Altering the strength of lectin binding interactions and controlling the amount of lectin clustering using mannose/hydroxyl-functionalized dendrimers

Protein-carbohydrate interactions play a critical role in many biological recognition events. Multivalent therapeutic agents that utilize protein-carbohydrate interactions have proven difficult to design, primarily because the fundamental requirements of protein-carbohydrate interactions are not well understood. Here, we report a systematic study of the effect on lectin binding of varying the loading of mannose surface residues on generations three through six PAMAM dendrimers. The degree of mannose functionalization was controlled by stoichiometric addition, and dendrimers were characterized using NMR and MALDI-TOF MS. Hemagglutination assays and quantitative precipitation assays were performed to determine the relative activity of the dendrimers. Using the mannose/hydroxyl-functionalized dendrimers reported here, we could systematically control both the degree of lectin clustering and the overall activity of the lectin with the dendrimer.     

Novel multivalent mannose compounds and their inhibition of the adhesion of type 1 fimbriated uropathogenic E. coli

A series of multivalent mannose containing compounds were prepared varying in size from small divalent, to 16-valent glycodenrimers and 21-valent glycopolymers. The molecules were approached via a common mannose building block. As scaffolds dendrimers and dendrons based on the 3,5-di-(2-aminoethoxy)-benzoic acid branching unit were used along with commercially available PAMAM dendrimers. To include larger structures, linear glycopolymers with varying amounts of mannose were prepared via radical polymerization. The compounds were tested for their biological activity using a newly developed ELISA based inhibition assay, for their ability to inhibit the binding of recombinant type I fimbriated E. coli to a monolayer of T24 cell line derived from human urinary bladder epithelium. All compounds showed enhanced affinity as compared to mannose with IC₅₀’s down to the low micromolar range.     

Globular carbosilane dendrimers with mannose groups at the periphery: synthesis, characterization and toxicity in dendritic cells

A synthetic strategy has been developed for the preparation of new globular carbosilane dendrimers with mannose groups at the periphery. It consists of hydrosilylation reaction of allyl tetraacetylmannose with carbosilane dendrimers containing monohydrosilane end groups and the subsequent deacetylation reaction. Evaluation of dendrimer toxicities in dendritic cells by MTT assay were carried out, and evidence a good biocompatibility.     

Cyanovirin-N binding to Manalpha1-2Man functionalized dendrimers

Manalpha1-2Man functionalized G(3) and G(4)-PAMAM dendrimers have been synthesized and characterized by MALDI-TOF MS and NMR spectroscopy. Precipitation assays to assess the binding of the dimannose-functionalized dendrimers to Cyanovirin-N, a HIV-inactivating protein that blocks virus-to-cell fusion through high mannose mediated interactions, are presented.     

Synthesis of carbosilane dendrimers having peripheral mannose and mannobiose

The mannose monosaccharide derivative, acetylthiopropyl 2,3,4,6-tetra-O-acetyl-α-d-mannopyranoside (Man), and the mannobiose derivative, acetylthiopropyl 2,4,6-tri-O-acetyl-3-O-(2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-α-d-mannopyranoside (α-1,3-Man), were synthesized respectively. These mannose derivatives were introduced into carbosilane dendrimer scaffolds of the zero and first generations. As a result, six carbosilane dendrimers were functionalized by Man and α-1,3-Man. Isothermal titration microcalorimetry was done to determine binding assay between mannose moieties of carbosilane dendrimer and concanavalin A. It was found that carbosilane dendrimers bound more efficiently to concanavalin A than free mannose (Me-α-Man) and mannobiose (Me-α-1,3-Man).     

