Hyaluronic acid (HA) is a linear high molecular weight polymer ubiquitously distributed in humans and animals. The D-glucuronic acid and N-acetyl-D-glucosamine repeating disaccharide backbone along with variable secondary and tertiary structures endows HA with unique rheological characteristics as well as diverse biological functions such as maintaining tissue homeostasis and mediating cell functions. Due to its excellent biocompatibility, biodegradability, viscoelasticity and moisturizing properties, natural HA and its chemically modified derivatives are widely used in medical, pharmaceutical, food and cosmetic industries. For broad application purposes, abundant HA-based biochemical products have been developed, including the methodologies for characterization of these products. This review provides an overview focusing on the methods used for determining HA structure as well as the strategies for constructing its derivatives. Apart from the analytical approaches for defining the physicochemical properties of HA (e.g., molecular weight, rheology and swelling capacity), quantitative methods for assessing the purity of HA-based materials are discussed. In addition, the biological functions and potential applications of HA and its derivatives are briefly embarked and perspectives in methodological development are discussed.

Natural heparin, a glycosaminoglycan consisting of repeating hexuronic acid and glucosamine linked by 1 → 4 glycosidic bonds, is the most widely used anticoagulant. To subvert the dependence on animal sourced heparin, alternative methods to produce heparin saccharides, i.e., either heterogenous sugar chains similar to natural heparin, or structurally defined oligosaccharides, are becoming hot subjects. Although the success by chemical synthesis of the pentasaccharide, fondaparinux, encourages to proceed through a chemical approach generating homogenous product, synthesizing larger oligos is still cumbersome and beyond reach so far. Alternatively, the chemoenzymatic pathway exhibited exquisite stereoselectivity of glycosylation and regioselectivity of modification, with the advantage to skip the tedious protection steps unavoidable in chemical synthesis. However, to a scale of drug production needed today is still not in sight. In comparison, a procedure of de novo biosynthesis in an organism could be an ultimate goal. The main purpose of this review is to summarize the current available/developing strategies and techniques, which is expected to provide a comprehensive picture for production of heparin saccharides to replenish or eventually to replace the animal derived products. In chemical and chemoenzymatic approaches, the methodologies are discussed according to the synthesis procedures: building block preparation, chain elongation, and backbone modification.

Heparanase is elevated in various pathological conditions, primarily cancer and inflammation. To investigate the significance and involvement of heparanase in liver fibrosis, we compared the susceptibility of wild-type (WT) and heparanase-overexpressing transgenic (Hpa-tg) mice to carbon tetrachloride (CCL4)-induced fibrosis. In comparison with WT mice, Hpa-tg mice displayed a severe degree of tissue damage and fibrosis, including higher necrotic tendency and intensified expression of smooth muscle actin. While damage to the WT liver started to recover after the acute phase, damage to the Hpa-tg liver was persistent. Recovery was attributed, in part, to heparanase-stimulated autophagic activity in response to CCL4, leading to increased apoptosis and necrosis. The total number of stellate cells was significantly higher in the Hpa-tg than the WT liver, likely contributing to the increased amounts of lipid droplets and smooth muscle actin. Our results support the notion that heparanase enhances inflammatory responses, and hence may serve as a target for the treatment of liver damage and fibrosis.

The spike (S) protein on the surface of the SARS-CoV-2 virus is critical to mediate fusion with the host cell membrane through interaction with angiotensin-converting enzyme 2 (ACE2). Additionally, heparan sulfate (HS) on the host cell surface acts as an attachment factor to facilitate the binding of the S receptor binding domain (RBD) to the ACE2 receptor. Aiming at interfering with the HS-RBD interaction to protect against SARS-CoV-2 infection, we have established a pentasaccharide library composed of 14,112 compounds covering the possible sulfate substitutions on the three sugar units (GlcA, IdoA, and GlcN) of HS. The library was used for virtual screening against RBD domains of SARS-CoV-2. Molecular modeling was carried out to evaluate the potential antiviral properties of the top-hit pentasaccharide focusing on the interactive regions around the interface of RBD-HS-ACE2. The lead pentasaccharide with the highest affinity for RBD was analyzed via drug-likeness calculations, showing better predicted druggable profiles than those currently reported for RBD-binding HS mimetics. The results provide significant information for the development of HS-mimetics as anti-SARS-CoV-2 agents.

Introduction: In this study, we used an approach by conjugating Fondaparinux Sodium (FS) with selected drugs to generate proteolysis-targeting chimeras (PROTACs).Methods: By applying bioprocess engineering principles, the direct amidation reaction was optimized –through precise control of pH, substrate ratios, and solvent selection –to reliably produce high‐purity (>99%) PROTAC molecules on a scalable platform. Surface plasmon resonance (SPR) analysis demonstrated that the synthesized PROTACs exhibit micromolar binding affinities (KD ≈ 10^–6 M) toward inflammatory mediators RANTES (CCL5) and interleukin-6 (IL-6). In vitro assays using peripheral blood mononuclear cells (PBMCs) revealed that two candidate compounds (Product 6 and Product 10) significantly inhibited lipopolysaccharide (LPS)‐induced interleukin‐1β (IL‐1β) release in a concentration-dependent manner, while FS and the drugs alone had no effect.Results: High-purity (>99%) PROTAC molecules were produced on a scalable platform. The synthesized PROTACs demonstrated micromolar binding affinities (KD ≈ 10^–6 M) toward RANTES (CCL5) and IL-6. Two candidate compounds (Product 6 and Product 10) significantly inhibited LPS-induced IL-1β release in PBMCs in a concentration-dependent manner; FS and the drugs alone showed no effect.Discussion: These findings not only provide an innovative strategy for targeting “undruggable” proteins but also establish a robust, scalable process for the production of PROTAC‐based anti-inflammatory agents.

Polyethyleneglycol diglycidyl ether (PEGDE) is a new generation of cross-linker with the advantages of low toxicity and improved elasticity. The physicochemical properties of PEGDE-HA are closely related to its degree of modification (MoD), effective cross-linker ratio (CrR), as well as modification types/positions. In this study, we developed a structural analysis method for comprehensive characterization of PEGDE cross-linked HA. Combined HPLC-MS/MS analysis accurately measures the parameters of MoD and CrR, and ambiguously locates modification sites/residues. Using the established method, we have, for the first time, identified the existence of intra-chain cross-linked structures in the hydrogel and revealed the difference in structures and rheological properties of hydrogels synthesized under different conditions. The insights obtained from this analytical method can be applied to guide the manufacturing process to generate HA hydrogel of “tailored” structures for different application scenarios.