Protein oxidative footprinting, using hydroxyl radical labeling detected by bottom-up proteomics, has progressed from an emerging method to a widely used approach in structural biology. Hydroxyl radicals generated from hydrogen peroxide (via photolysis, Fenton chemistry or electrochemistry) or directly from water (via X-rays, plasma or gamma rays) irreversibly encode structural information within protein side chains, which is read out using standard liquid chromatography-mass spectrometry workflows. Quantitative changes in labeling report on solvent accessibility and reveal effects of protein-protein interactions, ligand binding, protein folding, conformational changes or applied stress. Comparing labeling patterns between states provides detailed maps of structural changes and interaction sites. Over the past decade, oxidative footprinting has proven valuable as a solution-phase and in-cell method for protein structure analysis. This Perspective summarizes best practices for experimental design, sample processing, data analysis, interpretation and integration with orthogonal data, offering a consensus framework to guide application of oxidative footprinting in academic and biopharmaceutical research.

Abstract Hydroxyl radical protein footprinting (HRPF) is a method for protein higher-order structure analysis that oxidizes solvent-accessible side chains of amino acids. One major limitation of HRPF is that results represent an ensemble average of all conformers in solution, preventing resolution of conformationally heterogeneous systems. Here, we established an inline hydrophobic interaction chromatography (HIC)-HRPF workflow that enables conformer-specific labeling while correcting for gradient-dependent differences in radical scavenging. Significant radical scavenging disparity exists between ammonium sulfate and phosphate buffers, and we compensated for this with 7 mM HEPES supplementation, restoring effective radical dose parity across the gradient. Using apo and holomyoglobin as a benchmark, we achieved baseline chromatographic separation of conformers. Multi-dose HRPF revealed conformer-specific oxidation patterns: apomyoglobin displayed enhanced labeling in peptide 81–97, specifically residue H83, consistent with destabilization of the F-helix and opening of the heme pocket, while holomyoglobin exhibited increased oxidation in peptide 120–154 corresponding to M132, E137, and L138, suggesting hinge-like rearrangements in the H-helix upon heme incorporation. These results validate inline HIC-HRPF as a robust, conformer-specific probe of higher-order structure and highlight the necessity of scavenging parity across the gradient for reliable structural interpretation. The method establishes a framework for extending HRPF to dynamic conformationally heterogeneous systems resolvable by HIC.

Hydroxyl Radical Protein Footprinting is a powerful tool to probe protein higher-order structure, protein-protein and protein-ligand interactions. It is mostly performed in vitro, but recent advances have extended its use to live cells, nematodes, and 3D cultures. However, to the best of our knowledge, application in living mammalian tissues has not been accomplished. Here, we present the successful use of radical protein footprinting (RPF) in mammalian whole blood from wild-type and type 2 diabetes mellitus (T2DM) mice. Using persulfate photoactivated with the FOX Photolysis System, we achieve effective protein labeling without significant disruption to blood cell morphology. An optimized quenching protocol eliminates background labeling. We report oxidative modifications for the eleven most abundant proteins detected, revealing disease-associated conformational changes in multiple proteins. We validate that in-blood RPF identifies changes in protein structure resulting from complement activation and increased transferrin iron saturation in T2DM mice. These findings demonstrate the feasibility of RPF in mammalian blood and create opportunities for structural proteomics in preclinical models and clinical samples.

Hydroxyl radical protein footprinting (HRPF) is a well-established technique used to analyze the binding sites and conformational alterations of proteins. Inline LC-flash oxidation can efficiently capture and characterize structural changes between dynamic protein-ligand complexes as they elute from the LC column. The current work focuses on the development of multidose inline SEC-FOX (size-exclusion chromatography-flash oxidation) for the quantitative topographical analysis of the dynamic protein-ligand complex of human antithrombin III (ATIII) and unfractionated heparin (UFH). ATIII alone and the ATIII-UFH complex were separated by an inline SEC system and flash oxidized at multiple radical doses, followed by proteolytic digestion and LC-MS/MS analysis. A total of 21 oxidized peptides, including three glycopeptides with two major glycoforms each, were reproducibly detected. Six peptides including one glycopeptide (48-53, 58-70, 189-197 A2G2S2 glycoform, 108-114, 333-348, and 360-370) were significantly protected from oxidation upon UFH binding, and one peptide (242-257) showed a significant increase in oxidation upon UFH binding. Residue-level analysis showed that three amino acids (M49, E113, and F329) were significantly protected from modification in the ATIII-UFH complex compared to ATIII alone. Two of these three amino acids are proximal to the known pentasaccharide binding site, while the third is distal and suggests a potential extended interaction of the full-length UFH with ATIII, which was well supported by computational modeling data. We interpret these findings in the context of previously published biophysical data and present a computational model of UFH binding consistent with our findings.

