Heparin, the naturally occurring, highly sulfated glycosaminoglycan drug, acts as a regulator of diverse biological processes by interacting with diverse specific proteins through a variety of sulfated structural motifs with varying degrees of affinity. Despite its rarity, 3-O-sulfation of glucosamine plays a key role in several biological activities, especially anticoagulant activity mediated by interaction with antithrombin (AT), a mechanism that has been extensively studied. The present work primarily focuses on sequences containing 3-O-sulfated glucosamine that are not involved in anticoagulant activity, analysing their relative abundance and structural environments in heparins derived from distinct animal origins. Three heparin samples derived from bovine, ovine and porcine intestinal mucosa (BMH, OMH and PMH) were fractionated by affinity chromatography on AT-Sepharose into no affinity (NA) and high affinity (HA) fractions. Parent heparins and derived NA and HA fractions were enzymatically depolymerised using either a cocktail of heparinases I, II and III or heparinase II alone, and the resulting mixtures of di- and oligosaccharides were analysed by liquid chromatography coupled with mass spectrometry. Digestion with heparinase II, which preserves heparin sequences containing 3-O-sulfated glucosamine, produced a series of 3-O-sulfated trisaccharides containing two glucosamine residues either side of a uronic acid, located at the non-reducing end (NRE) of heparin chains. Treatment with a heparinase cocktail, which cleaves these trisaccharides, released NRE glucosamine monosaccharides, including 3-O-sulfated species. Notably, both the number of NRE trisaccharide species and the overall proportion of 3-O-sulfated monosaccharides were markedly higher in OMH compared to BMH or PMH. Using 1H/13C bi-dimensional nuclear magnetic resonance spectroscopy, the higher proportion of NRE 3-O-sulfated glucosamine in ovine heparin relative to bovine and porcine heparins was found to be preferentially located in chains lacking affinity for AT. These results offer new insights into the localisation of most 3-O-sulfated heparin sequences that are not associated with anticoagulant activity and, importantly, also reveal a possible structural marker that indicates the ovine origin of heparin. Additionally, a tetrasaccharide species was detected that may be indicative of a highly sulfated AT-binding site in BMH.

Pentosan polysulfate (PPS) is an approved drug for the treatment of interstitial cystitis in humans and osteoarthritis in animals. This semisynthetic highly sulfated polysaccharide shares structural similarities with heparin and also interacts with platelet factor 4 (PF4), the key protein implicated in thrombocytopenia, a serious side effect of heparin administration. Thrombocytopenia arises from an immune response to structural features of multimeric complexes of heparin and PF4, although the prediction of disease progression in patients is complicated by the variable polyclonal and polyspecific response. The potential risk of provoking a similar response to PPS or materials derivatized with PPS, which could include subcutaneous or intravenous applications for other therapeutic goals, therefore needs to be assessed. In the absence of a clear proxy measurement for the risk of PPS to induce HIT, the ability of PPS and its fractions to interact with PF4 was examined from a broad structural perspective, employing orthogonal techniques, which were compared with unfractionated heparins (UFHs) and low-molecular-weight heparins (LMWHs). Zeta potential analysis, isothermal titration microcalorimetry, and circular dichroism showed that PPS interacts with PF4 in a manner dependent on its molecular weight, exhibiting behavior intermediate between that of LMHW and UFH. The interaction of PPS size-separated fractions with PF4 also exhibited a dependence on M w; higher M w corresponding to stronger interactions, and the same trend was confirmed by atomic force microscopy. Interestingly, despite PPS forming complexes with PF4, and the complexes formed with PPS fractions being smaller than those formed with UFH and LMWH, enzyme immunoassay studies nevertheless demonstrated the formation of antigenic complexes. Since PPS provokes comparable interactions with PPS, the results suggest that close monitoring of potential thrombocytopenia effects will be necessary when considering PPS dosing, especially for intravenous applications.

