Manipulation of Electrostatic and Saccharide Linker Interactions in the Design of Efficient Glycopolypeptide‐Based Cholera Toxin Inhibitors

Multivalent, glycopolymer inhibitors designed for the treatment of disease and pathogen infection have shown improvements in binding correlated with general changes in glycopolymer architecture and composition. We have previously demonstrated that control of glycopolypeptide backbone extension and ligand spacing significantly impacts the inhibition of the cholera toxin B subunit pentamer (CT B₅) by these polymers. In the studies reported here, we elucidate the role of backbone charge and linker length in modulating the inhibition event. Peptides of the sequence AXPXG (where X is a positive, neutral or negative amino acid), equipped with the alkyne functionality of propargyl glycine, were designed and synthesized via solid‐phase peptide synthetic methods and glycosylated via Cu(I)‐catalyzed alkyne‐azide cycloaddition reactions. The capacity of the glycopeptides to inhibit the binding of the B₅ subunit of cholera toxin was evaluated. These studies indicated that glycopeptides with a negatively charged backbone show improved inhibition of the binding event relative to the other glycopeptides. In addition, variations in the length of the linker between the peptide and the saccharide ligand also affected the inhibition of CT by the glycopeptides. Our findings suggest that, apart from appropriate saccharide spacing and polypeptide chain extension, saccharide linker conformation and the systematic placement of charges on the polypeptide backbone are also significant variables that can be tuned to improve the inhibitory potencies of glycopolypeptide‐based multivalent inhibitors.

     

Amplification of bifunctional ligands for calmodulin from a dynamic combinatorial library

A well known strategy to prepare high affinity ligands for a biological receptor is to link together low affinity ligands. DCC (dynamic combinatorial chemistry) was used to select bifunctional protein ligands with high affinity relative to the corresponding monofunctional ligands. Thiol to disulfide linkage generated a small dynamic library of bifunctional ligands in the presence of calmodulin, a protein with two independently mobile domains. The binding constant of the bifunctional ligand (disulfide) most amplified by the presence of calmodulin is nearly two orders of magnitude higher than that of the corresponding monofunctional ligand (thiol).     

Dendrimeric Bisphosphonates for Multivalent Protein Surface Binding

A single weak‐binding event is multiplied into an efficient receptor site for protein surfaces (<10^?1 to >10⁶ M ^?1 in buffered aqueous solution) in a biomimetic fashion. This has hitherto been done with natural host/guest pairs, but not with artificial receptors. The organic reaction presented is one of very few that enable chemists to fuse multiple ionic building blocks covalently in highly polar solution; this one‐pot reaction proceeds with virtually quantitative yield. According to this concept, other building blocks with aldehyde groups can likewise be multiplied into monodisperse functional dendrimers. Small basic proteins are bound by octameric dendrimers in 1:1 or 1:2 complexes with millimolar to submicromolar affinities. The complexation event is studied independently in buffered aqueous solution by three different spectroscopic methods (PFG‐LED, UV/Vis, and fluorescence). Potential new applications include recombinant protein purification through Arg tags on immobilized dendrimers and on/off switching of protein function by reversible active‐site capping of enzymes.     

Supramolecular microcontact printing and dip-pen nanolithography on molecular printboards

The transfer of functional molecules onto self-assembled monolayers (SAMs) by means of soft and scanning-probe lithographic techniques-microcontact printing (muCP) and dip-pen nanolithography (DPN), respectively-and the stability of the molecular patterns during competitive rinsing conditions were examined. A series of guests with different valencies were transferred onto beta-cyclodextrin- (beta-CD-) terminated SAMs and onto reference hydroxy-terminated SAMs. Although physical contact was sufficient to generate patterns on both types of SAMs, only molecular patterns of multivalent guests transferred onto the beta-CD SAMs were stable under the rinsing conditions that caused the removal of the same guests from the reference SAMs. The formation of kinetically stable molecular patterns by supramolecular DPN with a lateral resolution of 60 nm exemplifies the use of beta-CD-terminated SAMs as molecular printboards for the selective immobilization of printboard-compatible guests on the nanometer scale through the use of specific, multivalent supramolecular interactions. Electroless deposition of copper on the printboard was shown to occur selectively on the areas patterned with dendrimer-stabilized gold nanoparticles.     

