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Carrageenan (CGN) belongs to the sulfated polysaccharides family that is commonly used in the food industry. For oligosaccharide analysis, a liquid chromatography quadrupole time-of-flight/mass spectrometry strategy was developed using a hypothetical database. There are 2100 structures in the developed hypothetical κ-CGN database. To eliminate false-positive results, three approaches were used, including size exclusion chromatography with mass spectrometry, which differentiates the loss of sulfated groups caused by the hydrolysis process or the ionization process. Profiling of acidic hydrolysis products of κ-CGN was found that after 12 h of HCl cultivation, the κ-CGN was hydrolyzed to oligosaccharides lower than the degree of polymerization 10, breaking the α-1,3-glycoside linkage and producing even-numbered oligosaccharides. Another finding was that the pH at which acidic hydrolysis is terminated affects the generation of even and odd oligosaccharides. Peeling reaction occurs at the reduction end 4-linked-3,6-anhydrous-d-galactose when adjusted to alkaline conditions, thus generating odd oligosaccharides., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

A water-soluble β-glucan from fruiting body of Hericium erinaceus was obtained after water extraction, purification and fractionation. Analyses of monosaccharide composition, molecular weight and linkage mode demonstrated that it is a β-glucan with 1→3 and 1→6 linkage modes and a molecular weight of 13.3 kDa. An endo-1,6-β-d-glucanase was used to digest the β-glucan and the digested products over time were analyzed with a HPAEC-PAD-MS platform. The linkage mode of each glycosidic bond in the digested oligosaccharides were confirmed with MS/MS. At the end of digestion, enzyme resistant oligosaccharides were observed as several 1→6 linked glucoses with one or two 1→3 linked glucose residues. All these domains are more like constructional pieces from a branch-on-branch glucan, in which multiple 1→6 linked glucan chains are hooked through one or two 1→3 linked glucose residues. Averagely, there is a 1→3 linkage per six 1→6 linked glucoses in this branch-on-branch β-glucan., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

We previously reported that β-glucosidase BGL1 at low concentration (15 μg mL -1 ) from Coprinopsis cinerea exhibited hydrolytic activity only toward laminarioligosaccharides but not toward cellooligosaccharides and gentiobiose. This study shows that BGL1 at high concentration (200 μg mL -1 ) also hydrolyzed cellobiose and gentiobiose, which accounted for only 0.83 and 2.05% of its activity toward laminaribiose, respectively. Interestingly, BGL1 at low concentration (1.5 μg mL -1 ) showed transglycosylation but BGL1 at high concentration (200 μg mL -1 ) did not. BGL1 utilizes only laminarioligosaccharides but not glucose, gentiobiose, and cellobiose to synthesize the higher oligosaccharides. BGL1 transferred one glucosyl residue from substrate laminarioligosaccharide to another laminarioligosaccharide as an acceptor in a β(1 → 3) or β(1 → 6) fashion to produce higher laminarioligosaccharides or 3-O-β-d-gentiobiosyl-d-laminarioligosaccharides. The BGL1-digested laminaritriose exhibited approximately 90% enhancement in the anti-oxidant activity compared to that of untreated laminaritriose, implying a potential application of BGL1-based transglycosylation for the production of high value-added rare oligosaccharides.

Porcine intestinal mucosa heparin was partially depolymerized by recombinant heparinase 1 (heparin lyase 1, originating from Flavobacterium heparinum and expressed in Escherichia coli) and then fractionated, leading to the isolation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel electrophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their structures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600 MHz. Five of the characterized oligosaccharides represent new compounds. The most prominent oligosaccharide comprises the common repeating unit of heparin, ΔUA2S-[-GlcNS6S-IdoA2S-](n)-GlcNS6S, where ΔUA is 4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-d-glucopyranose, IdoA is l-idopyranosyluronic acid, S is sulfate and n = 0-7. A second prominent heparin oligosaccharide motif corresponds to ΔUA2S-[GlcNS6S-IdoA2S](n)-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n = 0-5 and GlcA is d-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in heparin. The prominence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the -GlcNS3S6S-IdoA2S- linkage is particularly susceptible to heparinase 1.

