Rational design of heparin antagonist: Guanidine-based mimetics unveil key carbohydrate-carbohydrate interactions.

Glycosaminoglycans use positional sulfation to encode binding specificity onto its sequence. Understanding these sulfation patterns constitute a major challenge. Previous studies hinted that sulfate groups can migrate along glycans during collision-induced dissociation in mass spectrometry (MS) experiments, forming isomeric fragments that can lead to incorrect structural assignments. We use ion-mobility - mass spectrometry to investigate the mechanism of this phenomenon in heparin sulfate disaccharides. The sulfate group migrates from the non-reducing to reducing end of the sugar, and the degree of migration does not depend on the structure of the label. The migration product has a sulfate group attached to either 6O- or 3O-position of GlcNAc, and the migration mechanism consists of multiple steps, with the sulfate group first shifting from the iduronic acid to the 6O-position of GlcNAc, and next to the 3O-position. The presented data offer insight into the complexity and unpredictability of sulfated sugar fragmentation in tandem MS and extensive investigations is required to determine whether this represents a singular case or a general phenomenon characteristic of deprotonated sulfated glycans.

The conformation of a glycosaminoglycan (GAG) carbohydrate biopolymer is dependent upon the ring puckering states of its constituent monosaccharide residues and the dihedral angles (φ, ψ) of the glycosidic linkages connecting these residues. In the context of GAGs, the monosaccharide residue iduronate (IdoA; the conjugate base of iduronic acid) is able to take on both chair and boat-like ring pucker states. All-atom explicit-solvent molecular dynamics simulations were applied to determine the extent to which IdoA ring pucker state affects the conformational preferences of (φ, ψ) in 16 different IdoA-containing disaccharides derived from the GAGs heparin/heparan sulfate and dermatan sulfate. Using the extended-system adaptive biasing force (eABF) method, the complete free-energy surface ΔG(φ, ψ) was computed for each disaccharide with its IdoA ring restrained separately to the 1C4, 2SO, B3,O, or 4C1 ring pucker state. Global-minimum ΔG(φ, ψ) values resided within broad ΔG(φ, ψ) basins, and both ring pucker state and sulfation status influenced basin shape and size. Various sulfoforms of the disaccharide IdoAα1–4GlcNS had prominent secondary-minimum basins distinct from the global-minimum basins, and these secondary-minimum basins may manifest as metastable states in standard (nonbiased) molecular dynamics simulations on the 1-microsecond timescale. As such, the present results provide a reference for assessing (φ, ψ) sampling in nonbiased molecular dynamics simulations of GAGs and demonstrate the interplay between IdoA ring puckering, glycosidic linkage dihedral rotation, and sulfation status in contributing to GAG conformational preferences.

We herein report the synthesis and complete conformational analysis of a focused library of iminosugar analogues of l-idose and l-iduronic acid designed to probe the impact of N-substitution on ring dynamics. Molecular modelling, supported by experimental 1H NMR data, reveals that introducing alkyl or acyl substituents on the ring nitrogen profoundly alters the conformational equilibrium, inducing a clear shift from the preferred 1C4 chair to the 4C1 conformation. These findings highlight how targeted nitrogen functionalisation can be used to modulate the conformational landscape of iminosugars, offering new opportunities for the rational design of glycomimetic scaffolds.

The ongoing COVID-19 pandemic, caused by SARS-CoV-2, continues to pose major global health challenges despite extensive vaccination efforts. Variant escape, waning immunity, and reduced vaccine efficacy in immunocompromised populations underscore the urgent need for complementary antiviral therapeutics. Here, we report the design, synthesis, and biological evaluation of precision-engineered dermatan sulfate (DS)-mimetic glycopolymers as multi-targeted inhibitors of SARS-CoV-2. Guided by molecular docking and virtual screening, sulfation at the C2 and C4 positions of iduronic acid was identified as critical for binding to the viral spike protein and inhibiting host and viral enzymes, including heparanase (HPSE) and main protease (Mpro). Chemically synthesized DS disaccharides were covalently grafted onto polymer scaffolds via a post-modification strategy, yielding glycopolymers with well-defined assembly that form uniform nanoparticles under physiological conditions. Surface plasmon resonance and pseudovirus assays revealed strong binding to the viral spike protein (KD ≈ 177 nM), potent viral neutralization, and minimal cytotoxicity. Cellular uptake studies further demonstrated efficient internalization of nanoparticles and intracellular inhibition of HPSE and Mpro. These results establish a modular, non-anticoagulant, and glycosaminoglycan-mimetic platform for the development of broad-spectrum antiviral agents to complement vaccination and enhance preparedness against emerging coronavirus variants.

