A flexible tyrosine-attached chemical lid prevents premature disengagement of growing oligosaccharides and triggers the synthesis of a high molecular weight polymer.

Metabolic glycoengineering enables a directed modification of cell surfaces by introducing target molecules to surface proteins displaying new features. Biochemical pathways involving glycans differ in dependence on the cell type; therefore, this technique should be tailored for the best results. We characterized metabolic glycoengineering in telomerase-immortalized human mesenchymal stromal cells (hMSC‑TERT) as a model for primary hMSC, to investigate its applicability in TERT-modified cell lines. The metabolic incorporation of N‑azidoacetylmannosamine (Ac4ManNAz) and N‑alkyneacetylmannosamine (Ac4ManNAl) into the glycocalyx as a first step in the glycoengineering process revealed no adverse effects on cell viability or gene expression, and the in vitro multipotency (osteogenic and adipogenic differentiation potential) was maintained under these adapted culture conditions. In the second step, glycoengineered cells were modified with fluorescent dyes using Cu-mediated click chemistry. In these analyses, the two mannose derivatives showed superior incorporation efficiencies compared to glucose and galactose isomers. In time-dependent experiments, the incorporation of Ac4ManNAz was detectable for up to six days while Ac4ManNAl-derived metabolites were absent after two days. Taken together, these findings demonstrate the successful metabolic glycoengineering of immortalized hMSC resulting in transient cell surface modifications, and thus present a useful model to address different scientific questions regarding glycosylation processes in skeletal precursors.

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.

Metabolic glycoengineering involves the stimulation of cells with functionalized monosaccharides. Glucosamine, galactosamine, and mannosamine derivatives are commercially available, but their application may lead to undirected (i.e., chemical) incorporation into proteins. However, sialic acids are attached to the ends of complex sugar chains of glycoproteins, which might be beneficial for cell surface modification via click chemistry. Thus, we studied the incorporation of chemically synthesized unnatural alkyne modified sialic acid (SiaNAl) into glycoproteins of human telomerase-immortalized mesenchymal stromal cells (hMSC-TERT) and we show that SiaNAl can be efficiently incorporated in glycoproteins involved in signal transduction and cell junction.