Sulfate Hexasaccharide Research Articles

Well-Defined Heparin Mimetics Can Inhibit Binding of the Trimeric Spike of SARS-CoV-2 in a Length-Dependent Manner.

The emergence of new SARS-CoV-2 variants and the dangers of long-covid necessitate the development of broad-acting therapeutics that can reduce viral burden. SARS-CoV-2 employs heparan sulfate (HS) as an initial cellular attachment factor, and therefore, there is interest in developing heparin as a therapeutic for SARS-CoV-2. Its use is, however, complicated by structural heterogeneity and the risk of causing bleeding and thrombocytopenia. Here, we describe the preparation of well-defined heparin mimetics by a controlled head-to-tail assembly of HS oligosaccharides having an alkyne or azide moiety by copper-catalyzed azide-alkyne cycloaddition (CuAAC). Alkyne- and azide-containing sulfated oligosaccharides were prepared from a common precursor by modifying an anomeric linker with 4-pentynoic acid and by enzymatic extension with an N-acetyl-glucosamine having an azide moiety at C-6 (GlcNAc6N3), respectively, followed by CuAAC. The process of enzymatic extension with GlcNAc6N3 followed by CuAAC with the desired alkyne-containing oligosaccharides could be repeated to give compounds composed of 20 and 27 monosaccharides, respectively. The heparin mimetics could inhibit the binding of the SARS-CoV-2 spike or RBD to immobilized heparin or to Vero E6 cells. The inhibitory potency increased with increasing chain length, and a compound composed of four sulfated hexasaccharides linked by triazoles had a similar potency as unfractionated heparin. Sequence analysis and HS microarray binding studies with a wide range of RBDs of variants of concern indicate that they have maintained HS-binding capabilities and selectivities. The heparin mimetics exhibit no or reduced binding to antithrombin-III and platelet factor 4, respectively, which are associated with side effects.

Molecular dynamics-based descriptors of 3-O-Sulfated Heparan sulfate as contributors of protein binding specificity

Glycosaminoglycans are linear periodic and anionic polysaccharides found in the extracellular matrix, involved in a range of key biochemical processes as a result of their interactions with a variety of protein partners. Due to the template-less synthesis, high flexibility and charge of GAGs, as well as the multipose binding of GAG ligands to receptors, the specificity of GAG-protein interactions can be difficult to elucidate. In this study we propose a set of MD-based descriptors of unbound Heparan Sulfate hexasaccharides that can be used to characterize GAGs and explain their binding affinity to a set of protein receptors. With the help of experimental data on GAG-protein binding affinity, we were able to further characterize the nature of this interaction in addition to providing a basis for predictor functions of GAG-protein binding specificity.

Automated Synthesis of Chondroitin Sulfate Oligosaccharides.

Glycosaminoglycans (GAGs) are important sulfated carbohydrates prevalently found in the extracellular matrix that serve many biological functions. The synthesis of structurally diverse but defined GAGs is extremely challenging as one has to account for the various sulfation patterns. Described is the automated synthesis of chondroitin sulfate hexasaccharides on a solid support equipped with a photolabile linker. The linker cleavage from the resin is performed in a continuous-flow photoreactor under chemically mild conditions. This approach serves as a general scheme to access oligosaccharides of all GAG families.

Lipophilic Modification of an Anti‐Cancer Stem Cell Agent Improves Pharmacokinetic and Anti‐Cancer Properties

Current anti‐cancer agents target bulk tumor cells but do not eradicate the rare, quiescent, cancer stem cells (CSCs). CSCs are being increasingly recognized as the reason for cancer relapse. Targeting CSCs has become a major area of interest in the field of anti‐cancer drug discovery and several clinical trials focused on CSC targeting are in progress. Previously, we identified a CSC selective agent G2.2 (Fig. 1), which inhibited CSC spheroidal growth with a median IC50 (±1SEM) s of 193 μM±58 μM. G2.2 was found to exhibit robust anti‐CSC properties in colorectal cancer xenograft models in vivo. Mechanistically, G2.2 is a mimetic of heparan sulfate hexasaccharide (HS06) and is perhaps the only non‐saccharide heparan sulfate mimetic identified to date to exhibit considerable anti‐cancer promise. Preliminary pharmacokinetic (PK) studies showed that G2.2 exhibited a half‐life of nearly 4 hours following IV administration and 6% bioavailability following oral administration in rats.To improve upon G2.2's PK properties, we hypothesized lipid‐modification of G2.2 would enhance penetration across GI barrier while also simultaneously enhancing its potency through better targeting of plasma‐membrane growth factor receptors – the presumed targets of G2.2 and HS06. Several lipid‐modified agents were synthesized. These modifications resulted in improvement of PK properties (bioavailability by greater than 4‐fold). Further, the analogs also showed up to ~ 53‐fold (NCI‐H508 spheroids) enhanced inhibition potency against CSCs in in‐vitro spheroid cell‐based assays. In fact, these lipid‐modified G2.2 analogs inhibited several (>10) colorectal cancer spheroids with median IC50 values (±1SEM) ranging from 8 μM±3μM to 68 μM±14μM. Considering that G2.2 is a mimetic of heparan sulfate, these results present a simple avenue to enhance drug‐like properties of such highly charged agents. The results also suggest that the lipid‐modified analogs may also serve as probes of molecular processes governing the CSC phenotype.Support or Funding InformationThis work was funded by the NHLBI grant 1P01HL107152 given to Umesh R. Desai.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular dynamics simulations of the chemokine CCL2 in complex with pull down-derived heparan sulfate hexasaccharides

