Poly-N-acetyllactosamine (Poly-LacNAc, [3Galb1,4GlcNAcb1] n ) glycans play an essential role in carbohydrate-protein interactions. The synthesis of poly-LacNAc, both chemical and enzymatic, is typically characterized by high losses of product during sequential synthesis, due to deprotection and/ or purification steps. In this work we present a onepot synthesis of poly-LacNAc oligosaccharides by combining recombinant glycosyltransferases. By fractionation of the poly-LacNAc glycan mixture we were able to isolate glycans with up to six N-acetyllactosamine (LacNAc) units. Activity measurements of the involved recombinant b1,4-galactosyltransferase-1 (b4GalT-1) and b1,3-N-acetylglucosaminyltransferase (b3GlcNAcT) with isolated glycan substrates of up to eight sugar units revealed a preference of b3GlcNAcT for the tetrasaccharide and no preference of b4GalT-1 for a specific glycan length.These findings led us to the optimization of combinatorial one-pot synthesis by variation of substrate and enzyme ratios, as well as starting the synthesis with various poly-LacNAc chain lengths. Consequently, we present here an optimized poly-LacNAc synthesis by the combination of two glycosyltransferases and a uridine-diphospho-glucose/N-acetylglucosamine 4'-epimerase as one-pot strategy resulting in long polyLacNAc glycans with up to six LacNAc units in high yields while minimizing reaction time and product loss. The obtained products are important ligands for the biofunctionalization of biomaterial surfaces and the construction of an artificial extracellular matrix for tissue engineering.

Herein is demonstrated that a biomimetic, in‐vivo‐like presentation of extracellular matrix (ECM) proteins such as fibronectin, laminin, and collagen IV mediated by a fully functional poly‐sugar (polyLacNAc)–lectin complex can be achieved on a biomaterials surface. Cells recognize and bind to these immobilized proteins and can, due to the reversible protein presentation, remodel initially presented ECM proteins.

Precise determination of biomolecular interactions in high throughput crucially depends on a surface coating technique that allows immobilization of a variety of interaction partners in a non-interacting environment. We present a one-step hydrogel coating system based on isocyanate functional six-arm poly(ethylene oxide)-based star polymers for commercially available 96-well microtiter plates that combines a straightforward and robust coating application with versatile bio-functionalization. This system generates resistance to unspecific protein adsorption and cell adhesion, as demonstrated with fluorescently labeled bovine serum albumin and primary human dermal fibroblasts (HDF), and high specificity for the assessment of biomolecular recognition processes when ligands are immobilized on this surface. One particular advantage is the wide range of biomolecules that can be immobilized and convert the per se inert coating into a specifically interacting surface. We here demonstrate the immobilization and quantification of a broad range of biochemically important ligands, such as peptide sequences GRGDS and GRGDSK-biotin, the broadly applicable coupler molecule biocytin, the protein fibronectin, and the carbohydrates N-acetylglucosamine and N-acetyllactosamine. A simplified protocol for an enzyme-linked immunosorbent assay was established for the detection and quantification of ligands on the coating surface. Cell adhesion on the peptide and protein-modified surfaces was assessed using HDF. All coatings were applied using a one-step preparation technique, including bioactivation, which makes the system suitable for high-throughput screening in a format that is compatible with the most routinely used testing systems.

The formation and properties of glyco‐DNA–gold nanoparticles (NP) with a multivalent presentation of DNA‐glyco ligands is presented. These particles are equipped with two reversible binding modes, which enable reversible dissociation by two independent external stimuli: temperature‐induced DNA duplex melting and displacement of the DNA‐glyco ligands from the carbohydrate recognition domains with free sugar.

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