Synthesis and biological evaluation of mannose-6-phosphate-coated multivalent dendritic cluster glycosides

The synthesis of multivalent dendritic cluster glycosides of mannopyranosyl-6-phosphate is presented. Poly(amido amine)-based dendrimers of 0.5-3.5 generations, containing carboxylic acid peripheral functionalities, were utilized so as to install 4, 8, 16 and 32 mannopyranosyl-6-phosphate residues at the peripheries of the dendrimers. Amide bond formation between an amine-tethered mannopyranosyl-6-phosphate monomer unit and carboxylic acid-functionalized dendrimers was conducted to synthesize the dendritic cluster glycosides. The constitutions of the Man-6-P-containing dendrimers were assessed by 1H, 13C and 31P NMR spectroscopies and the sugar content analysis by a resorcinol assay. Preliminary biological studies with few newly synthesized Man-6-P-containing dendrimers showed that these compounds could bind the purified goat liver mannose 6-phosphate receptor (MPR 300) protein.     

EPR study of the interactions between dendrimers and peptides involved in Alzheimer's and prion diseases

Spin-probe and spin-label techniques were used to study the interactions of the Abeta 1-28 peptide involved in Alzheimer disease and the PrP 106-126 peptide suspected to be preferentially involved in spongiform encephalopathies with three different types of dendrimers. A computer-aided EPR analysis of a positively charged and a neutral spin probe was performed by comparing the pure dendrimer and peptide systems with the dendrimer-peptide ones. Also spin-labeled PAMAM dendrimers were used to test the interactions. The results show the interactions between dendrimer and peptide monomer to be stronger for Abeta 1-28 than for PrP 106-126. PAMAM dendrimers perturb the aggregation of the peptides more than PPI dendrimers do.     

Mannose/glucose-functionalized dendrimers to investigate the predictable tunability of multivalent interactions

G4-, G5-, and G6-PAMAM dendrimers were functionalized with mixtures of mannose and glucose in varying ratios, and the relative affinities of these compounds for Concanavalin A (Con A) were evaluated using the hemagglutination assay. As the ratio of mannose to glucose increases, the relative activity in the hemagglutination assay (on a per sugar basis) increases linearly. Methyl mannose binds to Con A with an affinity 4-fold higher than that of methyl glucose; multivalency amplifies this trend. The mannose/glucose-functionalized dendrimer results reported here suggest that the affinity of multivalent associations can be attenuated in predictable, reliable ways based on monovalent affinities of the ligands.     

Multiple functionalization of benzophenones inside polyphenylene dendrimers--toward entrapped ions and radicals

Polyphenylene dendrimers possessing a defined number of keto groups in the dendritic scaffold have been synthesized by using a benzophenone-functionalized tetraphenylcyclopentadienone branching unit. A postsynthetic functionalization of the polyphenylene backbone was achieved by reacting the entrapped keto groups with organolithium reagents yielding monodisperse alcohol products. To investigate the accessibility and reactivity of the embedded groups, many functions of different size and nature, for example, the chromophore pyrene, were introduced. Moreover, suitable precursors for the synthesis of dendrimer entrapped species, trityl cations, trityl radicals, and ketyl radical anions, were obtained. To gain insight into the structure of these newly functionalized dendrimers, UV/vis, EPR, and NMR measurements have been performed. They showed a delocalization of the charge/spin into the polyphenylene dendritic arms leading to a stabilization of the ions/radicals. Remarkably, for the ketyl radicals, EPR measurements indicated the occurrence of intermolecular metal-bridged biradicals. They suggest the existence of a dendritic radical network of the dendrimers themselves.     

Multivalent, bifunctional dendrimers prepared by click chemistry

Unsymmetrical dendrimers, containing both mannose binding units and coumarin fluorescent units, have been prepared using click chemistry and shown to be highly efficient, dual-purpose recognition/detection agents for the inhibition of hemagglutination.     