BackgroundBinding of chemokines to glycosaminoglycans (GAGs) is a crucial step in leukocyte recruitment to inflamed tissues. MethodsA disaccharide compositional analysis of the HS dp6 fraction in combination with MS analysis of the CCL2-depleted dp6 fraction was the basis for target GAG ligand structure suggestions. Four experimentally-derived heparan sulfate hexasaccharides, two potentially chemokine-specific and two unspecific, have been docked to CCL2. Subsequent 300 ns molecular dynamics simulations were used to improve the docked complexes. ResultsHexasaccharides with four sulfations and no acetylations are suggested for selective and high affinity chemokine binding. Using the Antithromin-III/heparin complex as positive control for docking, we were able to recover the correct complex structure only if the previously liganded ATIII structure was used as input. Since the liganded structure is not known for a CCL2-GAG complex, we investigated if molecular dynamics simulations could improve initial docking results. We found that all four GAG oligosaccharides ended up in close contact with the known binding residues after about 100 ns simulation time. ConclusionsA discrimination of specific vs. unspecific CCL2 GAG ligands is not possible by this approach. Long-time molecular dynamics simulations are, however, well suited to capture the delicate enthalpy/entropy balance of GAG binding and improve results obtained from docking. General significanceWith the comparison of two methods, MS-based ligand identification and molecular modelling, we have shown the current limitations of our molecular understanding of complex ligand binding which is could be due to the numerical inaccessibility of ligand-induced protein conformational changes.

Conformational Transitions in 3D Model of Bovine Testicular Hyaluronidase during Molecular Docking with Glycosaminoglycan Ligands

In silico molecular docking of the trimer repeating unit of chondroitin sulfate (sulfated hexasaccharide) and tetramer repeating unit of heparin (sulfated octasaccharide) to the 3D model of bovine testicular hyaluronidase by the methods of computational chemistry demonstrated the presence of eight significant binding sites for these ligands (cs1–cs8). The interaction of the active site of the enzyme with the heparin ligand, which inactivates the enzyme, and the protective effect of the chondroitin sulfate ligands bound to the surface sites of the biocatalyst molecule were theoretically studied using calculation approaches. We sequentially determined binding sites for the chondroitin sulfate ligands (in positions cs2, cs4, cs7, cs8 or cs1, cs2, cs4, cs7, cs8) critical for the protein structure stabilization, whose occupancy is theoretically sufficient to prevent irreversible deformations of the enzyme molecule when the heparin ligand is introduced into the cavity of its active site. Theoretical detection of these ‘sensibility points’ on the hyaluronidase globule indicates the possibility of regulating its functioning under the binding of the glycosaminoglycan ligands that initiate the fine formation of an effective type of the surface electrostatic potential. The interaction of the glycosaminoglycan ligands with hyaluronidase is mainly determined by electrostatic forces.

