembranes which allowed to evaluate the possible association between proteins or peptides with natural membranes. For example, the MIP of a membrane-anchored protein with a glycosylphosphatidylinositol (GPI) was found to be far below the estimated lateral pressure of biomembranes. This allowed us to conclude that this protein is probably unable to penetrate the membrane and should thus be hanged at the membrane surface by use of its GPI lipid anchor. Moreover, the values of MIP obtained with myristoylated and non-myristoylated forms of calcineurin suggest that the myristoyl group does not contribute to monolayer binding. However, the acylation of a peptide resulted in a large increase of MIP. Finally, the physical state of lipid monolayers can have a strong effect on the values of MIP such that it is preferable to perform measurements with lipids showing a single physical state. Altogether the data show that the measurement of the maximum insertion pressure provides very useful information on the membrane binding properties of proteins and peptides although uncertainties must be provided to make sure the observed differences are significant.     

Use of carbonyl group addition--elimination reactions for synthesis of nucleic acid conjugates

This review outlines the synthesis of covalent conjugates of oligonucleotides and their analogues that are obtained by reactions of carbonyl compounds with various nucleophiles such as primary amines, N-alkoxyamines, hydrazines, and hydrazides. The products linked by imino, oxime, hydrazone, or thiazolidine groups are shown to be useful intermediates for a wide range of chemical biology applications. Methods for their preparation, isolation, purification, and analysis are highlighted, and the comparative stabilities of the respective linkages are evaluated. The relative merits of incorporation of a carbonyl group, particularly an aldehyde group, into either the oligonucleotide or the ligand parts are considered. Examples of harnessing of aldehyde-nucleophile coupling for the labeling of nucleic acids are given, as well as their conjugation to various biomolecules (e.g. peptides and small molecule ligands), site-specific cross-linking of oligonucleotides to nucleic acid-binding proteins, assembly of multibranched supramolecular structures, and immobilization on functionalized surfaces. Future perspectives of bioconjugation and complex molecular engineering via carbonyl group addition-elimination reactions in nucleic acids chemistry are discussed.     

Automated synthesis and use of N-chloroacetyl-modified peptides for the preparation of synthetic peptide polymers and peptide-protein immunogens

A method to incorporate N-chloroacetyl moieties at the amino termini of synthetic peptides using a standard program with an automated peptide synthesizer has been developed. The N-chloroacetyl-modified peptides react well with sulfhydryl containing proteins such as 4-mercaptobutyrimide-modified bovine serum albumin to form stable protein-peptide conjugates. By incorporating cysteine into the synthetic peptide, autopolymerization or cyclization of the synthetic peptide occurs by reaction of the free sulfhydryl with the chloroacetyl group. N-Chloroacetyl-derivatized peptides may be useful as reagents for potential peptide immunogens and vaccines.     

Phosphorylation loops in synthetic peptides of the human neurofilament protein middle-sized subunit

Peptides containing 13 and 39 amino acid residues and serine-side-chain-phosphorylated (P) analogues thereof, corresponding to human neurofilament protein middle-sized subunit (NF-M), have been synthesized in order to localize the phosphorylation site of this protein. The secondary structure of the nonphosphorylated peptides, determined by circular dichroism (CD) measurements, predicted secondary structural calculations and energy conformational calculations, was suggested to be a series of alternating type I (III) -turns and 3₁₀ or -helices. By contrast, the phosphorylated peptides exhibit a unique conformation, probably due to salt bridges between the phosphoserine and the lysine residues. This has provided the first clear evidence that phosphorylation induces conformational changes among these synthetic peptides and presumably, in NF proteins as well. These phosphorylation loops might be the major recognition sites of the neurofilament protein-directed kinases.     

Synthesis of analogues of amaninamide, an amatoxin from the white Amanita virosa mushroom

Analogs of amaninamide, due to the absence of a 6-hydroxy group in the tryptophan moiety, are more easily accessible by synthesis than derivatives of alpha-amanitin. Syntheses of bicyclic octapeptide thioethers 1f-1m have been carried out starting from linear Hpi-S-trityl-octapeptides (3), cyclization by intramolecular 2'-indolylthioether formation yielding monocyclic peptides (2) and final cyclization by DCCI. One of the bicyclic thioethers was oxidized to yield the corresponding chromatographically separated (R)- and (S)-sulfoxides, respectively. The products were characterized by RF-values, u.v. and CD spectra as well as by mass (FAB) spectroscopy. The widely differing inhibitory activities on RNA polymerase II (or B) from calf thymus are listed.     

Synthesis of the flavour precursor, alliin, in garlic tissue cultures

The path of synthesis of alkyl cysteine sulphoxides, or flavour precursors, in the Alliums is still speculative. There are two proposed routes for alliin biosynthesis, one is from serine and allyl thiol while the other is from glutathione and an allyl source via gamma glutamyl peptides. The routes have been investigated by exposing undifferentiated callus cultures of garlic and onion to potential pathway intermediates. After a period of incubation of 2 days the callus was extracted, and analysed for flavour precursors and related compounds by HPLC. Standards of alliin, isoallin and propiin were synthesised and their identity confirmed by HPLC and NMR. Putative intermediates selected included the amino acids serine and cysteine, as well as more complex intermediates such as allylthiol, allyl cysteine and glutathione. Both garlic and onion tissue cultures were able to synthesize alliin following incubation with allylthiol, and cysteine conjugates such as allyl cysteine. The ability of the tissue cultures to form alliin from intermediates was compatible with the proposed routes of synthesis of alliin.     

Synthesis and protein binding properties of T-antigen containing GlycoPAMAM dendrimers.

Allyl O-(beta-D-galactopyranosyl)-(1-3)-2-acetamido-2-deoxy-alpha-D-galactopyranoside (8) was prepared in excellent yield from the corresponding galactosyl bromide (6, 7) and allyl 2-acetamido-4,6-benzylidene-2-deoxy-alpha-D-galactopyranoside (5) using Hg(CN)2 as a promoter. Compound 5 was obtained from N-acetylglucosamine 1 following sequential protecting group strategy and C-4 epimerization as a key step. Carboxylic acid functionalized T-antigen derivative 15, obtained by radical addition of 3-mercaptopropionic acid to allyl disaccharide 10, was conjugated to PAMAM dendritic cores 13-16 by an efficient amide coupling strategy using TBTU. GlycoPAMAM dendrimers having T-antigen residues with 4, 8, 16 and 32 valencies (17-20) were obtained in 73 to 99% yields. Their protein binding properties were demonstrated using peanut lectin from Arachis hypogaea and a mouse monoclonal IgG antibody. The higher valency conjugates generated stronger binding interactions indicating a cluster effect. The inhibitory potential of these glycoPAMAM conjugates toward antibody-coating antigen interactions was enhanced up to 3800 times over that of the monomeric T-antigen residue (10).