Heparan sulfate proteoglycans serve as initial attachment sites for several viruses and bacteria. Recent studies suggest that SARS-CoV-2 similarly exploits these glycosaminoglycans, facilitating conformational changes in the spike protein that promote the interaction between the receptor-binding domain (S1-RBD) and the cellular angiotensin-converting enzyme 2 receptor (ACE2), thereby triggering the virus internalization process. The molecular details that drive this process, particularly the co-receptor role of heparan sulfate (HS), remain incompletely understood. The interaction between an HS hexasaccharide (hexa) and the N343 glycosylated S1-RBD of the wild-type (WT) and Omicron variant of SARS-CoV-2 was investigated. The conformational properties of hexa with these S1-RBDs in unbound and bound states are explored using multiple independent MD simulations; the protein binding epitope of hexa, as well as the details of its interaction with S1-RBD of the Omicron variant, are characterized by comparing experimental and theoretical 1H STD NMR signals. This investigation identifies the role played by the glycosyl moiety at N343 in potentially affecting this interaction in both WT and Omicron S1-RBD, explaining the observed low specificity and multi-modal nature of the interaction between HS oligosaccharides and these S1-RBDs.

Among the structural proteins of SARS-CoV-2, the nucleocapsid (N) protein stands out for its pronounced structural heterogeneity and multifunctionality throughout the viral life cycle. Recent studies have demonstrated that the N protein localizes to the surface of infected and neighboring non-infected cells, by interacting with heparan sulfate in the extracellular matrix. The N protein (419 residues) comprises two folded domains (44NTD180 and 249CTD361) interspersed with three intrinsically disordered regions (1IDR143, 181IDR2248, 362IDR3419). The coexistence of ordered and disordered elements raises a key question: how does this structural heterogeneity influence N's interactions with biological partners? Here we employ high-resolution NMR spectroscopy as the primary technique to characterize the interaction of three N protein constructs (44NTD180, 1NTR248, and 1N419) with heparin-based ligands of increasing complexity. NMR provides atomic level information on the structured NTD domain and on the otherwise difficult to investigate flexible regions. Molecular dynamics simulations further probe the interaction between NTD and short heparin oligosaccharides. Our data reveal a clear correlation between ligand size and binding affinity: longer saccharide chains promote stronger binding. Additionally, the inclusion of intrinsically disordered regions in the NTR construct significantly enhances the interaction compared to NTD, highlighting the functional relevance of structural disorder. Finally, the full-length protein exhibits distinct spectral behavior with the investigated heparin-based ligand, potentially reflecting additional binding contributions and altered dynamics arising from its complex structure. These findings underscore the utility of NMR spectroscopy in elucidating the dynamic, multivalent nature of protein-polyanion interactions, particularly in highly flexible proteins with a modular domain organization.

Heparin manufacturing generates significant volumes of by-products, which are typically treated as waste, and whose potential as a source of alternative bioactive materials is yet to be fully developed. Here, we employed a circular economy approach to support the repurposing of this waste stream for potential anti-inflammatory applications. Glycosaminoglycan fractions were extracted and purified from porcine-derived heparin by-products and structurally characterised using nuclear magnetic resonance and high performance size exclusion chromatography. The fractions exhibited distinct saccharide compositions, sulfation patterns, and molecular weight profiles. Bioactivity assays demonstrated that selected fractions attenuated LPS-induced NF-κB activation in RAW-Blue™ cells and enhanced IL-10 production ex vivo, indicating immunomodulatory potential. Heparin by-products have negligible anticoagulant activity, which supports their safe use in non-anticoagulant biomedical applications. These findings illustrate the potential of heparin by-products as therapeutic agents, while contributing to sustainable pharmaceutical manufacturing.

The heparan sulfate (HS)-rich extracellular matrix (ECM) serves as an initial interaction site for the homotrimeric spike (S) protein of SARS-CoV-2 to facilitate subsequent docking to angiotensin-converting enzyme 2 (ACE2) receptors and cellular infection. More recent variants, notably Omicron, have evolved by swapping several amino acids to positively charged residues to enhance the interaction of the S-protein trimer with the negatively charged HS. However, these enhanced interactions may reduce Omicron’s ability to move through the HS-rich ECM to effectively find ACE2 receptors and infect cells, raising the question of how to mechanistically explain HS-associated viral movement. In this work, we show that Omicron S proteins have evolved to balance HS interaction stability and dynamics, resulting in enhanced mobility on an HS-functionalized artificial matrix. This property is achieved by the ability of Omicron S-proteins to cross-link at least two HS chains, allowing direct S-protein switching between chains as a prerequisite for cell surface mobility. Optimized HS interactions can be targeted pharmaceutically, as an HS mimetic significantly suppressed surface binding and cellular infection specifically of the Omicron variant. These findings suggest a robust way to interfere with SARS-CoV-2 Omicron infection and potentially future variants.