Heparan sulfate (HS) proteoglycans (PGs) interact with a number of extracellular signaling proteins, thereby playing an essential role in the regulation of many physiological processes. One major function of HS is to interact with fibroblast growth factors (FGFs) and their receptors (FGFRs) and form FGF.HS.FGFR signaling complexes. Past studies primarily examined the selectivity of HS for FGF or FGFR. In this report, we used a new strategy to study the structural specificity of HS binding to 10 different FGF.FGFR complexes. Oligosaccharide libraries prepared from heparin, 6-desulfated heparin, and HS were used for the interaction studies by solution competition surface plasmon resonance (SPR) and filter trapping assays. Specific oligosaccharides binding to FGF.FGFR complexes were subjected to polyacrylamide gel electrophoresis (PAGE) analysis and disaccharide compositional analysis using liquid chromatography and mass spectrometry. The competition SPR studies using sized oligosaccharide mixtures showed that binding of each of the tested FGFs or FGF.FGFR complexes to heparin immobilized to an SPR chip was size-dependent. The 6-desulfated heparin oligosaccharides exhibited a reduced level of inhibition of FGF and FGF.FGFR complex binding to heparin in the competition experiments. Heparin and the 6-desulfated heparin exhibited higher levels of inhibition of the FGF.FGFR complex binding to heparin than of FGF binding to heparin. In the filter trapping experiments, PAGE analysis showed different affinities between the FGF.FGFR complexes and oligosaccharides. Disaccharide analysis showed that HS disaccharides with a degree of polymerization of 10 (dp10) had high binding selectivity, while dp10 heparin and dp10 6-desulfated heparin showed reduced or no selectivity for the different FGF.FGFR complexes tested.

This study reports for the first time the complete liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and tandem mass spectrometry (MS/MS) analyses performed in negative ion mode of saturated unsulfated chondroitin oligosaccharides up to 16-mers and comparison with hyaluronic acid (HA) oligomers differing only in the nature of the hexosamine residue. MS/MS of the chondroitin disaccharide on the singly charged precursor at m/z 396.1 afforded a glycosidic cleavage C1 product ion at m/z 192.9. In the tetrasaccharide, C2 (m/z 396.0) and C3 (m/z 572.0) product anions were generated by glycosidic cleavage. A C5 [M-2H]2- product ion at m/z 475.1 was generated by the glycosidic cleavage of the hexasaccharide, and a C7 ion (m/z 664.6, charge state of -2) was produced from the octasaccharide. The same fragmentation pattern of deprotonated oligomers was observed for the largest oligosaccharides, from 10- to 16-mers. There has been no previous report of MS/MS spectra for unsulfated chondroitin oligomers of these sizes. Unsulfated saturated chondroitin oligosaccharides with x-mer units and larger than a tetrasaccharide dissociate to almost exclusively form CX-1-type ions. Saturated HA oligomers also afforded the same fragmentation pattern as deprotonated oligomers by ESI-MS and MS/MS analyses. Thus, under the experimental conditions used in the current study, we were unable to distinguish between unsulfated chondroitin and HA.

The solution conformation behavior of a dermatan-derived tetrasaccharide--Delta HexA-(1-->3)-GalNAc4S-beta-(1-->4)-IdoA-alpha-(1-->3)-red-GalNAc4S (S is a sulfate group)--has been explored by means of NMR spectroscopy, especially by NOE-based conformational analysis. The tetrasaccharide was present as four species, two of which are chemically different in the anomeric orientation of the reducing 2-deoxy-2-acetamido-galactose (red-GalNAc) residue, while the other two are the result of different conformations of the iduronic acid (IdoA) unit. The two alpha-beta-interconverting anomers were present in a 0.6:1 ratio. Ring conformations have been defined by analysis of (3)J(H,H) coupling constants and interresidual NOE contacts. Both 2-deoxy-2-acetamido-galactose (GalNAc) residues were found in the (4)C(1) chair conformation, the unsaturated uronic acid (Delta-Hex A) adopts a strongly predominant half-chair (1)H(2) conformation, while the IdoA residue exists either in the (1)C(4) chair or in the (2)S(0) skewed boat geometries, in a 4:1 ratio. There is a moderate flexibility of Phi and Psi torsions as suggested by nuclear Overhauser effects (NOEs), molecular modeling (MM), and molecular dynamics (MD) studies. This was further investigated by residual dipolar couplings (RDCs). One-bond C--H RDCs ((1)D(C,H)) and long-range H-H ((3)D(H,H)) RDCs were measured for the tetrasaccharide in a phage solution and interpreted in combination with restrained MD simulation. The RDC-derived data substantially confirmed the validity of the conformer distribution resulting from the NOE-derived simulations, but allowed an improved definition of the conformational behavior of the oligosaccharides in solution. In summary, the data show a moderate flexibility of the four tetrasaccharide species at the central glycosidic linkage. Differences in the shapes of species with the IdoA in skew and in chair conformations and in the distribution of the sulfate groups have also been highlighted.

