Here, we developed a general strategy for synthesizing homogeneous HA conjugates, and generated homogeneous HA–pNP, HA–biotin, and HA–oroxylin conjugates to investigate the relationships between HA chain length and its diverse biological functions.

A cation exchange assisted binding-elution (BE) strategy for enzymatic synthesis of human milk oligosaccharides (HMOs) was developed. An amino linker was used to provide the cation ion under acidic condition which can be readily bound to cation exchange resin and then eluted off by saturated ammonium bicarbonate. Ammonium bicarbonate in the collections was easily removed by vacuum evaporation. This strategy circumvented the incompatible issue between glycosyltransferases and solid support or large polymers, and no purification was needed for intermediate products. With current approach, polyLacNAc backbones of HMOs and fucosylated HMOs were synthesized smoothly.Download high-res image (151KB)Download full-size image

•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 × 105 Da) 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.

An enzymatic strategy was developed to generate asymmetrically branched N-glycans from natural sources by using a panel of glycosidases and glycosyltransferases. Briefly, LacZ β-galactosidase was employed to selectively trim symmetrically branched N-glycans isolated from bovine fetuin. The yielding structures were then converted to asymmetrically branched core structures by robust glycosyltransferase for further extension.

•An enzyme that specifically recognizes 6-deoxy-l-psicose was identified.•One-pot multienzyme system was used to convert l-rhamnose to 6-deoxy-l-psicose.•Product was obtained with high yield and purity without isomer separation step.Rare sugars offer a plethora of applications in the pharmaceutical, medicinal, and industries, as well as in synthetic chemistry. However, studies of rare sugars have been hampered by their relative scarcity. In this work, we describe a two-step strategy to efficiently and conveniently prepare 6-deoxy-l-psicose from l-rhamnose. In the first reaction step, the isomerization of l-rhamnose (6-deoxy-l-mannose) to l-rhamnulose (6-deoxy-l-fructose) catalyzed by l-rhamnose isomerase (RhaI), and the epimerization of l-rhamnulose to 6-deoxy-l-psicose catalyzed by d-tagatose 3-epimerase (DTE) were coupled with selective phosphorylation reaction by fructose kinase from human (HK), which selectively phosphorylate 6-deoxy-l-psicose at C-1 position. 6-deoxy-l-psicose 1-phosphate was purified by a silver nitrate precipitation method. In the second step, the phosphate group of the 6-deoxy-l-sorbose 1-phosphate was hydrolyzed with acid phosphatase (AphA) to produce 6-deoxy-l-psicose in 81% yield with respect to l-rhamnose. This method allows that the 6-deoxy-l-psicose to be obtained from readily available starting materials with high purity and without having to undergo isomer separation.

•Roles of highly conserved residues were investigated by site-directed mutagenesis.•tGlmU Q76E mutant with novel CDP-GlcNAc biosynthesis activity was identified.•Y103F and N169R mutants showed enhanced uridyltransferase activities.N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme that catalyzes both acetyltransfer and uridyltransfer reactions in the prokaryotic UDP-GlcNAc biosynthesis pathway. Our previous study demonstrated that the uridyltransferase domain of GlmU (tGlmU) exhibited a flexible substrate specificity, which could be further applied in unnatural sugar nucleotides preparation. However, the structural basis of tolerating variant substrates is still not clear. Herein, we further investigated the roles of several highly conserved amino acid residues involved in substrate binding and recognition by structure- and sequence-guided site-directed mutagenesis. Out of total 16 mutants designed, tGlmU Q76E mutant which had a novel catalytic activity to convert CTP and GlcNAc-1P into unnatural sugar nucleotide CDP-GlcNAc was identified. Furthermore, tGlmU Y103F and N169R mutants were also investigated to have enhanced uridyltransferase activities compared with wide-type tGlmU.

Nucleotide sugars are essential glycosyl donors for Leloir-type glycosyltransferases. The UDP-N-acetylgalactosamine pyrophosphorylase (UDP-GalNAc PP; AGX1) from Homo sapiens catalyzes the synthesis of UDP-N-acetylgalactosamine from N-acetylgalactosamine 1-phosphate and UTP. In this Letter, we systematically studied nucleotide substrate specificity of AGX1 during its uridyltransfer reaction, and described the capability of AGX1 to catalyze dUTP and dTTP to their corresponding nucleotide sugars for the first time. Furthermore, using such a eukaryotic enzyme, we synthesized dUDP-GalNAc and dTDP-GalNAc in multiple mg scale in vitro efficiently and rapidly.

Here, we developed a general strategy for synthesizing homogeneous HA conjugates, and generated homogeneous HA–pNP, HA–biotin, and HA–oroxylin conjugates to investigate the relationships between HA chain length and its diverse biological functions.