Toward Automated Enzymatic Synthesis of Oligosaccharides.

Oligosaccharides together with oligonucleotides and oligopeptides comprise the three major classes of natural biopolymers. Automated systems for oligonucleotide and oligopeptide synthesis have significantly advanced developments in biological science by allowing nonspecialists to rapidly and easily access these biopolymers. Researchers have endeavored for decades to develop a comparable general automated system to synthesize oligosaccharides. Such a system would have a revolutionary impact on the understanding of the roles of glycans in biological systems. The main challenge to achieving automated synthesis is the lack of general synthetic methods for routine synthesis of glycans. Currently, the two main methods to access homogeneous glycans and glycoconjugates are chemical synthesis and enzymatic synthesis. Enzymatic glycosylation can proceed stereo- and regiospecifically without protecting group manipulations. Moreover, the reaction conditions of enzyme-catalyzed glycosylations are extremely mild when compared to chemical glycosylations. Over the past few years methodology toward the automated chemical synthesis of oligosaccharides has been developed. Conversely, while automated enzymatic synthesis is conceptually possible, it is not as well developed. The goal of this survey is to provide a foundation on which continued technological advancements can be made to promote the automated enzymatic synthesis of oligosaccharides.

Cation exchange assisted binding-elution strategy for enzymatic synthesis of human milk oligosaccharides (HMOs).

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.

An enzymatic strategy to asymmetrically branched N-glycans.

An enzymatic strategy was developed to generate asymmetrically branched N-glycans from natural sources by using a panel of glycosidases and glycosyltransferases. Briefly, LacZ ß-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.

Two-step enzymatic synthesis of 6-deoxy-L-psicose.

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.

A General Chemoenzymatic Strategy for the Synthesis of Glycosphingolipids.

A concise, prototypical, and stereoselective strategy for the synthesis of therapeutically and immunologically significant glycosphingolipids has been developed. This strategy provides a universal platform for glycosphingolipid synthesis by block coupling of enzymatically prepared free oligosaccharideglycans to lipids using glycosyl N-phenyltrifluoroacetimidates as efficient activated intermediates. As demonstrated here, two different types of glycosphingolipids were obtained in excellent yields using the method.

Facile Enzymatic Synthesis of Ketoses.

Studies of rare ketoses have been hampered by a lack of efficient preparation methods. A convenient, efficient, and cost-effective platform for the facile synthesis of ketoses is described. This method enables the preparation of difficult-to-access ketopentoses and ketohexoses from common and inexpensive starting materials with high yield and purity and without the need for a tedious isomer separation step.

Chemoenzymatic synthesis of ADP-d-glycero-ß-d-manno-heptose and study of the substrate specificity of HldE.

An efficient one-pot three enzymes strategy for chemoenzymatic synthesis of ADP-d-glycero-ß-d-manno-heptose (ADP-d, d-heptose) was reported using chemically synthesized d, d-heptose-7-phosphate and the ADP-d, d-heptose biosynthetic enzymes HldE and GmhB. Moreover, the result of investigating substrate specificity of the kinase action of HldE revealed that HldE had highly restricted substrate specificity towards structurally modified heptose-7-phosphate analogs.

Injectable bio-multifunctional hyaluronic acid-based hydrogels loaded with poly ADP-ribose polymerase inhibitors for ovarian cancer therapy.