Heparan-6-O-endosulfatase 2 (Sulf-2) is a proteoglycan enzyme that modifies sulfation of heparan sulfate proteoglycans. Dysregulation of Sulf-2 is associated with various pathological conditions, including cancer, which makes Sulf-2 a potential therapeutic target. Despite the key pathophysiological roles of Sulf-2, inhibitors remain insufficiently developed. In previous work, a fucosylated chondroitin sulfate from the sea cucumber Holothuria floridana (HfFucCS) exhibited potent Sulf-2 inhibition. This study investigates the structural basis of HfFucCS-mediated Sulf-2 inhibition, examines the binding profile of HfFucCS to Sulf-2, and explores the mode of inhibition. Additionally, a structurally diverse library of sulfated poly/oligosaccharides, including common glycosaminoglycans and unique marine sulfated glycans, was screened for Sulf-2 inhibition. Results from a high-throughput arylsulfatase assay and specific 6-O-desulfation assay have proved that HfFucCS is the most potent among the tested sulfated glycans, likely due to the presence of the unique 3,4-disulfated fucose structural motif. HfFucCS demonstrated non-competitive inhibition, and inhibitory analysis of its low-molecular-weight fragments suggests a minimum length of ~7.5 kDa for effective inhibition. Surface plasmon resonance analyses revealed that Sulf-2 binds to surface heparin with high affinity (KD of 0.817 nM). HfFucCS and its derivatives effectively disrupt this interaction. Results from mass spectrometry-hydroxyl radical protein footprinting and repulsive scaling replica exchange molecular dynamics indicate similarities in the binding of heparin and HfFucCS oligosaccharides to both the catalytic and hydrophilic domains of Sulf-2. These findings reveal the unique inhibitory properties of a structurally distinct marine glycosaminoglycan, supporting its further investigation as a selective and effective inhibitor for Sulf-2-associated cancer events.

Despite decades of research, the successful clinical translation of intravenously administered nanomedicines is underwhelming. One significant barrier to progress is the opsonization and rapid clearance from the bloodstream due to protein corona formation as an innate immune response. Biocompatible nanoparticle coatings that act as a barrier between the nanomaterial and the physiological environment are being continuously explored to elevate delivery success. However, the formation and composition of protein coronae, especially across species, are still poorly understood, which hinders the progress of translation from preclinical animal models to human applications. Here, we use quantitative protein assays, LC-MS proteomics, and machine learning to catalog the protein coronae from human and mouse serum formed on poly(lactic-co-glygolic) acid (PLGA) nanoparticles and explore the impact of a large library of coatings comprised of cholinium fatty acid-based ionic liquids (ILs) from 4- to 10-carbon chains with varying degrees of unsaturation. We discover that the species matters, with vast changes in the coronae being observed, depending on the source of the serum sample. Additionally, even very small changes in the ionic liquid anion structure result in the formation of diverse hard coronae. We identify several ILs that show enriched dysopsonins and depleted opsonins relative to serum that are promising candidates for future development as therapeutics or in biosensing.

Targeted drug delivery seeks to revolutionize disease treatment by enhancing therapeutic efficacy and specificity. However, developing and achieving precisely targeted delivery remains a significant challenge, particularly in cancers such as triple-negative breast cancer (TNBC), which lack traditional markers for targeted delivery. TNBC cells overexpress glucose transport proteins (GLUTs) on their surfaces, providing an opportunity for targeting. Herein, sugar-based ionic liquids (Glyco-ILs or GILs) are developed and used to modify poly (lactic-co-glycolic acid) (PLGA) nanocarriers (NPs), show enhanced affinity and selectivity towards TNBC cells and human and mouse erythrocytes. Inhibition assays, molecular docking simulations, and liquid chromatography-mass spectrometry (LCMS) analysis data show that the enhanced nanoparticle affinity for TNBC cells is likely due to a combination of specific binding interactions with GLUT transporters and endocytosis. The observed RBC affinity is evidenced to be driven by interactions with RBC membrane GLUTs along with their unique serum protein corona on the nanoparticle surface. In vivo, experiments in a healthy BALB/c mouse model show that Glyco-IL-NPs demonstrate longer retention time in the bloodstream and a significant reduction in liver accumulation relative to the control. These findings suggest that Glyco-IL-modified PLGA nanoparticles (GIL-NPs) hold a promising approach for selective drug delivery, particularly in cells that overexpress GLUTs.