Cladolosides I1, I2, J1, K1, K2 and L1, monosulfated triterpene glycosides with new carbohydrate chains from the sea cucumber Cladolabes schmeltzii

Six new monosulfated triterpene glycosides, cladolosides I1 (1), I2 (2), J1 (3), K1 (4), K2 (5) and L1 (6) were isolated from the tropical Indo-West Pacific sea cucumber Cladolabes schmeltzii (Cladolabinae, Sclerodactylidae, Dendrochirotida). Structures of these glycosides were elucidated by 2D NMR spectroscopy and mass spectrometry. Four new types of carbohydrate chains have been found in 1–6. Cladolosides of the groups I and J are characterized by pentasaccharide carbohydrate chains sulfated at a terminal 3-O-methylglucose residue and branched by the position 4 of the first xylose residue, but differing from each other in the lengths of the main and side carbohydrate chains. Cladolosides of the groups K and L contain hexasaccharide chains with different positions of a sulfated terminal 3-O-methylglucose residue (as the fourth or the sixth monosaccharide residue). Sulfated hexasaccharide carbohydrate chains were found in the sea cucumbers glycosides for the first time. A pentasaccharide carbohydrate chain of cladoloside J1 (3) having a disaccharide moiety of glucose and a sulfated 3-O-methylglucose linked to the first xylose residue in a linear trisaccharide fragment is also unusual. All substances studied demonstrated strong or moderate hemolytic and cytotoxic effects.

A Hexasaccharide Containing Rare 2‐O‐Sulfate‐Glucuronic Acid Residues Selectively Activates Heparin Cofactor II

AbstractGlycosaminoglycan (GAG) sequences that selectively target heparin cofactor II (HCII), a key serpin present in human plasma, remain unknown. Using a computational strategy on a library of 46 656 heparan sulfate hexasaccharides we identified a rare sequence consisting of consecutive glucuronic acid 2‐O‐sulfate residues as selectively targeting HCII. This and four other unique hexasaccharides were chemically synthesized. The designed sequence was found to activate HCII ca. 250‐fold, while leaving aside antithrombin, a closely related serpin, essentially unactivated. This group of rare designed hexasaccharides will help understand HCII function. More importantly, our results show for the first time that rigorous use of computational techniques can lead to discovery of unique GAG sequences that can selectively target GAG‐binding protein(s), which may lead to chemical biology or drug discovery tools.

A Hexasaccharide Containing Rare 2-O-Sulfate-Glucuronic Acid Residues Selectively Activates Heparin Cofactor II.

Glycosaminoglycan (GAG) sequences that selectively target heparin cofactor II (HCII), a key serpin present in human plasma, remain unknown. Using a computational strategy on a library of 46 656 heparan sulfate hexasaccharides we identified a rare sequence consisting of consecutive glucuronic acid 2‐O‐sulfate residues as selectively targeting HCII. This and four other unique hexasaccharides were chemically synthesized. The designed sequence was found to activate HCII ca. 250‐fold, while leaving aside antithrombin, a closely related serpin, essentially unactivated. This group of rare designed hexasaccharides will help understand HCII function. More importantly, our results show for the first time that rigorous use of computational techniques can lead to discovery of unique GAG sequences that can selectively target GAG‐binding protein(s), which may lead to chemical biology or drug discovery tools.

In Sanfilippo syndrome, heparan sulfate hexasaccharides are the most pathogenic fractions involved in glia activation.

In Sanfilippo syndrome, heparan sulfate hexasaccharides are the most pathogenic fractions involved in glia activation.

Heparan sulfate hexasaccharide selectively inhibits cancer stem cells self-renewal by activating p38 MAP kinase.

Heparan sulfate (HS) plays a role in the majority of essential hallmarks of cancer, yet its ability to modulate self-renewal, especially of cancer stem cells (CSCs), remains unknown. We have discovered that a non-anticoagulant HS hexasaccharide (HS06) sequence, but not other shorter or longer sequences, selectively inhibited CSC self-renewal and induced apoptosis in colorectal, pancreatic, and breast CSCs suggesting a very general phenomenon. HS06 inhibition of CSCs relied upon early and sustained activation of p38α/β mitogen activated protein kinase (MAPK) but not other MAPKs family members i.e. ERK and JNK. In contrast, polymeric HS induced exactly opposite changes in MAPK activation and failed to inhibit CSCs. In fact, TCF4 signaling, a critical regulator of CSC self-renewal, was inhibited by HS06 in a p38 activation dependent fashion. In conclusion, HS06 selectively inhibits CSCs self-renewal by causing isoform specific activation of p38MAPK to inhibit TCF4 signaling. These observations on chain length-induced specificity carry major mechanistic implications with regard to HS in cancer biology, while also presenting a novel paradigm for developing novel anti-CSC hexasaccharides that prevent cancer relapse.

ChemInform Abstract: Automated Synthesis of Chondroitin Sulfate Oligosaccharides

AbstractReview: 15 refs.

Nuclear Magnetic Resonance Insight into the Multiple Glycosaminoglycan Binding Modes of the Link Module from Human TSG-6.

Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA)-binding protein that is essential for stabilizing and remodeling the extracellular matrix (ECM) during ovulation and inflammatory disease processes such as arthritis. The Link module, one of the domains of TSG-6, is responsible for binding hyaluronan and other glycosaminoglycans found in the ECM. In this study, we used a well-defined chondroitin sulfate (CS) hexasaccharide (ΔC444S) to determine the structure of the Link module, in solution, in its chondroitin sulfate-bound state. A variety of nuclear magnetic resonance techniques were employed, including chemical shift perturbation, residual dipolar couplings (RDCs), nuclear Overhauser effects, spin relaxation measurements, and paramagnetic relaxation enhancements from a spin-labeled analogue of ΔC444S. The binding site for ΔC444S on the Link module overlapped with that of HA. Surprisingly, ΔC444S binding induced dimerization of the Link module (as confirmed by analytical ultracentrifugation), and a second weak binding site that partially overlapped with a previously identified heparin site was detected. A dimer model was generated using chemical shift perturbations and RDCs as restraints in the docking program HADDOCK. We postulate that the molecular cross-linking enhanced by the multiple binding modes of the Link module might be critical for remodeling the ECM during inflammation/ovulation and might contribute to other functions of TSG-6.

Interactions of the Chemokine CCL5/RANTES with Medium-Sized Chondroitin Sulfate Ligands

Interactions of the chemokine CCL5 (RANTES) with glycosaminoglycans (GAGs) are crucial to the CCL5-mediated inflammation process. However, structural information on interactions between CCL5 and longer GAG fragments is lacking. In this study, the interactions between oligosaccharides derived from chondroitin sulfate and a dimeric variant of CCL5 were investigated using solution nuclear magnetic resonance. The data indicate that, in addition to the BBXB motif in the 40s loop, GAGs also contact residues in the N loop in a manner similar to interactions between chemokine and the receptor N terminus, leading to possible stabilization of the dimer. Using 2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl-tagged hexasaccharides, the binding orientation of the hexasaccharides was shown to be highly dependent on thesulfation pattern of the N-acetyl galactosamine groups. Finally, a model of the CCL5 dimer complexed to chondroitin sulfate hexasaccharides was constructed using paramagnetic relaxation enhancement and intra- and intermolecular nuclear Overhauser effect constraints.

Automated synthesis of chondroitin sulfate oligosaccharides.

Glycosaminoglycans (GAGs) are important sulfated carbohydrates prevalently found in the extracellular matrix that serve many biological functions. The synthesis of structurally diverse but defined GAGs is extremely challenging as one has to account for the various sulfation patterns. Described is the automated synthesis of two chondroitin sulfate hexasaccharides. The oligosaccharides are prepared on a solid support that is equipped with a photolabile linker. The linker cleavage from the resin is performed in a continuous-flow photoreactor under chemically mild conditions. The described approach will serve as a general scheme to systematically access oligosaccharides of all GAG families.

Chemoenzymatic synthesis and structural characterization of 2-O-sulfated glucuronic acid-containing heparan sulfate hexasaccharides.

Heparan sulfate and heparin are highly sulfated polysaccharides that consist of a repeating disaccharide unit of glucosamine and glucuronic or iduronic acid. The 2-O-sulfated iduronic acid (IdoA2S) residue is commonly found in heparan sulfate and heparin; however, 2-O-sulfated glucuronic acid (GlcA2S) is a less abundant monosaccharide (∼<5% of total saccharides). Here, we report the synthesis of three GlcA2S-containing hexasaccharides using a chemoenzymatic approach. For comparison purposes, additional IdoA2S-containing hexasaccharides were synthesized. Nuclear magnetic resonance analyses were performed to obtain full chemical shift assignments for the GlcA2S- and IdoA2S-hexasaccharides. These data show that GlcA2S is a more structurally rigid saccharide residue than IdoA2S. The antithrombin (AT) binding affinities of a GlcA2S- and an IdoA2S-hexasaccharide were determined by affinity co-electrophoresis. In contrast to IdoA2S-hexasaccharides, the GlcA2S-hexasaccharide does not bind to AT, confirming that the presence of IdoA2S is critically important for the anticoagulant activity. The availability of pure synthetic GlcA2S-containing oligosaccharides will allow the investigation of the structure and activity relationships of individual sites in heparin or heparan sulfate.