In Halobacterium salinarum, halophilic archaea that grow in saturated salt solutions, glycoproteins are modified by an N-linked tetrasaccharide that includes iduronic acid (IdoA). This represents the only known example of IdoA, a sugar better known as a component of glycosaminoglycans found in various eukaryal tissues, in N-glycosylation. Although much of the pathway used to assemble this tetrasaccharide is defined, the epimerase that presumably converts glucuronic acid (GlcA) into IdoA had yet to be described. In silico predictions assign this role to VNG1058H. In Hbt. salinarum deleted of VNG1058H, an N-linked tetrasaccharide appeared in which IdoA is replaced by GlcA and greatly reduced sulfation of the last two tetrasaccharide sugars is noted. The absence of VNG1058H, moreover, affects cell physiology. Furthermore, Hbt. salinarum differentially transcribe VNG1058H as a function of growth temperature, suggestive of a response to environmental change. VNG1058H is the first D-glucuronyl C5-epimerase to be identified in Archaea.

ABSTRACTAscidians (Chordata, Tunicata) are model organisms for studying molecules, particularly sulfated glycosaminoglycans (GAGs), due to their phylogenetic proximity to vertebrates and unique GAG variants. Over‐sulfated dermatan sulfate (DS) contributes to proteoglycan diversity and cellular processes in metazoans. Research has identified highly sulfated DS in six ascidian species across two orders: Phlebobranchia (Phallusia nigra and Ciona intestinalis) and Stolidobranchia (Halocynthia pyriformis, Styela plicata, Herdmania pallida, and Halocynthia roretzi). These polymers share a common disaccharide backbone structure [→4 Iduronic Acid (2‐SO₄) β‐1→3 N‐Acetylgalactosamine β‐1→], but show distinct sulfation patterns at N‐acetylgalactosamine residues, suggesting differences in biosynthetic pathways. To investigate DS sulfation patterns, we analyzed sulfotransferase (SULT) gene families through genomic analysis. We sequenced draft genomes of S. plicata (Stolidobranchia), which produces 2,4‐disulfated DS, and P. nigra (Phlebobranchia), which produces 2,6‐disulfated DS. We conducted comparative genomic analysis of 19 additional ascidian species using C. intestinalis as a reference for its SULT repertoire. Phylogenetic analyses revealed 154 novel putative dermatan SULT genes across three enzyme families: uronyl 2‐O‐SULTs, and carbohydrate 4‐O‐ and 6‐O‐SULTs. Analysis of mitochondrial cytochrome c oxidase subunit I (COI) sequences provided insights into evolutionary relationships among taxa. Results indicate that Phlebobranchia represents a basal lineage within Ascidiacea, while Stolidobranchia comprises more recently diverged species. The DS 6‐O‐SULTs gene family originated recently in ascidian evolution, with diversification potentially influenced by lineage‐specific selective pressure. These findings advance our understanding of SULT evolution in tunicates and provide a foundation for future functional studies. SULT genes represent targets for expression profiling and biotechnological applications, including enzyme cloning and characterization in DS biosynthesis. This study provides a genetic framework for understanding and harnessing the biomedical potential of unique sulfated polysaccharides in ascidians.

Rheological behavior of ulvan-based networks: Influence of cations, pH and chemical composition.

Glycosaminoglycans (GAGs) are linear, high molecular weight polydisperse heteropolysaccharides consisting of repeating disaccharide units, which always contain a uronic acid building block (e.g., d-glucuronic acid or l-iduronic acid). Their analogues containing d-mannuronic acid were not known until now. Another important class of the linear negatively charged polisaccharides are alginates, which are also present in the cell surface in the cell wall. They are composed of blocks of 1,4-linked β-d-mannuronic acid and its C-5 epimer α-l-guluronic acid in alternating or random order. Both groups of molecules have significant biological activity (e.g., cell growth inhibitory activity, anti-inflammatory effect, etc.). In the course of our research, we combined the structural characteristics of these two groups of molecules and produced a series of heparan sulphate analogue trisaccharides containing d-mannuronic acid, with a simplified structure, in which α- and β-mannosidic bonds are also found. Since trisaccharides may exert diverse biological effects and alginate derivatives can influence wound healing processes, we investigated the effects of the synthesized compounds on primary human dermal fibroblasts. We found that, when applied at 10 μM, none of the compounds influenced viability or spontaneous collagen production; however, some derivatives exhibited anti-inflammatory activity and suppressed the poly(I:C)-induced release of interleukin 6.

Mechanisms and key factors influencing ulvan gelation.