Bola-Amphiphilic Glycodendrimers: New Carbohydrate-Mimicking Scaffolds to Target Carbohydrate-Binding Proteins

Dendrimers are appealing scaffolds for creating carbohydrate mimics with unique multivalent cooperativity. We report here novel bola-amphiphilic glycodendrimers bearing mannose and glucose terminals, and a hydrophobic thioacetal core responsive to reactive oxygen species. The peculiar bola-amphiphilic feature enabled stronger binding to lectin compared to conventional amphiphiles. In addition, these dendrimers are able to target mannose receptors and glucose transporters expressed at the surface of cells, thus allowing effective and specific cellular uptake. This highlights their great promise for targeted delivery.     

Structures and parameters of the EPR spectra of dendrimeric iron(III) complexes

The structures of high-spin iron(III) complexes with poly(propylenimine) dendrimers were examined by EPR spectroscopy. The pseudotetrahedral distortion of the coordination unit (CU) was considered. The fine structure parameters (FSPs) of the EPR spectra were analyzed. A dependence of the FSPs on the CU distortion and the covalent bonding was established. The influence of the axial Cl atoms on FSPs was considered. Splitting of the quartet term caused by the orthorhombic CU distortion with a pseudotetrahedral structure was estimated from the EPR data.     

Copper-containing dendromesogens: the influence of the metal on the mesomorphism

The synthesis and liquid crystalline behaviour of the first and second generations of a dendrimeric structure based on poly(propyleneimine)(DAB-dendr(NH₂)_x) are reported. 4-(4-n-Alkoxybenzoyloxy)salicylaldehydes are used as mesogenic moieties attached at the peripheral amino groups of the dendrimers giving rise to dendromesogens with four and eight mesogenic branches. From these dendromesogens, considered as organic ligands, were prepared six metal-containing dendrimers which incorporate two or four copper atoms in their structures. All the dendrimeric ligands and three of the metal-containing dendrimers exhibit liquid crystalline properties which were studied by optical microscopy, DSC, X-ray diffraction and EPR spectroscopy.     

A Bifunctional Spin Label for Ligand Recognition on Surfaces

In situ monitoring of biomolecular recognition, especially at surfaces, still presents a significant technical challenge. Electron paramagnetic resonance (EPR) of biomolecules spin‐labeled with nitroxides can offer uniquely sensitive and selective insights into these processes, but new spin‐labeling strategies are needed. The synthesis and study of a bromoacrylaldehyde spin label (BASL), which features two attachment points with orthogonal reactivity is reported. The first examples of mannose and biotin ligands coupled to aqueous carboxy‐functionalized gold nanoparticles through a spin label are presented. EPR spectra were obtained for the spin‐labeled ligands both free in solution and attached to nanoparticles. The labels were recognized by the mannose‐binding lectin, Con A, and the biotin‐binding protein avidin‐peroxidase. Binding gave quantifiable changes in the EPR spectra from which binding profiles could be obtained that reflect the strength of binding in each case.     

Characterization of heterogeneously functionalized dendrimers by mass spectrometry and EPR spectroscopy

Starburst dendrimers are receiving considerable attention as templates for the assembly of structured arrays of molecular components. This research motivates the development of improved methods for dendrimer characterization-specifically, for determining the numbers, distributions of numbers, and spatial distribution of molecular species synthetically attached to macromolecular templates. Such information provides the basis for advancing strategies aimed at controlling dendrimer functionalization, and thus represents enabling technology for tailoring the composition and structure of molecular arrays fashioned on dendrimer templates. Moreover, this information is vital to the proper interpretation of ongoing experiments in which dendrimers sparsely functionalized with reporter groups are used as probes. In this article, we report MALDI-TOF mass spectrometry and EPR spectroscopy of heterogeneously functionalized G(4)-PAMAM dendrimers bearing nitroxide spin-labels.     

Copper-containing dendromesogens: the influence of the metal on the mesomorphism

The synthesis and liquid crystalline behaviour of the first and second generations of a dendrimeric structure based on poly(propyleneimine)(DAB-dendr(NH₂)_x) are reported. 4-(4-n-Alkoxybenzoyloxy)salicylaldehydes are used as mesogenic moieties attached at the peripheral amino groups of the dendrimers giving rise to dendromesogens with four and eight mesogenic branches. From these dendromesogens, considered as organic ligands, were prepared six metal-containing dendrimers which incorporate two or four copper atoms in their structures. All the dendrimeric ligands and three of the metal-containing dendrimers exhibit liquid crystalline properties which were studied by optical microscopy, DSC, X-ray diffraction and EPR spectroscopy.     