Carbohydrates are found in various forms in living organisms, both as free-standing glycans as well as glycoconjugates including glycoproteins, glycolipids, and glycosaminoglycans. These structures play crucial roles in many biological processes, often mediated or influenced by interactions of carbohydrates with other biomolecules. However, studying these interactions is particularly challenging due to the structural complexity of carbohydrates, their dynamic conformational behavior, and the low binding affinities often involved. To address these challenges, we are developing a novel method that leverages mass spectrometry-based radical carbohydrate footprinting (RCF). We monitored changes in the solvent accessibility of specific regions within oligosaccharides by measuring variations in the apparent rate of hydroxyl radical and trifluoromethyl radical-mediated oxidation. In our studies, a collection of trisaccharide isomers and N,N',N″-triacetylchitotriose (NAG3) shows no significant change in modification in nonbinding protein solutions. However, in the presence of two proteins that bind NAG3 specifically, NAG3 oxidation is reduced. We find that the free reducing end is the primary site of hydroxyl radical oxidation under covalent labeling conditions, allowing it to distinguish interactions at the glycan reducing end. Trifluoromethyl radicals, conversely, label broadly across the trisaccharide by substitution into a C-H bond. Overall, this approach offers a powerful novel approach for identifying glycan-protein interactions and mapping the binding interface of glycans.

O-GalNAc glycans on glycoproteins with eight different core structures sharing a common α-glycosidic linkage (O-GalNAc-α-Ser/Thr) are critical in various physiological and pathological processes. Among the eight O-GalNAc glycan cores, core 2 characterized by a GlcNAcβ1-6(Galβ1-3)GalNAc structural motif plays a significant role in regulating diverse biological processes, such as immune response modulation, adhesive properties of selectins, and gastrointestinal tract protection. However, the large-quantity synthesis of core 2 containing glyco-amino acids for downstream solid-phase peptide synthesis is challenging. In this work, we successfully employed a silver oxide for coupling a 2-azido-galactosyl chloride donor with two acceptors, Fmoc-Ser/Thr-OtBu, respectively, for the large-scale synthesis of the two important intermediates, α-GalN3-Fmoc-Ser/Thr-OtBu, which can be further utilized for the large-scale synthesis of core 2 containing glyco-amino acids. The two intermediates, α-GalN3-Fmoc-Ser/Thr-OtBu, were utilized for synthesizing core 2 containing Fmoc-Ser/Thr-COOH. The synthesis of core 2 containing Fmoc-Ser-COOH was achieved on a 1.95 g scale, while the synthesis of core 2 containing Fmoc-Thr-COOH was achieved on a 0.38 g scale. Additionally, the synthesis of the 2-azido-galactosyl chloride donor was optimized into a three-step process with only one column chromatography purification. Finally, core 2 containing Fmoc-Ser/Thr-COOH were applied for the synthesis of glycosylated CCR1 and CCR5 N-terminal peptides.

The cation-independent mannose 6-phosphate receptor (CI-MPR) is clinically significant in the treatment of patients with lysosomal storage diseases because it functions in the biogenesis of lysosomes by transporting mannose 6-phosphate (M6P)-containing lysosomal enzymes to endosomal compartments. CI-MPR is multifunctional and modulates embryonic growth and fetal size by downregulating circulating levels of the peptide hormone insulin-like growth factor 2 (IGF2). The extracellular region of CI-MPR comprises 15 homologous domains with binding sites for M6P-containing ligands located in domains 3, 5, 9, and 15, whereas IGF2 interacts with residues in domain 11. How a particular ligand affects the receptor’s conformation or its ability to bind other ligands remains poorly understood. To address these questions, we purified a soluble form of the receptor from newborn calf serum, carried out glycoproteomics to define the N-glycans at its 19 potential glycosylation sites, probed its ability to bind lysosomal enzymes in the presence and absence of IGF2 using surface plasmon resonance, and assessed its conformation in the presence and absence of IGF2 by negative-staining electron microscopy and hydroxyl radical protein footprinting studies. Together, our findings support the hypothesis that IGF2 acts as an allosteric inhibitor of lysosomal enzyme binding by inducing global conformational changes of CI-MPR.