Heparan sulfate (HS) is a linear, highly sulfated, and heterogeneous polysaccharide that covalently attaches to core proteins to form heparan sulfate proteoglycans (HSPGs). HSPGs are widely expressed in mammalian cells and are found on the cell surface and within the extracellular matrix (ECM). Structurally, HS consists of repeating disaccharide units composed of hexuronic acid (HexA) (either glucuronic acid (GlcA) or iduronic acid (IdoA)) linked to glucosamine (GlcN) units. The HS chain undergoes extensive post-polymerization modifications, including N-deacetylation of GlcN, C5-epimerization of HexA, and sulfation at various positions like 2-O-sulfation of HexA, as well as 3-O-, 6-O-, and N-sulfation of GlcN. Among these modifications, 3-O-sulfation of HS, produced by HS 3-O-sulfotransferase (HS3OST), is the rarest and most functionally significant. While 3-O-sulfated HS is well known for its anticoagulant properties through the activation of antithrombin, it also plays a critical role in various physiological and pathological processes, including cell differentiation, cancer progression, herpes simplex virus entry, and neuronal development. However, the precise mechanisms underlying these functions and their pathological implications remain inadequately characterized. This knowledge gap is primarily due to the low abundance of 3-O-sulfated HS and the lack of standardized analytical methods for its detection in biological samples. In this review, we summarize recent advancements in analytical techniques for the analysis of 3-O-sulfated HS and highlight potential future directions to improve its characterization and advance our understanding of its biological roles.

The carbon and oxygen atoms of tetrahydropyran form the common substructure of pyranose monosaccharides in vertebrate glycans. This substructure can assume various ring puckering chair and skew-boat conformations, and can thereby impact glycan conformations relevant for biomolecular structure and signaling. The Protein Data Bank (PDB) provides a wealth of experimental glycan structural biology data that can be useful in the development and validation of molecular mechanics force fields for these molecules. However, these experimental data are typically from solvent-depleted crystalline environments at very low temperatures, in contrast to biological conditions that are aqueous and near ambient temperature, which is the regime targeted by biomolecular force fields. To determine if these PDB X-ray crystal data can be of utility as references for carbohydrate force fields, we compared ring puckering conformations from these experimental data to both vacuum and explicit aqueous solvent puckering free energy data from extended-system adaptive biasing force (eABF) molecular dynamics simulations using the previously validated CHARMM36 force field. We found that, for monosaccharides that are not charged (glucose, N-acetylglucosamine, galactose, N-acetylgalactosamine, mannose, xylose, and fucose), both the vacuum and aqueous simulation puckering preferences strongly correlate with PDB data, and therefore with each other. In contrast, all charged monosaccharides that were considered (the conjugate bases of N-acetylneuraminic acid, glucuronic acid, and iduronic acid) had puckering preferences correlating with PDB data only in aqueous simulations and not in vacuum simulations. These results suggest that comparing puckering preferences from aqueous simulations to PDB X-ray crystal puckering conformation data can be a valid and useful component of carbohydrate force field development and validation.

The study evaluated the chemical composition, molecular weight, and antioxidant activity of ulvan extracted from Ulva papenfussii and Ulva lactuca. The results revealed distinct differences in the monosaccharide composition, sulfate content, and uronic acid levels between ulvans from both species, with ulvan from U. papenfussii exhibiting higher levels of iduronic acid and sulfate (16.39 ± 0.45 %w/w and 28.46 + 3.63 %w/w). Ulvan was fractionated using anion exchange chromatography (DEAE) based on charge differences, yielding three main fractions from each species. This study is the first to analyze different ulvan fractions from Vietnamese green algae using the anion exchange chromatography method. The antioxidant activity of the ulvan fractions was determined by their ability to DPPH radical scavenging activity, reducing power activity, and antioxidant activity, with UL2 (29.5 ± 0.94 Sc%) and UP2 (25.9 ± 1.77 Sc%) demonstrating the most prominent results. These active fractions were characterized by higher sulfate and uronic acid content, suggesting a potential structure–activity relationship. These findings highlight the potential of ulvan in the food and pharmaceutical industries.

Heparan sulfate 6-endosulfatases (SULFs) remove 6-O-sulfo groups from heparan sulfate polysaccharide chains. SULFs modify the functions of heparan sulfate and contribute to the development of cancers, organ development and endothelial inflammatory responses. However, direct measurement of the activity of SULFs from human and mouse plasma is not currently possible. Here, we report a liquid chromatography coupled with tandem mass spectrometry (LS-MS/MS) assay to measure the activity of SULFs. The method uses a structurally homogeneous heparan sulfate dodecasaccharide (12-mer) in which the glucuronic and iduronic acid residues are labeled with both 13C- and 2H-atoms. The 12-mers desulfated by the SULFs is subjected to degradation with heparin lyases to yield disaccharides, which is followed by LC-MS/MS. The amount of two specific disaccharides, ΔIIIS and ΔIVS, quantified by LC-MS/MS reports the activity of the SULFs with high sensitivity and specificity. This method allows for the determination of the activity from conditioned cell media and mouse plasma. Our findings offer an essential novel tool to delineate many roles of SULFs in biological processes.