Heparanase is the only known endo-β-glucuronidase able to cleave heparan sulfate, participating in degradation and remodelling of the extracellular matrix. Heparanase upregulation promotes tumor growth and metastasis, therefore, its inhibition is a target for anticancer therapies. Heparan sulfate mimetics bearing glycol-split (gs) units are one of the most promising class of heparanase inhibitors. Herein we describe a total synthesis of two trisaccharides (MeO-GlcNS6S-IdoA/GlcA-GlcNS6S-OMe) differing in epimeric uronic acid residues and one tetrasaccharide (MeO-IdoA-GlcNS6S-IdoA-GlcNS6S-OMe), together with their corresponding glycol-split versions, prepared by periodate oxidation and further modified either via reduction or Pinnick oxidation to obtain gs or tricarboxylated saccharides. An intermediate imine was observed during periodate oxidation, which causes formation of byproducts. Evaluation of the heparanase inhibitory activity showed that the glycol-split trisaccharides were more potent than their intact uronic acid congeners. The binding interactions of the glycol-split trisaccharides with heparanase were investigated by a combined STD NMR and molecular docking approach, with good agreement obtained between the STD NMR experimental data, docking calculations and the in vitro activity results, helping to rationalize the observed inhibition data.

Sodium hyaluronate (HA), a derivative of hyaluronan, is a natural and biocompatible polysaccharide that interacts with cluster of differentiation-44 receptor to promote fine-tuning of inflammation, fibrosis, and tissue remodeling. HA has a smaller molecular weight than hyaluronan and is overall more stable being less prone to oxidation. In this study, we report a novel lactose-functionalized sodium hyaluronate, named HYLACH®. Functionalization with multiple β-galactose residues facilitates its interaction with galectin-3, a β-galactose binding lectin implicated in various pathological processes including inflammation, host defense, and fibrosis, especially critical in idiopathic pulmonary fibrosis (IPF). Our strategy was to modify HA, to varying extents, at carboxyl sites with 1-amino-1-deoxy-lactitol, in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl morpholinium chloride in aqueous media. We characterized the chemical structure, molecular weight, and degree of substitution of HYLACH® using NMR spectroscopy and size exclusion chromatography. We further determined several key parameters including its stability toward enzymatic degradation and the binding affinity and conformational changes of galectin-3 interaction with HYLACH®. Collectively, the generation of a novel functionalized HA with an ability to bind and suppress galectin-3 function, in combination with safety and biocompatibility, offers the opportunity to test this compound in therapeutic trials of devastating fibrotic diseases such as IPF.

The interaction of heparin with antithrombin (AT) involves a specific sequence corresponding to the pentasaccharide GlcNAc/NS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S (AGA*IA). Recent studies have revealed that two AGA*IA-containing hexasaccharides, which differ in the sulfation degree of the iduronic acid unit, exhibit similar binding to AT, albeit with different affinities. However, the lack of experimental data concerning the molecular contacts between these ligands and the amino acids within the protein-binding site prevents a detailed description of the complexes. Differential epitope mapping (DEEP)-STD NMR, in combination with MD simulations, enables the experimental observation and comparison of two heparin pentasaccharides interacting with AT, revealing slightly different bound orientations and distinct affinities of both glycans for AT. We demonstrate the effectiveness of the differential solvent DEEP-STD NMR approach in determining the presence of polar residues in the recognition sites of glycosaminoglycan-binding proteins.

The high transmissibility and mutation ability of coronaviruses enable them to easily escape existing immune protection and also pose a challenge to existing antiviral drugs. Moreover, drugs only targeting viruses cannot always attenuate the "cytokine storm". Herein, a synthetic heparan sulfate (HS) mimetic, HMSA-06 is reported, that exhibited antiviral activities against both the SARS-CoV-2 prototype and Omicron strains by targeting viral entry and replication. Of particular note, HMSA-06 demonstrated more potent anti-SARS-CoV-2 effects than PG545 and Roneparstat. SARS-CoV-2 is reported to hijack autophagy to facilitate its replication, therefore boosting autophagy can attenuate SARS-CoV-2 infection. It is revealed that HMSA-06, but not a similar HS mimetic that failed to inhibit SARS-CoV-2, can upregulate cellular autophagy flux. In addition, HMSA-06 was found to robustly block the NLRP3-mediated inflammatory reaction in SARS-CoV-2 infected THP-1 derived macrophages as evidenced by a reduction in inflammasome formation and the subsequent decreased secretion of mature caspase-1 and IL-1β. The HMSA-06's inflammation inhibitory function is further confirmed using a LPS/ATP-stimulated THP-1 macrophage model. Altogether, this study has identified a promising HS mimetic to combat SARS-CoV-2-associated diseases by inhibiting viral infection and attenuating viral-induced inflammatory reaction, providing insights into the development of novel anti-coronavirus drugs in the future.