Chemoenzymatic synthesis and structural characterization of 2-O-sulfated glucuronic acid-containing heparan sulfate hexasaccharides

Chemoenzymatic synthesis and structural characterization of 2-O-sulfated glucuronic acid-containing heparan sulfate hexasaccharides

Highly sulfated hexasaccharide sequences isolated from chondroitin sulfate of shark fin cartilage: Insights into the sugar sequences with bioactivities

Chondroitin sulfate (CS) chains regulate the development of the central nervous system in vertebrates and are linear polysaccharides consisting of variously sulfated repeating disaccharides, [-4GlcUAβ1-3GalNAcβ1-](n), where GlcUA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively. CS chains containing D-disaccharide units [GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)] are involved in the development of cerebellar Purkinje cells and neurite outgrowth-promoting activity through interaction with a neurotrophic factor, pleiotrophin, resulting in the regulation of signaling. In this study, to obtain further structural information on the CS chains containing d-disaccharide units involved in brain development, oligosaccharides containing D-units were isolated from a shark fin cartilage. Seven novel hexasaccharide sequences, ΔO-D-D, ΔA-D-D, ΔC-D-D, ΔE-A-D, ΔD-D-C, ΔE-D-D and ΔA-B-D, in addition to three previously reported sequences, ΔC-A-D, ΔC-D-C and ΔA-D-A, were isolated from a CS preparation of shark fin cartilage after exhaustive digestion with chondroitinase AC-I, which cannot act on the galactosaminidic linkages bound to D-units. The symbol Δ stands for a 4,5-unsaturated bond of uronic acids, whereas A, B, C, D, E and O represent [GlcUA-GalNAc(4-O-sulfate)], [GlcUA(2-O-sulfate)-GalNAc(4-O-sulfate)], [GlcUA-GalNAc(6-O-sulfate)], [GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)], [GlcUA-GalNAc(4-O-, 6-O-sulfate)] and [GlcUA-GalNAc], respectively. In binding studies using an anti-CS monoclonal antibody, MO-225, the epitopes of which are involved in cerebellar development in mammals, novel epitope structures, ΔA-D-A, ΔA-D-D and ΔA-B-D, were revealed. Hexasaccharides containing two consecutive D-units or a B-unit will be useful for the structural and functional analyses of CS chains particularly in the neuroglycobiological fields.

Understanding Glycosaminoglycan – Protein Specificity Through Computational Approaches

Glycosaminoglycans – protein interactions modulate a number of physiological processes such as coagulation, immune response, angiogenesis, morphogenesis and pathogen infection. Yet, a majority of these interactions involve allosteric regulation of protein and are poorly understood. We have developed a genetic algorithm‐based combinatorial virtual screening approach to understand the nature of interaction of glycosaminoglycans with proteins. A dual‐filter strategy employing an ‘affinity’ filter followed by a ‘specificity’ filter rapidly sorted the combinatorial library into distinct groups of sequences. Application of this strategy on a library of 46,000 sequences resulted in identification of only five sequences that recognize heparin cofactor II with ‘high‐affinity and high‐specificity’. Likewise, the strategy led to the prediction of a novel geometry for dermatan sulfate hexasaccharide onto heparin cofactor II, which explained the formation of the ternary glycosaminoglycan – heparin cofactor II – protease complex. The strategy represents a general approach for understanding specificity of allosteric glycosaminoglycan – protein interactions and may lead to the design of novel allosteric agonists or antagonists of proteins.

Sulfated Hexasaccharides Attenuate Metastasis by Inhibition of P-selectin and Heparanase

Development of compounds that target both heparanase and selectins is emerging as a promising approach for cancer therapy. Selectins are vascular cell adhesion molecules that mediate tumor cell interactions with platelets, leukocytes, and the vascular endothelium. Heparanase is an endoglycosidase that degrades heparan sulfate in the tumor microenvironment, cell surfaces, and vessel wall. Acting together, these molecules facilitate tumor cell arrest, extravasation, and metastasis. Here, we report the preparation of novel semisynthetic sulfated tri mannose C-C-linked dimers (STMCs) endowed with heparanase and selectin inhibitory activity. The P-selectin specificity of the STMC was defined by the anomeric linkage of the C-C bond. This STMC hexasaccharide is an effective inhibitor of P-selectin in vivo. We show that selective inhibition of heparanase attenuates metastasis in B16-BL6 melanoma cells, expressing high levels of this endoglycosidase, but has no effect on the metastasis of MC-38 carcinoma cells that express little or no heparanase activity. P-selectin-specific STMC attenuated metastasis in both animal models, indicating that inhibition of tumor cell interaction with the vascular endothelium is critical for cancer dissemination. Thus, the small size, the stability of the C-C bond, and the chemically defined structure of the newly generated STMCs make them superior to heparin derivatives and signify STMCs as valuable candidates for further evaluation.