Poly-N-acetyllactosamine (poly-LacNAc) structures have been identified as important ligands for galectin-mediated cell adhesion to extra-cellular matrix (ECM) proteins. We here present the biofunctionalization of surfaces with poly-LacNAc structures and subsequent binding of ECM glycoproteins. First, we synthesized beta-GlcNAc glycosides carrying a linker for controlled coupling onto chemically functionalized surfaces. Then we produced poly-LacNAc structures with defined lengths using human beta1,4-galactosyltransferase-1 and beta1,3-N-acetylglucosaminyltransferase from Helicobacter pylori. These compounds were also used for kinetic characterization of glycosyltransferases and lectin binding assays. A mixture of poly-LacNAc-structures covalently coupled to functionalized microtiter plates were identified for best binding to our model galectin His(6)CGL2. We further demonstrate for the first time that these poly-LacNAc surfaces are suitable for further galectin-mediated binding of the ECM glycoproteins laminin and fibronectin. This new technology should facilitate cell adhesion to biofunctionalized surfaces by imitating the natural ECM microenvironment.

Galectin-3 (Gal-3), a member of the β-galactoside-binding lectin family, is a tumor biomarker and involved in tumor angiogenesis and metastasis. Gal-3 is therefore considered as a promising target for early cancer diagnosis and anticancer therapy. We here present the synthesis of a library of tailored multivalent neo-glycoproteins and evaluate their Gal-3 binding properties. By the combinatorial use of glycosyltransferases and chemo-enzymatic reactions, we first synthesized a set of N-acetyllactosamine (Galβ1,4GlcNAc; LacNAc type 2)-based oligosaccharides featuring five different terminating glycosylation epitopes, respectively. Neo-glycosylation of bovine serum albumin (BSA) was accomplished by dialkyl squarate coupling to lysine residues resulting in a library of defined multivalent neo-glycoproteins. Solid-phase binding assays with immobilized neo-glycoproteins revealed distinct affinity and specificity of the multivalent glycan epitopes for Gal-3 binding. In particular, neo-glycoproteins decorated with N',N″-diacetyllactosamine (GalNAcβ1,4GlcNAc; LacdiNAc) epitopes showed high selectivity and were demonstrated to capture Gal-3 from human serum with high affinity. Furthermore, neo-glycoproteins with terminal biotinylated LacNAc glycan motif could be utilized as Gal-3 detection agents in a sandwich enzyme-linked immunosorbent assay format. We conclude that, in contrast to antibody-based capture steps, the presented neo-glycoproteins are highly useful to detect functionally intact Gal-3 with high selectivity and avidity. We further gain novel insights into the binding affinity of Gal-3 using tailored multivalent neo-glycoproteins, which have the potential for an application in the context of cancer-related biomedical research.

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.