Sequential one-pot multienzyme synthesis of hyaluronan and its derivative

•A sequential one-pot multienzyme synthetic strategy was developed which combined in situ sugar nucleotides generation with HA polymerization.•Partial labeled HA derivative was synthesized by metabolic incorporation of monosaccharide analogues into the sequential OPME system.•Cross-linked HA hydrogel was achieved by azide-alkyne click chemistry reaction and exhibited high porous microstructures. Hyaluronan (HA) is a linear polysaccharide composed of repeating disaccharide units. It has been well documented to play an array of biological functions in cancer events. Here, we reported a sequential one-pot multienzyme (OPME) strategy for in vitro synthesis of HA and its derivatives. The strategy, which combined in situ sugar nucleotides generation with HA chain polymerization, could convert cheap monosaccharides into HA polymers without consuming exogenous sugar nucleotide donors. HA polymers (number-average molecular weight ranged from 1.5×104 to 5.5×105Da) with over 70% yields were efficiently synthesized and purified from this one-pot system. More importantly, partial labeled HA derivative was further synthesized by metabolic incorporation of unnatural monosaccharide analogues into the sequential OPME system. Cross-linked HA hydrogel was achieved via copper (I)-catalyzed azide-alkyne cycloaddition and exhibited novel networks consisting of both inter- and intra-connected HA chains, which could facilitate the potential applications of this unique polysaccharide.