Hyaluronic acid (HA) is a natural and biocompatible polysaccharide that is able to interact with CD44 receptors to regulate inflammation, fibrosis, and tissue reconstruction. It is a suitable chemical scaffold for drug delivery that can be functionalized with pharmacophores and/or vectorizable groups. The derivatization of HA is achieved to varying extents by reacting 1-amino-1-deoxy-lactitol via the carboxyl group to form amide linkages, giving rise to the grafted polymer, HYLACH. This retains the broad properties of HA, even though, as in most HA-grafted polymers, the detailed conformational effects of such substitutions, while crucial in the design or optimization of drug delivery systems, remain unknown. Here, the conformation, size, secondary structure, hydrogen bond network, and hydration features of lactosylated HA derivatives were evaluated by using multiple independent molecular dynamics simulations. This revealed subtle but nevertheless significant changes in the HA scaffold, establishing the density of grafting as the key parameter determining its properties.

Tamarind seed polysaccharide (TSP) is a neutral water-soluble galactoxyloglucan isolated from the seed kernel of Tamarindus indica with average molecular weight (Mw) 600–800 kDa. The high viscosity of TSP slows solubilisation, and the absence of charged substituent hinders the formation of electrostatic interactions with biomolecules. TSP was sulphated in a one-step process using dimethylformamide as a solvent, and sulphur trioxide-pyridine complex as a sulphating reagent. Studies of chemical structure, molecular weight distribution and viscosity were conducted to characterise the synthesised products. The sulphation degree was established by conductimetric titration; the sulphate group distribution was studied by NMR spectroscopy and liquid chromatography-mass spectrometry, and sulphated TSP oligomers were obtained by enzymatic degradation with cellulase and/or xyloglucanase. Sulphated products showed higher solubility than TSP, Mws in the range of 700–1000 kDa, a sulphation degree of two to four per subunit and pseudoplastic behaviour. A preliminary study of mucoadhesion revealed the unexpected interaction of S-TSP with mucin, providing a route by which sulphated TSP interactions with biomolecules may be influenced.

The N1 neuraminidases (NAs) of avian and pandemic human influenza viruses contain tyrosine and asparagine, respectively, at position 347 on the rim of the catalytic site; the biological significance of this difference is not clear. Here, we used molecular dynamics simulation to model the effects of amino acid 347 on N1 NA interactions with sialyllacto-N-tetraoses 6'SLN-LC and 3'SLN-LC, which represent NA substrates in humans and birds, respectively. Our analysis predicted that Y347 plays an important role in the NA preference for the avian-type substrates. The Y347N substitution facilitates hydrolysis of human-type substrates by resolving steric conflicts of the Neu5Ac2-6Gal moiety with the bulky side chain of Y347, decreasing the free energy of substrate binding, and increasing the solvation of the Neu5Ac2-6Gal bond. Y347 was conserved in all N1 NA sequences of avian influenza viruses in the GISAID EpiFlu database with two exceptions. First, the Y347F substitution was present in the NA of a specific H6N1 poultry virus lineage and was associated with the substitutions G228S and/or E190V/L in the receptor-binding site (RBS) of the hemagglutinin (HA). Second, the highly pathogenic avian H5N1 viruses of the Gs/Gd lineage contained sporadic variants with the NA substitutions Y347H/D, which were frequently associated with substitutions in the HA RBS. The Y347N substitution occurred following the introductions of avian precursors into humans and pigs with N/D347 conserved during virus circulation in these hosts. Comparative evolutionary analysis of site 347 revealed episodic positive selection across the entire tree and negative selection within most host-specific groups of viruses, suggesting that substitutions at NA position 347 occurred during host switches and remained under pervasive purifying selection thereafter. Our results elucidate the role of amino acid 347 in NA recognition of sialoglycan substrates and emphasize the significance of substitutions at position 347 as a marker of host range and adaptive evolution of influenza viruses.

