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.

No abstract

No abstract