Negative-ion electrospray tandem mass spectrometry (ES-MS/MS) with collision-induced dissociation (CID) is attempted for sequence determination of alginate oligosaccharides, derived from polyanionic alginic acid, polymannuronate, and polyguluronate by partial depolymerization using either alginate lyase or mild acid hydrolysis. Sixteen homo- and hetero-oligomeric fragments were obtained after fractionation by gel-filtration and strong anion exchange high performance liquid chromatography. The product-ion spectra of these alginate oligosaccharides were dominated by intense B-, C-, Y-, and Z-type ions together with (0,2)A- and (2,5)A-ions of lower intensities. Internal mannuronate residues (M) produce weak but specific decarboxylated Z(int)-ions (Z(int) - 44 Da; int: denotes internal), which can be used for distinction of M and a guluronate residue (G) at an internal position. A reducing terminal M or G, although neither gives rise to a specific ion, can be identified by differences in the intensity ratio of fragment ions of the reducing terminal residue [(2,5)A(red)]/[(0,4)A(red)] (red: denotes reducing terminal).

Alginate that was purified from the fermentation solution of marine bacteria Vibro sp. 510 under specific reaction conditions was hydrolyzed by alginate lyase. Seven oligosaccharides, including di-, tri- and tetrasaccharides, were isolated through low-pressure, gel-permeation chromatography (LP-GPC) and semipreparative strong-anion exchange (SAX) fast-protein liquid chromatography (FPLC). The oligosaccharide structures were elucidated based on ESIMS and 2D NMR spectral analysis. The hydrolytic specificity of this alginate lyase to alginate is discussed., (Copyright 2004 Elsevier Ltd.)

Porcine intestinal mucosa heparin was partially depolymerized by recombinant heparinase 1 (heparin lyase 1, originating from Flavobacterium heparinum and expressed in Escherichia coli) and then fractionated, leading to the isolation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel electrophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their structures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600 MHz. Five of the characterized oligosaccharides represent new compounds. The most prominent oligosaccharide comprises the common repeating unit of heparin, ΔUA2S-[-GlcNS6S-IdoA2S-]n-GlcNS6S, where ΔUA is 4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-d-glucopyranose, IdoA is l-idopyranosyluronic acid, S is sulfate and n = 0–7. A second prominent heparin oligosaccharide motif corresponds to ΔUA2S-[GlcNS6S-IdoA2S]n-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n = 0–5 and GlcA is d-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in heparin. The prominence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the -GlcNS3S6S-IdoA2S- linkage is particularly susceptible to heparinase 1. [ABSTRACT FROM AUTHOR]

Isobaric oligosaccharides enzymatically prepared from hyaluronic acid (HA) and N-acetylheparosan (NAH), were distinguished using tandem mass spectrometry. The only difference between the two series of oligosaccharides was the linkage pattern (in HA 1→3 and in NAH 1→4) between glucuronic acid and N-acetylglucosamine residues. Tandem mass spectrometry afforded spectra in which glycosidic cleavage fragment ions were observed for both HA and NAH oligosaccharides. Cross-ring cleavage ions 0,2An and 0,2An-h (n is even number) were observed only in GlcNAc residues of NAH oligosaccharides. One exception was an 0,2A2 ion fragment observed for the disaccharide from HA. These cross-ring cleavage fragment ions are useful to definitively distinguish HA and NAH oligosaccharides. [Copyright &y& Elsevier]