Glycosaminoglycans (GAGs) are complex polysaccharides exhibiting a vast structural diversity and fulfilling various functions mediated by thousands of interactions in the extracellular matrix, at the cell surface, and within the cells where they have been detected in the nucleus. It is known that the chemical groups attached to GAGs and GAG conformations comprise "glycocodes" that are not yet fully deciphered. The molecular context also matters for GAG structures and functions, and the influence of the structure and functions of the proteoglycan core proteins on sulfated GAGs and vice versa warrants further investigation. The lack of dedicated bioinformatic tools for mining GAG data sets contributes to a partial characterization of the structural and functional landscape and interactions of GAGs. These pending issues will benefit from the development of new approaches reviewed here, namely (i) the synthesis of GAG oligosaccharides to build large and diverse GAG libraries, (ii) GAG analysis and sequencing by mass spectrometry (e.g., ion mobility-mass spectrometry), gas-phase infrared spectroscopy, recognition tunnelling nanopores, and molecular modeling to identify bioactive GAG sequences, biophysical methods to investigate binding interfaces, and to expand our knowledge and understanding of glycocodes governing GAG molecular recognition, and (iii) artificial intelligence for in-depth investigation of GAGomic data sets and their integration with proteomics.

The clinically important anticoagulant heparin, a member of the glycosaminoglycan family of carbohydrates that is extracted predominantly from porcine and bovine tissue sources, has previously been shown to inhibit the β-site amyloid precursor protein cleaving enzyme 1 (BACE-1), a key drug target in Alzheimer's Disease. In addition, heparin has been shown to exert favourable bioactivities through a number of pathophysiological pathways involved in the disease processes of Alzheimer's Disease including inflammation, oxidative stress, tau phosphorylation and amyloid peptide generation. Despite the multi-target potential of heparin as a therapeutic option for Alzheimer's disease, the repurposing of this medically important biomolecule has to-date been precluded by its high anticoagulant potential. An alternative source to mammalian-derived glycosaminoglycans are those extracted from marine environments and these have been shown to display an expanded repertoire of sequence-space and heterogeneity compared to their mammalian counterparts. Furthermore, many marine-derived glycosaminoglycans appear to retain favourable bioactivities, whilst lacking the high anticoagulant potential of their mammalian counterparts. Here we describe a sulphated, marine-derived glycosaminoglycan extract from the Atlantic Sea Scallop, Placopecten magellanicus that displays high inhibitory potential against BACE-1 (IC50 = 4.8 μg.mL−1) combined with low anticoagulant activity; 25-fold less than that of heparin. This extract possesses a more favourable therapeutic profile compared to pharmaceutical heparin of mammalian provenance and is composed of a mixture of heparan sulphate (HS), with a high content of 6-sulphated N-acetyl glucosamine (64%), and chondroitin sulphate.

The risk of heparin shortage opens up the possibility of diversifying the sources of heparin by introducing products of other animal/organ origins. In addition to bovine heparin, already used in the past in the United States and Europe, ovine heparin can become another alternative to the widely used pig source. It is therefore appropriate to compare the anticoagulant activity of pig heparin with that of products of different animal origin and to verify whether blended heparin obtained from different sources can give rise to anticoagulant effects comparable to those of heparin of a single origin.

Several publications have recently proposed NMR spectroscopy to evaluate the critical quality attributes (CQA) of pentosan polysulfate sodium (PPS), the active ingredient of ElmironTM approved to treat interstitial cystitis. PPS is a polymer of sulfated 13(1-4)-D-xylopyranose residues randomly substituted by 4-O-methyl-glucopyranosyluronic acid, containing, beyond the main xylose-2,3-O-disulfate repetitive unit, some minor residues that can be marker of both the starting material and preparation process. In the present study we assigned some previously unknown cross-peaks in 1H-13C HSQC NMR of PPS related to its minor sequences adding additional details to its CQA. Four anomeric cross-peaks related to glucuronate-branched xylose and different sulfation pattern as well as the preceding xyloses were identified. Two minor process-related signals of monosulfated xyloses (unsubstituted in position 2 or 3) were also assigned. The isolation of a disaccharide fraction allowed the assignment of the reducing end xylose-& alpha;/13 as well as the preceding xylose residues to be corrected. Additionally, the oversulfation of PPS allowed detection of the reducing end xylose-tri-1,2,3-O-sulfate. The newly identified cross-peaks were integrated into an updated quantitative NMR method. Finally, we demonstrated that an indepth PPS analysis can be obtained using NMR instruments at medium magnetic fields (500 MHz/600 MHz), commonly available in pharmaceutical industries.