Dendralene-type TTF vinylogs containing a 1,3-diselenole ring.

2-Diformylmethylene-1,3-diselenole was prepared and condensed with dithiolium phoshonium bromides and dithiolium phosphonates in the presence of base to give dendralene-type vinylogs of TTF bearing a 1,3-diselenole moiety. The electrochemistry of these dendrimers was studied. SEEPR measurements were also carried out. Calculations were carried out on the radical cations and correlated with the EPR values.     

Spectral characterization,antimicrobial and antibiofilm activity of poly(propylene imine) metallodendrimers in solution and applied onto cotton fabric

Abstract

Poly(propylene imine) dendrimers from first and third generation modified with 1,8-naphthalimide units and their Cu(II) complexes have been characterized by fluorescence and EPR spectroscopy. Cotton fabric has been modified with these dendrimers and their color characteristics were determined. The antimicrobial activity of dendrimer ligands and their Cu(II) complexes in solution and after their deposition on a cotton fabric was investigated. Good antibacterial effect of dendrimer ligands has been obtained, which is enhanced at their Cu(II) complexes. After their deposition on cotton fabric metallodendrimers exhibit good antibiofilm activity.     

Interactions of dendrimers with selected amino acids and proteins studied by continuous wave EPR and Fourier transform EPR

Interactions of polyamidoamine dendrimers, termed Gn, where n indicates the generation (=number of amidoamine layers), at different protonation levels with selected amino acids and proteins have been investigated by means of continuous wave electron paramagnetic resonance (cw-EPR) and pulsed-EPR (electron spin-echo = ESE) analyses. A low-generation dendrimer (G2) and a high-generation one (G6) were labeled with nitroxides for the EPR measurements. Gly, Glu, Arg, and Leu were selected as representative of neutral(zwitterionic)-polar, acidic, basic, and low-polar amino acids, respectively. The water-soluble proteins alpha-chymotrypsin and albumin were selected on the basis of a basic and an acidic isoelectric point, respectively. The cw-EPR spectra were analyzed by computing the line shapes to extract information about the dendrimer-biomolecule interactions. In general, dendrimers at a high protonation level interact stronger with amino acids than those at a low level of protonation. However, even for highly protonated dendrimers, a synergistic effect between hydrophilic and hydrophobic interactions promoted the formation of stable Gn-amino acid adducts, as demonstrated by the enhanced interactions with Leu. As expected from acid-base interactions, stable adducts were formed between Arg and highly protonated dendrimers and between Glu and low level protonated dendrimers. The relatively strong dendrimer interactions with the protein chymotrypsin and the poor interactions of dendrimers with albumin demonstrated that the protonated amino groups of the dendrimers are predominantly involved in the interactions with these proteins and indicated a significant role in the interactions with the dendrimers of the hydrophobic external residues of chymotrypsin. Computer-aided analysis of the ESE experiments was consistent with the cw-EPR results and supported the conclusion of a partial complexation of the nitroxides of the dendrimer with Leu and alpha-chymotrypsin.     

Functionalization of PAMAM dendrimers with nitronyl nitroxide radicals as models for the outer‐sphere relaxation in dentritic potential MRI contrast agents

PAMAM dendrimers functionalized with nitronyl nitroxide radicals were characterized. Quantitative determination of substitution with radicals was performed using EPR and electrochemical methods. The study of the ¹H NMR relaxation of the surrounding water showed how the outer‐sphere contribution to the relaxivity may be limited by the presence of the dendrimer core. Copyright © 2003 John Wiley & Sons, Ltd.