A novel homogenous polysaccharide LPWF together with its three acid hydrolysis products LPWF1–3 were isolated and prepared from lotus plumule (germs of Nelumbo nucifera). LPWF was composed of rhamnose (Rha), arabinose (Ara), galactose (Gal), xylose (Xyl), and galacturonic acid (GalA) in the molar ratio of 7.3: 34.0: 7.0: 19.1: 32.6 with a molecular weight of 567.6 kDa. The structure of LPWF was elucidated by methylation and NMR analysis of LPWF1–3 and a follow-up structural assembling aided by high-resolution mass spectrometry mapping of oligosaccharides and ROSEY spectra. LPWF was characterized as an unusual pectin linked by rhamnogalacturonan I (RGI, composed of LPWF1–2) and xylogalacturonan (XGA, LPWF3). LPWF1 was an arabinan peeled from the RGI part with a 1,5-linked backbone branching on the O -2 position, while LPWF2 was the remaining part of RGI composed of Rha (36.1%), Gal (17.8%), and GalA (43.7%). LPWF3 was identified as the XGA part with a backbone of α-1,4-linked GalA and branches of mono-xylose substitutions on the O -3 of GalA. LPWF (25 μg/mL) demonstrated significant inhibitions on the expression of IL-1β, IL-6, and TNF-α in LPS-stimulated primary murine microglia cultures. LPWF1 and 2 showed selectively and significantly inhibitory activity against the expression of IL-1β. • Structure features of the pectin LPWF was determined by NMR and methylation. • Structural characterization of LPWF was elucidated by assembling three stepwise hydrolyzed fragments. • LPWF is speculated to be a linearly linked RGI and XGA pectin. • The pectin LPWF could significantly inhibit the expression of IL-1β, IL-6, and TNF-α. [ABSTRACT FROM AUTHOR]

An endo -β-1,3(4)-glucanase AnENG16A from Aspergillus nidulans shows distinctive catalytic features for hydrolysis of β-glucans. AnENG16A hydrolyzed Eisenia bicyclis laminarin to mainly generate 3-O-β-gentiobiosyl- d -glucose and hydrolyzed barley β-glucan to mainly produce 3-O-β-cellobiosyl- d -glucose. Using molecular exclusion chromatography, we isolated and purified 3-O-β-cellobiosyl- d -glucose and 3-O-β-gentiobiosyl- d -glucose, respectively, from AnENG16A-hydrolysate of barley β-glucan and E. bicyclis laminarin. Further study reveals that 3-O-β-cellobiosyl- d -glucose had 8.99-fold higher antioxidant activity than barley β-glucan and 3-O-β-gentiobiosyl- d -glucose exhibited 43.0% higher antioxidant activity than E. bicyclis laminarin. Notably, 3-O-β-cellobiosyl- d -glucose and 3-O-β-gentiobiosyl- d -glucose exhibited 148.9% and 116.0% higher antioxidant activity than laminaritriose, respectively, indicating that β-1,4-linkage or -1,6-linkage at non-reducing end of β-glucotrioses had enhancing effect on antioxidant activity compared to β-1,3-linkage. Furthermore, 3-O-β-cellobiosyl- d -glucose showed 237.9% higher antioxidant activity than cellotriose, and laminarin showed 5.06-fold higher antioxidant activity than barley β-glucan, indicating that β-1,4-linkage at reducing end of β-glucans or oligosaccharides resulted in decrease of antioxidant activity compared to β-1,3-linkage. • A distinctive β-glucanase for preparation of glucooligosaccharide from β-glucan • Superdex™ 30 open column can efficiently purification of glucooligosaccharides. • Antioxidant activity increases with increase of DP of glucooligosaccharides. • β-1,4/1,6-Linkage at non-reducing end enhance antioxidant activity of oligosaccharide. • β-1,4-Linkage at reducing end decreases antioxidant activity of glucooligosaccharide. [ABSTRACT FROM AUTHOR]

Hyaluronic acid (HA), a glycosaminoglycan, is a linear polysaccharide with negative charge, composed of a repeating disaccharide unit [→4)-β- d -glucopyranosyluronic acid (1 → 3)-β- d - N -acetyl- d -glucoaminopyranose (1 → ] n ([→4) GlcA (1 → 3) GlcNAc 1 → ] n ). It is widely used in different applications based on its physicochemical properties associated with its molecular weight. Enzymatic digestion by polysaccharides lyases is one of the most important ways to decrease the molecular weight of HA. Thus, it is important to understand the action patterns of lyases acting on HA. In this study, the action patterns of two common lyases, Flavobacterial chondroitinase AC I and Streptomyces hyaluronidase, were investigated by analyzing HA oligosaccharide digestion products. HA oligosaccharides having an odd-number of saccharide residues were observed in the products of both lyases, but their distributions were quite different. Chondroitinase AC acted more efficiently at the β 1–4 glycosidic bond linking GlcNAc and GlcA. Oligosaccharides, having an even number of saccharide residues, and with an unsaturated uronic acid (4-deoxy-α- l - threo -hex-4-enepyranosyluronic acid, △UA) residue at their non-reducing end represent the major product. A minor amount of oligosaccharides having an odd number of saccharide residues resulted from the irregular terminal residues of HA substrate chains. Hyaluronidase showed a more complicated product mixture. Its minimum recognition and digestion domain is HA heptasaccharide and it could cleave both β 1–4 and β 1–3 glycosidic linkages. The HA oligosaccharides, generated with a 2-acetamido-2,3-di-deoxy-β- d - erythro -hex-2-enopyranose (△HexNAc) at the non-reducing end, are believed to be unstable and undergo breakdown immediately after their generation, and the oligosaccharides with △UA residue at the non-reducing end are formed. Thus, oligosaccharides having both an even and odd-number saccharide residues with a △UA residue at their non-reducing ends, represent the major products of hyaluronidase acting on HA. [ABSTRACT FROM AUTHOR]