Glyconanocavities: Cyclodextrins and Beyond

Cyclodextrins (CDs) represent a unique example of complementarity between nanotechnology and biotechnology. Their molecular nanocavity character and the possibility of selective functionalization offer an excellent opportunity for chemical elaboration of unique nanostructures in a three-dimensional network. Several approaches from our laboratories, aimed at endowing CDs with biorecognition properties by incorporation of saccharide ligands, are discussed. Applications range from site-specific drug delivery systems to more fundamental studies on carbohydrate–protein interactions. Results on the de novo synthesis of a new family of glyconanocavities constructed from α,α-trehalose building blocks, namely cyclotrehalans (CTs), and on their complexing properties are also presented.     

Designing Multivalent Ligands to Control Biological Interactions: From Vaccines and Cellular Effectors to Targeted Drug Delivery

Multivalent interactions in which multiple ligands on one object bind to multiple receptors on another are commonly found in natural biological systems. In addition, these interactions can lead to increased strength and selectivity when compared to the corresponding monovalent interaction. These attributes have also guided the design of synthetic multivalent ligands to control biological interactions. This review will highlight the recent literature describing the use of multivalent ligand display in the design of vaccines, immunomodulators, cell signaling effectors, and vehicles for targeted drug delivery.     

Chemoselectively template-assembled glycopeptide presenting clustered cancer related T-antigen

The first synthesis of the tumor-associated α-aminooxy T-antigen 1, a relevant recognition motif for the direct construction of multitopic carbohydrate architecture of biological interest is described. The usefulness of this building block is emphasized with the efficient preparation through oxime ligation of a neoglycopeptide cluster, which is readily suitable for evaluating the role of multivalency in antigen presentation to the immune system from an anticancer vaccine perspective.     

The role of dimension in multivalent binding events: structure-activity relationship of dendritic polyglycerol sulfate binding to L-selectin in correlation with size and surface charge density

L ‐, P ‐, and E ‐Selectin are cell adhesion molecules that play a crucial role in leukocyte recruitment from the blood stream to the afflicted tissue in an acute and chronic inflammatory setting. Since selectins mediate the initial contact of leukocytes to the vascular endothelium, they have evolved as a valuable therapeutic target in diseases related to inflammation by inhibition of the physiological selectin–ligand interactions. In a previous study, it was demonstrated that dPGS, a fully synthetic heparin analogue, works as an efficient inhibitor towards L ‐ and P ‐selectin in vitro as well as in vivo. Herein, the focus is directed towards the effect of size and charge density of the polyanion. The efficiency of L ‐selectin inhibition via an SPR‐based in vitro assay and a cell‐based flow chamber assay is investigated with dPGS ranging from approximately 4 to 2000 kDa. SPR measurements show that the inhibitory potential of highly sulfated dPGS increases with size and charge density. Thereby, IC₅₀ values from the micromolar to the low picomolar range are determined. The same tendency could be observed in a cell‐based flow chamber assay with three representative dPGS samples. This structure–affinity relationship of dPGS suggests that the strong inhibitory potential of dPGS is not only based on the strong electrostatic interaction with areas of cationic surface potential on L ‐selectin but is also due to a steric shielding of the carbohydrate binding site by large, flexible dPGS particles.

     

A synthetic "tour de force": well-defined multivalent and multimodal dendritic structures for biomedical applications

Dendrimers have several unique properties that make them attractive scaffolds for use in biomedical applications. To date, multivalent and multimodal dendritic structures have been synthesized predominantly by statistical modification of peripheral groups. However, the potential application of such probes in patients demands well-defined and monodisperse materials that have unique structures. Current progress in the field of chemical biology, in particular chemoselective ligation methods, renders this challenge possible. In this Minireview, we outline the different available synthetic strategies, some applications that already make use of this new generation of multivalent and multimodal architectures, and the challenges for future developments.