Daniela Ponader

Sequence-defined glycopolymer segments presenting mannose: synthesis and lectin binding affinity.

We present for the first time the synthesis of sequence-defined monodisperse glycopolymer segments via solid-phase polymer synthesis. Functional building blocks displaying alkyne moieties and hydrophilic ethylenedioxy units were assembled stepwise on solid phase. The resulting polymer segments were conjugated with mannose sugars via 1,3-dipolar cycloaddition. The obtained mono-, di-, and trivalent mannose structures were then subject to Con A lectin binding. Surface plasmon resonance studies showed a nonlinear increase in binding regarding the number and spacing of sugar ligands. The results of Con A lectin binding assays indicate that the chemical composition of the polymeric scaffold strongly contributes to the binding activities as well as the spacing between the ligands and the number of presented mannose units. Our approach now allows for the synthesis of highly defined glycooligomers and glycopolymers with a diversity of properties to investigate systematically multivalent effects of polymeric ligands.

Carbohydrate-Lectin Recognition of Sequence-Defined Heteromultivalent Glycooligomers

Multivalency as a key principle in nature has been successfully adopted for the design and synthesis of artificial glycoligands by attaching multiple copies of monosaccharides to a synthetic scaffold. Besides their potential in various applied areas, e.g. as antiviral drugs, for the vaccine development and as novel biosensors, such glycomimetics also allow for a deeper understanding of the fundamental aspects of multivalent binding of both artificial and natural ligands. However, most glycomimetics so far neglect the purposeful arranged heterogeneity of their natural counterparts, thus limiting more detailed insights into the design and synthesis of novel glycomimetics. Therefore, this work presents the synthesis of monodisperse glycooligomers carrying different sugar ligands at well-defined positions along the backbone using for the first time sequential click chemistry and stepwise assembly of functional building blocks on solid support. This approach allows for straightforward access to sequence-defined, multivalent glycooligomers with full control over number, spacing, position, and type of sugar ligand. We demonstrate the synthesis of a set of heteromultivalent oligomers presenting mannose, galactose, and glucose residues. All heteromultivalent structures show surprisingly high affinities toward Concanavalin A lectin receptor in comparison to their homomultivalent analogues presenting the same number of binding ligands. Detailed studies of the ligand/receptor interaction using STD-NMR and 2fFCS indeed indicate a change in binding mechanism for trivalent glycooligomers presenting mannose or combinations of mannose and galactose residues. We find that galactose residues do not participate in the binding to the receptor, but they promote steric shielding of the heteromultivalent glycoligands and thus result in an overall increase in affinity. Furthermore, the introduction of nonbinding ligands seems to suppress receptor clustering of multivalent ligands. Overall these results support the importance of heteromultivalency specifically for the design of novel glycoligands and help to promote a fundamental understanding of multivalent binding modes.

Mechanical carbohydrate sensors based on soft hydrogel particles.

Elastic sensors: A simple method is presented for the measurement of specific biomolecular interactions with soft colloidal hydrogel particles (SCPs) as sensors. Carbohydrate/lectin interactions (see picture; green: carbohydrate molecules) were studied by optical detection of the mechanical deformation of the particles on a lectin surface. The affinity of various carbohydrate inhibitors could also be readily determined.

Photoswitchable precision glycooligomers and their lectin binding

Summary The synthesis of photoswitchable glycooligomers is presented by applying solid-phase polymer synthesis and functional building blocks. The obtained glycoligands are monodisperse and present azobenzene moieties as well as sugar ligands at defined positions within the oligomeric backbone and side chains, respectively. We show that the combination of molecular precision together with the photoswitchable properties of the azobenzene unit allows for the photosensitive control of glycoligand binding to protein receptors. These stimuli-sensitive glycoligands promote the understanding of multivalent binding and will be further developed as novel biosensors.

Precise redox-sensitive cleavage sites for improved bioactivity of siRNA lipopolyplexes

Lipo-oligomers have been proven as potent siRNA carriers based on stable electrostatic and hydrophobic complex formation and endosomal membrane destabilization. Although high stability of siRNA polyplexes is desirable in the extracellular space and cellular uptake, intracellular disassembly is important for the cytosolic release of siRNA and RNA-induced silencing complex formation. To improve the release, bioreducible sequence-defined lipo-oligomers were synthesized by solid-phase assisted synthesis using the disulfide building block Fmoc-succinoyl-cystamine for precise positioning of a disulfide unit between a lipophilic diacyl (bis-myristyl, bis-stearyl or bis-cholestanyl) domain and an ionizable oligocationic siRNA binding unit. Reducible siRNA polyplexes show higher gene silencing efficacy and lower cytotoxicity than their stable analogs, consistent with glutathione-triggered siRNA release and reduced lytic activity.

Combinatorial Screening for Specific Drug Solubilizers with Switchable Release Profiles

Polymer-block-peptide conjugates are tailored to render hydrophobic small molecule drugs water soluble. The combinatorial strategy selects for bioconjugates that exhibit sequence-specific solubilization and switchable release profiles of the cargo through incorporation of a disulfide linker moiety into the peptide-library design. While the study focused on the photosensitizer m-THPC and reductive carrier cleavage, the approach is generic and might be expanded toward a broad range of poorly soluble small-molecule drugs and other selective cleavage mechanisms to disassemble a peptide binding domain of the bioconjugate-based solubilizer.

Exploiting oligo(amido amine) backbones for the multivalent presentation of coiled-coil peptides

The investigation of coiled coil formation for one mono- and two divalent peptide-polymer conjugates is presented. Through the assembly of the full conjugates on solid support, monodisperse sequence-defined conjugates are obtained with defined positions and distances between the peptide side chains along the polymeric backbone. A heteromeric peptide design was chosen, where peptide K is attached to the polymer backbone, and coiled-coil formation is only expected through complexation with the complementary peptide E. Indeed, the monovalent peptide K-polymer conjugate displays rapid coiled-coil formation when mixed with the complementary peptide E sequence. The divalent systems show intramolecular homomeric coiled-coil formation on the polymer backbone despite the peptide design. Interestingly, this intramolecular assembly undergoes a conformational rearrangement by the addition of the complementary peptide E leading to the formation of heteromeric coiled coil-polymer aggregates. The polymer backbone acts as a template bringing the covalently bound peptide strands in close proximity to each other, increasing the local concentration and inducing the otherwise nonfavorable formation of intramolecular helical assemblies.