Like essentially all Archaea, the halophile Halobacterium salinarum performs N-glycosylation, namely, the covalent attachment of a glycan moiety to select asparagine residues in a target protein. Moreover, Hbt. salinarum represents one of the few current archaeal examples in which the pathway of N-glycosylation has been largely defined. Still, several components of this pathway remain to be defined, including the sulfotransferase(s) responsible for modifying the iduronic acid and glucuronic acid corresponding to the third and final sugars of the N-linked tetrasaccharide that decorates glycoproteins in this haloarchaeon. In the present report, a series of bioinformatics, genetic, biochemical, and structural approaches served to reveal how membrane-associated VNG1056C and soluble VNG1057C respectively sulfate the iduronic acid at tetrasaccharide position three and the terminal glucuronic acid, seemingly independent of each other. The need for two different enzymes reflects the sulfation of these sugars at distinct positions.

Herein, we report the synthesis of heparan sulfate (HS) proteoglycan mimetics bearing iduronic acid (IdoA) and sulfated L-idose (Ido) hexasaccharides to assess how these isostructural sugars with similar charge density influence neoproteoglycan display on the cell membrane. PG@I2, carrying sulfated L-idose, showed rapid internalization in both cancerous and normal cells, whereas PG@I1, containing native IdoA expressed on the cell membrane and slowly internalized, underscoring the role of IdoA in HSPG cell surface engineering.

Amphiphilic glycopeptides and glycopolymers have been characterized as potential glycocalyx engineering probes to regulate growth factors mediated stem cell differentiation and neural plasticity. Here, we describe a rational and facile method to synthesize fluorescent tagged amphiphilic multivalent glycoprobes using solid-phase peptide synthesizer and copper-free click chemistry. As a prototype, we report the synthesis of fluorescently tagged penta-valent sulfated L-iduronic acid glycopeptide for cell surface engineering and imaging.

Neural net analysis of NMR spectra from strongly-coupled spin systems

The Triggering Receptor Expressed on Myeloid Cells-2 (TREM2), a pivotal innate immune receptor, orchestrates functions such as inflammatory responses, phagocytosis, cell survival, and neuroprotection. TREM2 variants R47H and R62H have been associated with Alzheimer's disease, yet the underlying mechanisms remain elusive. Our previous research established that TREM2 binds to heparan sulfate (HS) and variants R47H and R62H exhibit reduced affinity for HS. Building upon this groundwork, our current study delves into the interplay between TREM2 and HS and its impact on microglial function. We confirm TREM2's binding to cell surface HS and demonstrate that TREM2 interacts with HS, forming HS-TREM2 binary complexes on microglia cell surfaces. Employing various biochemical techniques, including Surface Plasmon Resonance, low molecular weight HS microarray screening, and serial HS mutant cell surface binding assays, we demonstrate TREM2's robust affinity for HS, and the effective binding requires a minimum HS size of approximately 10 saccharide units. Notably, TREM2 selectively binds specific HS structures, with 6-O-sulfation and, to a lesser extent, the iduronic acid residue playing crucial roles. N-sulfation and 2-O-sulfation are dispensable for this interaction. Furthermore, we reveal that 6-O-sulfation is essential for HS-TREM2 ternary complex formation on the microglial cell surface, and HS and its 6-O-sulfation are necessary for TREM2-mediated ApoE3 uptake in microglia. By delineating the interaction between HS and TREM2 on the microglial cell surface and demonstrating its role in facilitating TREM2-mediated ApoE uptake by microglia, our findings provide valuable insights that can inform targeted interventions for modulating microglial functions in Alzheimer's disease.

Herein, we report the synthesis and biological evaluation of a novel series of heparinoid amphiphiles as inhibitors of heparanase and SARS-CoV-2. By employing a tailor-made synthetic strategy, a library of highly sulfated homo-oligosaccharides bearing d-glucose or a C5-epimer (i.e., l-idose or l-iduronic acid) conjugated with various lipophilic groups was synthesized and investigated for antiviral activity. Sulfated higher oligosaccharides of d-glucose or l-idose with lipophilic aglycones displayed potent anti-SARS-CoV-2 and antiheparanse activity, similar to or better than pixatimod (PG545), and were more potent than their isosteric l-iduronic acid congeners. Lipophilic groups such as cholestanol and C18-aliphatic substitution are more advantageous than functional group appended lipophilic moieties. These findings confirm that fine-tuning of higher oligosaccharides, degree of sulfation, and lipophilic groups can yield compounds with potent anti-SARS-CoV-2 activity.