Combinatorial One‐Pot Synthesis of Poly‐N‐acetyllactosamine Oligosaccharides with Leloir‐Glycosyltransferases

Poly-N-acetyllactosamine (Poly-LacNAc, [3Galβ1,4GlcNAcβ1]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 one-pot 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 β1,4-galactosyltransferase-1 (β4GalT-1) and β1,3-N-acetylglucosaminyltransferase (β3GlcNAcT) with isolated glycan substrates of up to eight sugar units revealed a preference of β3GlcNAcT for the tetrasaccharide and no preference of β4GalT-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 poly-LacNAc 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.