The recent use of PARP inhibitors (PARPi) in the maintenance treatment of ovarian tumor has significantly improved the survival rates of cancer patients. However, the current oral administration of PARP inhibitors fails to realize optimal therapeutic effects due to the low bioavailability in cancerous tissues, and often leads to a range of systemic adverse effects including hematologic toxicities, digestive system reactions, and neurotoxicities. Therefore, the demand for an advanced drug delivery system that can ensure effective drug administration while minimizing these unfavorable reactions is pressing. Injectable hydrogel emerges as a promising solution for local administration with the capability of sustainable drug release. In this study, we developed an injectable hydrogel made from aminated hyaluronic acid and aldehyde-functionalized pluronic127 via Schiff base reaction. This hydrogel exhibits excellent injectability with short gelation time and remarkable self-healing ability, and is applied to load niraparib. The drug-loaded hydrogel (HP@Nir hydrogel) releases drugs sustainably as tested in vitro as well as displays significant anti-proliferation and anti-migratory properties on human epithelial ovarian cancer cell line. Notably, HP@Nir hydrogel effectively suppresses the growth of ovarian cancer, without significant adverse reactions as demonstrated in animal studies. Additionally, the developed hydrogel is gradually degraded in vivo for around 20 d, while maintaining good biocompatibility. Overall, the injectable hydrogel loaded with niraparib provides a secure and efficient strategy for the treatment and management of ovarian cancer.

Enzymatic modular synthesis of asymmetrically branched human milk oligosaccharides.

Human milk oligosaccharides (HMOs) are intricate glycans that promote healthy growth of infants and have been incorporated into infant formula as food additives. Despite their importance, the limited availability of asymmetrically branched HMOs hinders the exploration of their structure and function relationships. Herein, we report an enzymatic modular strategy for the efficient synthesis of these HMOs. The key branching enzyme for the assembly of branched HMOs, human ß1,6-N-acetylglucosaminyltransferase 2 (GCNT2), was successfully expressed in Pichia pastoris for the first time. Then, it was integrated with six other bacterial glycosyltransferases to establish seven glycosylation modules. Each module comprises a one-pot multi-enzyme (OPME) system for in-situ generation of costly sugar nucleotide donors, combined with a glycosyltransferase for specific glycosylation. This approach enabled the synthesis of 31 branched HMOs and 13 linear HMOs in a stepwise manner with well-programmed synthetic routes. The binding details of these HMOs with related glycan-binding proteins were subsequently elucidated using glycan microarray assays to provide insights into their biological functions. This comprehensive collection of synthetic HMOs not only serves as standards for HMOs structure identification in complex biological samples but also significantly enhances the fields of HMOs glycomics, opening new avenues for biomedical applications.

A chemoenzymatic route to synthesize unnatural sugar nucleotides using a novel N-acetylglucosamine-1-phosphate pyrophosphorylase from Camphylobacter jejuni NCTC 11168.

A novel N-acetylglucosamine-1-phosphate pyrophosphorylase was identified from Campylobacter jejuni NCTC 11168. An unprecedented degree of substrate promiscuity has been revealed by systematic studies on its substrate specificities towards sugar-1-P and NTP. The yields of the synthetic reaction of seven kinds of sugar nucleotides catalyzed by the enzyme were up to 60%. In addition, the yields of the other nine were around 20%. With this enzyme, three novel sugar nucleotide analogs were synthesized on a preparative scale and well characterized.

Recent Progress in antibacterial hydrogel coatings for targeting biofilm to prevent orthopedic implant-associated infections.

The application of orthopedic implants for bone tissue reconstruction and functional restoration is crucial for patients with severe bone fractures and defects. However, the abiotic nature of orthopedic implants allows bacterial adhesion and colonization, leading to the formation of bacterial biofilms on the implant surface. This can result in implant failure and severe complications such as osteomyelitis and septic arthritis. The emergence of antibiotic-resistant bacteria and the limited efficacy of drugs against biofilms have increased the risk of orthopedic implant-associated infections (OIAI), necessitating the development of alternative therapeutics. In this regard, antibacterial hydrogels based on bacteria repelling, contact killing, drug delivery, or external assistance strategies have been extensively investigated for coating orthopedic implants through surface modification, offering a promising approach to target biofilm formation and prevent OIAI. This review provides an overview of recent advancements in the application of antibacterial hydrogel coatings for preventing OIAI by targeting biofilm formation. The topics covered include: (1) the mechanisms underlying OIAI occurrence and the role of biofilms in exacerbating OIAI development; (2) current strategies to impart anti-biofilm properties to hydrogel coatings and the mechanisms involved in treating OIAI. This article aims to summarize the progress in antibacterial hydrogel coatings for OIAI prevention, providing valuable insights and facilitating the development of prognostic markers for the design of effective antibacterial orthopedic implants.

