Polyoxazolines with a Vicinally Double-Bioactivated Terminus for Biomacromolecular Affinity Assessment.

Interactions between proteins and carbohydrates with larger biomacromolecules, e.g., lectins, are usually examined using self-assembled monolayers on target gold surfaces as a simplified model measuring setup. However, most of those measuring setups are either limited to a single substrate or do not allow for control over ligand distance and spacing. Here, we develop a synthetic strategy, consisting of a cascade of a thioesterification, native chemical ligation (NCL) and thiol-ene reaction, in order to create three-component polymer conjugates with a defined double bioactivation at the chain end. The target architecture is the vicinal attachment of two biomolecule residues to the α telechelic end point of a polymer and a thioether group at the ω chain end for fixating the conjugate to a gold sensor chip surface. As proof-of-principle studies for affinity measurements, we demonstrate the interaction between covalently bound mannose and ConA in surface acoustic wave (SAW) and surface plasmon resonance (SPR) experiments.

Tuning the Product Spectrum of a Glycoside Hydrolase Enzyme by a Combination of Site-Directed Mutagenesis and Tyrosine-Specific Chemical Modification.

Selective chemical modification of proteins plays a pivotal role for the rational design of enzymes with novel and specific functionalities. In this study, a strategic combination of genetic and chemical engineering paves the way for systematic construction of biocatalysts by tuning the product spectrum of a levansucrase from Bacillus megaterium (Bm-LS), which typically produces small levan-like oligosaccharides. The implementation of site-directed mutagenesis followed by a tyrosine-specific modification enabled control of the product synthesis: depending on the position, the modification provoked either enrichment of short oligosaccharides (up to 800 % in some cases) or triggered the formation of high molecular weight polymer. The chemical modification can recover polymerization ability in variants with defective oligosaccharide binding motifs. Molecular dynamic (MD) simulations provided insights into the effect of modifying non-native tyrosine residues on product specificity.

Metabolic Glycoengineering of Cell-Derived Matrices and Cell Surfaces: A Combination of Key Principles and Step-by-Step Procedures.

Metabolic glycoengineering allows insertion of non-natural monosaccharides into glycan structures during biosynthesis thereby enabling extracellular matrices (ECMs), cell surfaces, or tissues for decoration with functional cues with ultimate spatial control while deploying aqueous and toxicologically benign coupling chemistries. In this work, we discuss relevant methods in the design of metabolic glycoengineered systems, ranging from synthetic procedures to decoration of cell surfaces and ECM components by bioorthogonal chemistries for widespread biomedical applications. As representative example, we chose a tetra-acetylated azide-bearing monosaccharide as model compound to be metabolically incorporated into glycans of the glycocalyx and ECM components generated by NIH 3T3 cells. Detailed guidance in fabrication and functionalization of azide-bearing glycan structures via bioorthogonal click chemistries in glycoengineered extracellular matrices is provided. In addition, a biocompatible design space of the copper(I)-catalyzed azide-alkyne cycloaddition due to the toxicity of the copper catalyst is detailed enabling effective and safe modification of living cell systems. Thereby, this set of methods provides the blueprint enabling the design and characterization of metabolically glycoengineered systems for novel applications in drug delivery and tissue engineering.

Exploring the Structural Space of the Galectin-1-Ligand Interaction.

Galectin-1 is a tumor-associated protein recognizing the Galß1-4GlcNAc motif of cell-surface glycoconjugates. Herein, we report the stepwise expansion of a multifunctional natural scaffold based on N-acetyllactosamine (LacNAc). We obtained a LacNAc mimetic equipped with an alkynyl function on the 3'-hydroxy group of the disaccharide facing towards a binding pocket adjacent to the carbohydrate-recognition domain. It served as an anchor motif for further expansion by the Sharpless-Huisgen-Meldal reaction, which resulted in ligands with a binding mode almost identical to that of the natural carbohydrate template. X-ray crystallography provided a structural understanding of the galectin-1-ligand interactions. The results of this study enable the development of bespoke ligands for members of the galectin target family.