Investigation of the action pattern of enzymes acting on carbohydrates is challenging, as both the substrate and the digestion products are complex mixtures. Dextran and its enzyme-derived oligosaccharides are widely used for many industrial applications. In this work, a new method relying on ultra-performance hydrophilic interaction liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UP-HILIC- Q/TOF-MS/MS) was developed to analyze a complex mixture of dextran oligosaccharide products to determine the action pattern of dextranase. No derivatization of oligosaccharides was required and the impact of the α- and β-configurations of the native oligosaccharides on the chromatographic separation was eliminated. The 1→6, 1→3, 1→4 backbone linkages and the branch linkages of these oligosaccharides were all distinguished from diagnostic ions in their MS/MS spectra, including fragments corresponding to A, A, A, B-HO, A, and A. The sequences of the oligosaccharide products were similarly established. Thus, the complex oligosaccharide mixtures in dextran digestion products were profiled and identified using this method. The more enzyme-resistant structures in dextran were established using much less sample, labor, time, and uncertainty than in previous studies. This method provides an efficient, sensitive, and straightforward way to monitor the entire process of digestion, establish the action pattern of the dextranase from Penicillium sp., and to support the proper industrial application of dextranase. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The TEMPO/NaBr/NaClO system was used to modify konjac glucomannan and prepare β-1,4-linked D-mannuronic/glucuronic acid oligosaccharides. The impact of oxidant amount on the degree of oxidation, molecular weight, terminal structure and the ratio of sugar units were systematically investigated. Mannoses were transformed to mannuronic acids quantitatively, while some glucose were transformed to glucuronic acids, some degraded. The unspecific oxidation causes strong degradation of konjac glucomannan sugar chains. Multiple terminal structures were observed in the oxidized KGM products. According to those observations, the oxidation pattern of konjac glucomannan with the TEMPO/NaBr/NaClO system was speculated. It is suggested that the axial hydroxyl group at position 2 in mannose might form a hydrogen bond with the oxygen on the sugar ring. It would against the unspecific oxidation. While, both specific and unspecific oxidations were observed at glucose units with an equatorial hydroxyl group at position 2, and the degradation occurs mostly on glucose during the oxidation. [Display omitted] • TEMPO/NaBr/NaClO system was used to modify konjac glucomannan. • The structure of oxidized konjac glucomannan was fully characterized. • Mannose is more stable than glucose in the KGM during oxidation. • The oxidation pattern of KGM with the TEMPO/NaBr/NaClO system was speculated. [ABSTRACT FROM AUTHOR]

Low molecular weight heparins (LMWHs), depolymerized from unfractionated heparin (UFH), are widely used as anticoagulant drugs in clinic. The variable degradation methods result the different types of LMWHs, such as enoxaparin prepared by alkaline degradation following benzylation and nadroparin degraded by nitrous acid and subsequent reduction. They have different anticoagulant activities, molecular weight and special oligosaccharide sequences. Oligosaccharide analysis of the heparinase-catalyzed digestion products of heparin and LMWHs is an important way to explore the fine structural composition. In this work, a MHC-2D-LC-MS system using SAX followed by SEC and tandem to MS was applied to analyze the heparinase-products of LMWHs. 15 components of enoxaparin and 20 components of nadroparin were separated and unambiguously characterized with mass spectrum, including eight common disaccharides, and the special structural domains resistant to enzyme digestion which have the 3- O sulfated residue and/or characteristic terminal residues and the linkage region tetrasaccharides. • The platform of MHC 2D-LC linked to MS was applied to analyze digested LMWHs. • SAX is the first dimensional, SEC is the second dimensional chromatography. • 15 and 20 structural domains in enoxaparin and nadroparin were identified. • Common structural framework and special structural domains were observed. [ABSTRACT FROM AUTHOR]