Investigation of the nucleotide triphosphate substrate specificity of Homo sapiens UDP-N-acetylgalactosamine pyrophosphorylase (AGX1).

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.

NahK/GlmU fusion enzyme: characterization and one-step enzymatic synthesis of UDP-N-acetylglucosamine.

The availability of uridine 5'-diphosphate N-acetylglucosamine (UDP-GlcNAc) is a prerequisite for the GlcNAc-transferase-catalyzed glycosylation reaction. UDP-GlcNAc has already been synthesized using an N-acetylhexosamine 1-kinase (NahK) and a GlcNAc-1-P uridyltransferase (truncated GlmU) and here, a fusion enzyme was constructed with truncated GlmU and NahK. After determination of the optimum catalytic condition (pH 8.0 at 40 °C), the fusion enzyme was used to synthesize UDP-GlcNAc in a single step with a yield of 88 % from GlcNAc, ATP and UTP. Furthermore, a simplified purification method was demonstrated using separation by gel filtration after by-product digestion with alkaline phosphatase. An overall yield of 77 % and a purity of over 90 % were achieved.

Enzymatic and chemoenzymatic synthesis of human milk oligosaccharides and derivatives.

Human milk oligosaccharides (HMOs) are complex glycans that are the third largest solid component in human milk. It has attracted great interest in recent years due to their critical role in boosting infant health. These oligosaccharides play an important role in a variety of physiological processes, such as shaping the infant gut microbiome, preventing pathogenic infections and promoting the development of immune system. However, limited availability of HMOs hampered their use in food and medical areas. Moreover, most of the HMOs are unique to human milk and difficult to isolate. The strategies, chemical synthesis, whole-cell fermentation, and purification from human milk, have their advantages and come with their own challenges. In this review, we examined the remarkable progress that has been made in the enzymatic and chemoenzymatic synthesis of HMOs, and discussed the challenges and opportunities in large-scale synthesis of HMOs.

Anti-inflammatory effects of a SERP 30 polysaccharide from the residue of Sarcandra glabra against lipopolysaccharide-induced acute respiratory distress syndrome in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Sarcandra glabra (Thunb.) Nakai, a valuable dietetic Chinese herb, is still widely used today. Multiple ingredients of S. glabra with a variety of activities such as anti-inflammatory, antiviral, and antitumor were studied. However, the Sarcandra glabra (Thunb.) Nakai polysaccharide hasn't been reported for its anti-inflammatory effect. AIM OF THE STUDY: In this study, the anti-inflammatory activity of Sarcandra glabra (Thunb.) Nakai polysaccharide was assessed in LPS-induced ARDS mice. MATERIALS AND METHODS: A polysaccharide coded as SERP 30 was obtained by water extraction, alcohol precipitation, and gel filtration. After the physicochemical properties determination and structural characterization, LPS induced-mice ARDS model was used to evaluate the anti-inflammatory and associated antioxidant activities of SERP 30. H&E staining was used to observe the seriousness of lung injury in mice. The ELISA method was used to measure the expression of inflammatory factors (TNF-α and IL-6) in the serum of the mice. The TBA method and the WST-1 method were used to evaluate the oxidative stress injury. Immunohistochemistry was used to distinguish the expression of metalloproteinase-9 (MMP-9), heparinase (HPA), syndecan-1, and decorin in ARDS-mice lung tissue. Western blotting was used to confirm the expression of related proteins in mouse lung tissue. RESULTS: SERP 30 had a potential role in improving lung damage, reducing inflammation, and preventing oxidative stress. Moreover, SERP 30 significantly attenuated the damage to the endothelial glycocalyx and maintained the integrity of the glycocalyx. The western blotting result implied that the main anti-inflammatory mechanism is directed towards NF-κB and MAPK signaling pathways with inhibiting the activation of associated proteins. CONCLUSION: This research provides a theoretical basis for treating ARDS by using a byproduct from food resource.