Nanoparticles (NPs) offer significant promise as drug delivery vehicles; however, their in vivo efficacy is often hindered by the formation of a protein corona (PC), which influences key physiological responses such as blood circulation time, biodistribution, cellular uptake, and intracellular localization. Understanding NP-PC interactions is crucial for optimizing NP design for biomedical applications. Traditional approaches have utilized hydrophilic polymer coatings like polyethylene glycol (PEG) to resist protein adsorption, but glycopolymer-coated nanoparticles have emerged as potential alternatives due to their biocompatibility and ability to reduce the adsorption of highly immunogenic proteins. In this study, we synthesized and characterized glycopolymer-based poly[2-(diisopropylamino)ethyl methacrylate-b-poly(methacrylamidoglucopyranose) (PDPA-b-PMAG) NPs as an alternative to PEGylated NPs. We characterized the polymers using a range of techniques to establish their molecular weight and chemical composition. PMAG and PEG-based NPs showed equivalent physicochemical properties with sizes of similar to 100 nm, spherical morphology, and neutral surface charges. We next assessed the magnitude of protein adsorption on both NPs and catalogued the identity of the adsorbed proteins using mass spectrometry-based techniques. The PMAG NPs were found to adsorb fewer proteins in vitro as well as fewer immunogenic proteins such as Immunoglobulins and Complement proteins. Flow cytometry and confocal microscopy were employed to examine cellular uptake in RAW 264.7 macrophages and MDA-MB-231 tumor cells, where PMAG NPs showed higher uptake into tumor cells over macrophages. In vivo studies in BALB/c mice with orthotopic 4T1 breast cancer xenografts showed that PMAG NPs exhibited prolonged circulation times and enhanced tumor accumulation compared to PEGylated NPs. The biodistribution analysis also revealed greater selectivity for tumor tissue over the liver for PMAG NPs. These findings highlight the potential of glycopolymeric NPs to improve tumor targeting and reduce macrophage uptake compared to PEGylated NPs, offering significant advancements in cancer nanomedicine and immunotherapy.

Zwitterionic-based systems offer promise as next-generation drug delivery biomaterials capable of enhancing nanoparticle (NP) stimuli-responsiveness, biorecognition, and biocompatibility. Further, imidazole-functionalized amphiphilic zwitterions are able to readily bind to various biological macromolecules, enabling antifouling properties for enhanced drug delivery efficacy and bio-targeting. Herein, we describe structurally tuned zwitterionic imidazole-based ionic liquid (ZIL)-coated PEG-PLGA nanoparticles made with sonicated nanoprecipitation. Upon ZIL surface modification, the hydrodynamic radius increased by nearly 20 nm, and the surface charge significantly shifted closer to neutral. 1H NMR spectra suggests that the amount of ZIL on the nanoparticle surface is controlled by the structure of the ZIL and that the assembly occurs as a result of non-covalent interactions of ZIL-coated nanoparticle with the polymer surface. These nanoparticle-zwitterionic liquid (ZIL) constructs demonstrate selective affinity towards red blood cells in whole mouse blood and show relatively low human hemolysis at similar to 5%. Additionally, we observe higher nanoparticle accumulation of ZIL-NPs compared with unmodified NP controls in human triple-negative breast cancer cells (MDA-MB-231). Furthermore, although the ZIL shows similar protein adsorption by SDS-PAGE, LC-MS/MS protein analysis data demonstrate a difference in the relative abundance and depletion of proteins in mouse and human serum. Hence, we show that ZIL-coated nanoparticles provide a new potential platform to enhance RBC-based drug delivery systems for cancer treatments. Zwitterionic liquid surface-modified PEG-PLGA nanoparticles are developed to explore their potential as intravenous drug delivery carriers via RBC hitchhiking and affinity towards triple-negative breast cancer cells.