Heparin is a polydisperse, heterogeneous polysaccharide of the glycosaminoglycan (GAG) class that has found widespread clinical use as a potent anticoagulant and is classified as an essential medicine by the World Health Organization. The importance of rigorous monitoring and quality control of pharmaceutical heparin was highlighted in 2008, when the existing regulatory procedures failed to identify a life-threatening adulteration of pharmaceutical heparin with oversulfated chondroitin sulfate (OSCS). The subsequent contamination crisis resulted in the exploration of alternative approaches for which the use of multidimensional nuclear magnetic resonance (NMR) spectroscopy techniques and multivariate analysis emerged as the gold standard. This procedure is, however, technically demanding and requires access to expensive equipment. An alternative approach, utilizing attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) combined with multivariate analysis, has been developed. The method described enables the differentiation of diverse GAG samples, the classification of samples of distinct species provenance, and the detection of both established heparin contaminants and alien polysaccharides. This methodology has sensitivity comparable to that of NMR and can facilitate the rapid, cost-effective monitoring and analysis of pharmaceutical heparin. It is therefore suitable for future deployment throughout the supply chain.

The clinically important anticoagulant heparin, a member of the glycosaminoglycan family of carbohydrates that is extracted predominantly from porcine and bovine tissue sources, has previously been shown to inhibit the 0-site amyloid precursor protein cleaving enzyme 1 (BACE-1), a key drug target in Alzheimer's Disease. In addition, heparin has been shown to exert favourable bioactivities through a number of pathophysiological pathways involved in the disease processes of Alzheimer's Disease including inflammation, oxidative stress, tau phosphorylation and amyloid peptide generation. Despite the multi-target potential of heparin as a therapeutic option for Alzheimer's disease, the repurposing of this medically important biomolecule has to-date been precluded by its high anticoagulant potential. An alternative source to mammalian-derived glycosaminoglycans are those extracted from marine environments and these have been shown to display an expanded repertoire of sequence-space and heterogeneity compared to their mammalian counterparts. Furthermore, many marinederived glycosaminoglycans appear to retain favourable bioactivities, whilst lacking the high anticoagulant potential of their mammalian counterparts. Here we describe a sulphated, marine-derived glycosaminoglycan extract from the Atlantic Sea Scallop, Placopecten magellanicus that displays high inhibitory potential against BACE-1 (IC50 = 4.8 mu g.mL-1) combined with low anticoagulant activity; 25-fold less than that of heparin. This extract possesses a more favourable therapeutic profile compared to pharmaceutical heparin of mammalian provenance and is composed of a mixture of heparan sulphate (HS), with a high content of 6-sulphated N-acetyl glucosamine (64%), and chondroitin sulphate.

Objective Danaparoid sodium is a biopolymeric complex drug composed of the most abundant heparan sulfate (HS) followed in descending order by dermatan sulfate (DS) and chondroitin sulfate (CS). This composite nature explains its peculiar antithrombotic and anticoagulant properties and make it particularly advantageous when the risk of heparin-induced thrombocytopenia occurs. A specific control of the danaparoid composition is required by the Ph. Eur. The monograph includes the CS and DS limit contents and describes the method for their quantification through selective enzymatic degradations.Materials and Methods In this study, a quantitative two-dimensional nuclear magnetic resonance (NMR) method is proposed as a new method suitable for CS and DS quantification. Statistical comparison of the results provided by the analysis of a series of danaparoid samples with both NMR and enzymatic methods highlights a small systematic difference, likely derived from lyase-resistant sequences bearing oxidized terminals. Some modified structures, whose survival to the enzymatic action was confirmed by mass spectrometry, can be detected and quantified by NMR.Conclusion and Results The proposed NMR method can serve for the determination of DS and CS contents, is an easy-to-apply method with no dependence from enzymes and standards, and provides extensive structural information on the overall glycosaminoglycans mixture.