Docking-guided rational engineering of a macrolide glycosyltransferase glycodiversifies epothilone B.

Glycosyltransferases typically display acceptor substrate flexibility but more stringent donor specificity. BsGT-1 is a highly effective glycosyltransferase to glycosylate macrolides, including epothilones, promising antitumor compounds. Here, we show that BsGT-1 has three major regions significantly influencing the glycodiversification of epothilone B based on structural molecular docking, "hot spots" alanine scanning, and site saturation mutagenesis. Mutations in the PSPG-like motif region and the C2 loop region are more likely to expand donor preference; mutations of the flexible N3 loop region located at the mouth of the substrate-binding cavity produce novel epothilone oligosaccharides. These "hot spots" also functioned in homologues of BsGT-1. The glycosides showed significantly enhanced water solubility and decreased cytotoxicity, although the glycosyl appendages of epothilone B also reduced drug permeability and attenuated antitumor efficacy. This study laid a foundation for the rational engineering of other GTs to synthesize valuable small molecules.

Robust one-pot multi-enzyme polysaccharide remodeling strategy for the synthesis of uniform chondroitin fragments and derivatives.

The non-sulfated chondroitin backbone (CH) is the synthetic precursor of chondroitin sulfate, a linear polysaccharide with dramatic biological functions. Owing to the intrinsic characteristics of the polysaccharide biosynthetic pathway, it is still a challenge to obtain structural-defined glycans via microbial fermentation or enzymatic synthesis, which hindering the illustration of CH polysaccharide functions. Herein, we report a robust one-pot multi-enzyme polysaccharide remodeling strategy to synthesize uniform CH fragments and their derivatives. CH tetrasaccharide, which was obtained from the digestion of heterogeneous CH fragments, was used as the starting material to trigger the assembly of uniform CH fragments in a one-pot multi-enzyme system. This strategy, which combined heteropolymer digestion, sugar nucleotide in situ generation, and sugar chain synchronized polymerization, provides a robust toolbox for structural-defined polysaccharides synthesis.

Chemoenzymatic synthesis of homogeneous chondroitin polymers and its derivatives.

Chondroitin sulfate is a linear glycosaminoglycan widely distributed as an important extracellular matrix component of mammalian cells. It participates in numerous pathological processes, however, illustration of its diverse biological roles is hampered by the unavailability of structurally defined chondroitin polymers and their derivatives. Herein, we report a novel homogeneous chondroitin polymers synthetic strategy which combines stepwise oligosaccharides synthesis with one-pot homogeneous chondroitin chain polymerization. Exogenous trisaccharide was proved to be the necessary acceptor for PmCS-catalyzed homogeneous chondroitin polymers synthetic reactions. The strategy exhibited a well-controlled relationship between the final sugar chain length and the molar ratios of reaction substrates that could synthesize homogenous chondroitin polymers with unprecedented narrow molecular weight distribution. More importantly, the strategy was further expanded to synthesis of unnatural zwitterionic and N-sulfonated chondroitin polymers by incorporation of sugar nucleotide derivatives into the synthetic approach.

Systematic study on the broad nucleotide triphosphate specificity of the pyrophosphorylase domain of the N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12.

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) from Escherichia coli K12 is a bifunctional enzyme that catalyzes both the acetyltransfer and uridyltransfer reactions in the prokaryotic UDP-GlcNAc biosynthetic pathway. In this study, we report the broad substrate specificity of the pyrophosphorylase domain of GlmU during its uridyltransfer reaction and the substrate priority is ranked in the following order: UTP > dUTP > dTTP >> CTP > dATP/dm(6) ATP. This pyrophosphorylase domain of GlmU is also a tool to synthesize UDP-GlcNAc analogs, two examples of which were synthesized herein in multiple mg scale in vitro.

A general strategy for the synthesis of homogeneous hyaluronan conjugates and their biological applications.

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.