Dried leech (Whitmania pigra whitman) has been widely used as a traditional animal-based Chinese medicine. Dried leech extracts have been reported to have various biological activities that are often associated with mammalian glycosaminoglycans. However, their presence and possible structural characteristics within dried leech were previously unknown. In this study, glycosaminoglycans were isolated from dried leech for the first time and their structures were analyzed by the combination of Fourier-transform infrared spectroscopy, liquid chromatography-ion trap/time-of-flight mass spectrometry and polyacrylamide gel electrophoresis. Heparan sulfate and chondroitin sulfate/dermatan sulfate were detected in dried leech with varied disaccharide compositions and possess a heterogeneous structure. Heparan sulfate species possess an equal amount of total 2-O-sulfated, N-sulfated and acetylated disaccharides, while chondroitin sulfate /dermatan sulfate contain high content of 4-O-sulfated disaccharides. Also, the quantitative analysis revealed that the contents of heparan sulfate and chondroitin/dermatan sulfate in dried leech varied significantly, with chondroitin/dermatan sulfate being by far the most abundant. This novel structural information could help clarify the possible involvement of these polysaccharides in the biological activities of the dried leech. Furthermore, leech glycosaminoglycans showed a strong ABTS radical scavenging ability, which suggests the potential of leech polysaccharides for exploitation in the nutraceutical and pharmaceutical industries.

In biological systems, proteins can bind to nanoparticles to form a “corona” of adsorbed molecules. The nanoparticle corona is of high interest because it impacts the organism’s response to the nanomaterial. Understanding the corona requires knowledge of protein structure, orientation, and dynamics at the surface. Ultimately, a residue-level mapping of protein behavior on nanoparticle surfaces is needed, but this mapping is difficult to obtain with traditional approaches. Here, we have investigated the interaction between R2ab and polystyrene nanoparticles (PSNPs) at the level of individual residues. R2ab is a bacterial surface protein from Staphylococcus epidermidis and is known to interact strongly with polystyrene, leading to biofilm formation. We have used mass spectrometry after lysine methylation and hydrogen-deuterium exchange (HDX) NMR spectroscopy to understand how the R2ab protein interacts with PSNPs of different sizes. Through lysine methylation, we observe subtle but statistically significant changes in methylation patterns in the presence of PSNPs, indicating altered protein surface accessibility. HDX measurements reveal that certain regions of the R2ab protein undergo faster exchange rates in the presence of PSNPs, suggesting conformational changes upon binding. Both results support a recently proposed “adsorbotope” model, wherein adsorbed proteins consist of unfolded anchor points interspersed with regions of partial structure. Our data also highlight the challenges of characterizing complex protein-nanoparticle interactions using these techniques, such as fast exchange rates. While providing insights into how proteins respond to nanoparticle surfaces, this research emphasizes the need for advanced methods to comprehend these intricate interactions fully at the residue level. TOC Image Lysine methylation and hydrogen-deuterium exchange can reveal useful structural details about protein adsorption to nanoparticle surfaces.

The structure of the sulfated galactan from the red alga Botryocladia occidentalis (BoSG) was originally proposed as a simple repeating disaccharide of alternating 4-linked α-galactopyranose (Galp) and 3-linked β-Galp units with variable sulfation pattern. Abundance was estimated only for the α-Galp units: one-third of 2,3-disulfation and one-third of 2-monosulfation. Here, we isolated again the same BoSG fractions from the anion-exchange chromatography, obtaining the same NMR profile of the first report. More careful NMR analysis led us to revise the structure. A more complex sulfation pattern was noted along with the occurrence of 4-linked α-3,6-anhydro-Galp (AnGalp) units. Interestingly, the more sulfated BoSG fraction showed slightly reduced in vitro anti-SARS-CoV-2 activities against both wild-type and delta variants, and significantly reduced anticoagulant activity. The BoSG fractions showed no cytotoxic effects. The reduction in both bioactivities is attributed to the presence of the AnGalp unit. Docking scores from computational simulations using BoSG disaccharide constructs on wild-type and delta S-proteins, and binding analysis through competitive SPR assays using blood (co)-factors (antithrombin, heparin cofactor II and thrombin) and four S-proteins (wild-type, delta, gamma, and omicron) strongly support the conclusion about the deleterious impact of the AnGalp unit.

Sulfated fucans (SFs) from echinoderms, such as sea cucumbers and sea urchins, present linear and regular sulfation patterns within defined oligosaccharide building blocks. The high molecular weights of these polymers pose a problem in advanced structure-activity relationship studies for which derived oligosaccharides are more appropriate tools for investigation. However, enzymes capable of specifically depolymerizing SFs, fucanases, are not very common. Scarce abundance and unknown catalytic activities are additional barriers to exploiting fucanases. Oligosaccharide production by controlled chemical reactions such as mild acid hydrolysis then becomes a convenient strategy. As a consequence, physicochemical studies are necessary to understand the structural modifications caused on SFs by this chemical hydrolysis. Hence, in this work, we subjected three tetrasaccharide-repeating SFs from sea cucumbers, Isostichopus badionotus (IbSF), Holothuria floridana (HfSF), and Lytechinus variegatus (LvSF) to mild acid hydrolysis for oligosaccharide production. Interestingly, selective 2-desulfation reaction was observed in all three SFs. Through our study, we indicate that selective 2-desulfation is a common and expected phenomenon in oligosaccharide production by mild acid hydrolysis of SFs, including those composed of tetrasaccharide-repeating units.

Achieving safe and efficacious drug delivery is still an outstanding challenge. Herein we have synthesized 20 biocompatible good's buffer-based ionic liquids (GBILs) with a range of attractive properties for drug delivery applications. The synthesized GBILs were used to coat the surface of poly(lactic-co-glycolic acid) (PLGA) by nanoprecipitation-sonication and characterized by dynamic light scattering (DLS) and proton nuclear magnetic resonance (1H NMR) spectroscopy. The GBIL-modified PLGA NPs were then tested for their interaction with bio-interfaces such as serum proteins (using SDS-PAGE and LCMS) and red blood cells (RBCs) isolated from human and BALB/c mouse blood. In this report, we show that surface modification of PLGA with certain GBILs led to modulation of preferential cellular uptake towards human triple-negative breast cancer cells (MDA-MB-231) compared to human normal healthy breast cells (MCF-10A). For example, cholinium N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonate (CBES) coated PLGA NPs were found to be selective for MDA-MB-231 cells (60.7 +/- 0.7%) as compared to MCF-10A cells (27.3 +/- 0.7%). In this way, GBIL-coatings have increased PLGA NP uptake in the cancer cells by 2-fold while decreasing the uptake towards normal healthy breast cells. Therefore, GBIL-modified nanoparticles could be a versatile platform for targeted drug delivery and gene therapy applications, as their surface properties can be tailored to interact with specific cell receptors and enhance cellular uptake. This formulation technique has shown promising results for targeting specific cells, which could be explored further for other cell types to achieve site-specific and efficient delivery of therapeutic agents. Schematic representation of preferential uptake of good's buffer-coated PLGA nanoparticles into human breast cancer cells. Created with biorender.com.

Hydroxyl radical protein footprinting (HRPF) is a mass-spectrometry-based method for studying protein structures, interactions, conformations, and folding. This method is based on the irreversible labeling of solvent-exposed amino acid side chains by hydroxyl radicals. While catalase is commonly used as a quencher after the labeling of a protein by the hydroxyl radicals to efficiently remove the remaining hydrogen peroxide, it has some disadvantages. Catalase quenching adds a relatively high amount of protein to the sample, limiting the sensitivity of the method due to dynamic range issues and causing significant issues when dealing with more complex samples. We evaluated dimethylthiourea (DMTU) as a replacement for catalase in the quenching HRPF reactions. We observed that DMTU is highly effective at quenching HRPF oxidation. DMTU does not cause the background protein issues that catalase does, resulting in an increased number of protein identifications from complex mixtures. We recommend the replacement of catalase quenching with DMTU for all HRPF experiments.

In this work, we isolated two new sulfated glycans from the body wall of the sea cucumber Thyonella gemmata: one fucosylated chondroitin sulfate (TgFucCS) (17.5 ± 3.5% kDa) and one sulfated fucan (TgSF) (383.3 ± 2.1% kDa). NMR results showed the TgFucCS backbone composed of [→3)-β-N-acetylgalactosamine-(1→4)-β-glucuronic acid-(1→] with 70% 4-sulfated and 30% 4,6-disulfated GalNAc units and one-third of the GlcA units decorated at the C3 position with branching α-fucose (Fuc) units either 4-sulfated (65%) or 2,4-disulfated (35%) and the TgSF structure composed of a tetrasaccharide repeating unit of [→3)-α-Fuc2,4S-(1→2)-α-Fuc4S-(1→3)-α-Fuc2S-(1→3)-α-Fuc2S-(1→]n. Inhibitory properties of TgFucCS and TgSF were investigated using SARS-CoV-2 pseudovirus coated with S-proteins of the wild-type (Wuhan-Hu-1) or the delta (B.1.617.2) strains and in four different anticoagulant assays, comparatively with unfractionated heparin. Molecular binding to coagulation (co)-factors and S-proteins was investigated by competitive surface plasmon resonance spectroscopy. Among the two sulfated glycans tested, TgSF showed significant anti-SARS-CoV-2 activity against both strains together with low anticoagulant properties, indicating a good candidate for future studies in drug development.