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A new glycosaminoglycan (LF-GAG) was purified from the slug Limacus flavus. Its unique chemical structure and heparanase inhibitory activity were studied in this work. The native LF-GAG was composed of L-iduronic acid (L-IdoA) and N-acetyl-D-glucosamine (D-GlcNAc), with a Mw of 22,700 Da. To elucidate the precise structure and structure-activity relationship, its deacetylation-deaminative depolymerized product (dLF-GAG) was prepared, and from which four oligosaccharides were purified. Combining the NMR spectral analysis of LF-GAG and its derived oligosaccharides, the structure of LF-GAG was deduced to be -4)-L-IdoA2R-(α1,4)-D-GlcNAc-(α1-, in which R was −OH (˜80%) or –OSO3− (˜20%). Bioactivity assays showed that LF-GAG could potently inhibit human heparanase (IC50, 0.10 μM). dLF-GAG and LF-3 were less potent but also active for heparanase inhibition. Structure-activity relationship analysis indicated that the chain length and sulfate substitution of LF-GAG are essential for its heparanase inhibitory activity.

Enoxaparin, widely used antithrombotic drug, is a polydisperse glycosaminoglycan with highly microheterogeneous structure dictated by both parent heparin heterogeneity and depolymerization conditions. While the process-related modifications of internal and terminal sequences of enoxaparin have been extensively studied, very little is known about the authentic non-reducing ends (NRE). In the present study a multi-step isolation and thorough structural elucidation by NMR and LC/MS allowed to identify 16 saturated tetramers along with 23 unsaturated ones in the complex enoxaparin tetrasaccharide fraction. Altogether the elucidated structures represent a unique enoxaparin signature, whereas the composition of saturated tetramers provides a structural readout strictly related to the biosynthesis of parent heparin NRE. In particular, both glucuronic and iduronic acids were detected at the NRE of macromolecular heparin. The tetrasaccharides bearing glucosamine at the NRE are most likely associated with the heparanase hydrolytic action. High sulfation degree and 3-O-sulfation are characteristic for both types of NRE.

Green seaweeds of the genus Ulva are rich in the bioactive sulfated polysaccharide ulvan. Herein we characterise ulvan from Ulva species collected from the Bay of Plenty, Aotearoa New Zealand. Using standardised procedures, we quantified, characterised, and compared ulvans from blade (U. australis, U. rigida, U. sp. B, and Ulva sp.) and filamentous (U. flexuosa, U. compressa, U. prolifera, and U. ralfsii) Ulva species. There were distinct differences in composition and structure of ulvans between morphologies. Ulvan isolated from blade species had higher yields (14.0-19.3 %) and iduronic acid content (IdoA = 7-18 mol%), and lower molecular weight (Mw = 190-254 kDa) and storage moduli (G' = 0.1-6.6 Pa) than filamentous species (yield = 7.2-14.6 %; IdoA = 4-7 mol%; Mw = 260-406 kDa; G' = 22.7-74.2 Pa). These results highlight the variability of the physicochemical properties of ulvan from different Ulva sources, and identifies a morphology-based division within the genus Ulva.

Crude anionic polysaccharides extracted from the Pacific starfish Lysastrosoma anthosticta were separated by anion-exchange chromatography into fractions LA-F1 and LA-F2. The main fraction LA-F1 was solvolytically desulfated giving rise to preparation LA-F1-DS with a structure of dermatan core [→3)-β-d-GalNAc-(1→4)-α-l-IdoA-(1→]n. Reduction of LA-F1 afforded preparation LA-F1-RED composed mainly of the repeating disaccharide units →3)-β-d-GalNAc4R-(1→4)-α-l-Ido2S3S-(1→, where R was SO3- or H. Analysis of the NMR spectra of the parent fraction LA-F1 led to determine the main component as the oversulfated dermatan sulfate LA-Derm bearing sulfate groups at O-2 and O-3 of α-l-iduronic acid, as well as at O-4 of some N-acetyl-d-galactosamine residues. The minor fraction LA-F2 contained a mixture of LA-Derm and heparinoid LA-Hep, the latter being composed of the fragments →4)-α-d-GlcNS3S6S-(1→4)-α-l-IdoA2S3S-(1→ and →4)-α-d-GlcNS3S-(1→4)-α-l-IdoA2S3S-(1→. The presence of 2,3-di-O-sulfated iduronic acid residues is very unusual both for natural dermatan sulfate and heparinoid. Preparations LA-F1, LA-F2 and LA-F1-RED demonstrated significant anticoagulant effect in vitro.

Diabetic foot ulcer (DFU) is a common high-risk complication in patients with diabetes mellitus, but current drugs and therapies in management of this disease cannot meet the urgent clinical needs. In this study, a snail glycosaminoglycan (SGAG) from the cultured China white jade snail was purified and structurally clarified. This snail glycosaminoglycan is a regular sulfated polysaccharide, composed of iduronic acid (IdoA) and N-acetyl-glucosamine (GlcNAc) with the repeating sequence of →4)-α-GlcNAc (1→4)-α-IdoA2S (1→. The biological assays showed that SGAG had no anticoagulant activity for lacking specific heparin pentasaccharide sequence. The pharmacological experiments suggested that SGAG markedly accelerated the healing of full-thickness wounds in diabetic mice skin. Histologic and immunohistochemical analysis revealed that SGAG treatment alleviated the inflammation and dermal edema, and promoted angiogenesis. This is the first report applying the snail glycosaminoglycan to favor diabetic wound healing.

The cell wall polysaccharide ulvan was isolated from two species of the seaweed Ulva collected along the Swedish west coast. Acidic extraction was benchmarked against hot water extraction with enzymatic purification and against commercial ulvan. Extracted ulvan contained 11–18 % g/g of ash, some protein (up to 1.3 % g N/g) but minimal colored impurities. The ulvans had high molecular weights (660,000–760,000 g/mol) and were composed of 77–79 % g/g carbohydrates, mainly rhamnose, xylose, glucose, glucuronic acid, and iduronic acid. The extraction protocol and the ulvan source strongly impact the molecular weight and the chemical composition. Acidic extraction caused almost complete desulfation of the isolated ulvan while the other method preserved a significant degree of SO3 substituents. Elemental analysis of ash remaining after thermal degradation showed presence of common mineral elements such as Na, Ca, Mg, Al, and K, but none of the heavy metals Pb, Hg, or As.

Contamination in heparin batches during early 2008 has resulted in a significant effort to develop a safer bioengineered heparin using bacterial capsular polysaccharide heparosan and recombinant enzymes derived from the heparin/heparan sulfate biosynthetic pathway. This requires controlled chemical N-deacetylation/ N-sulfonation of heparosan followed by epimerization of most of its glucuronic acid residues to iduronic acid and O-sulfation of the C2 position of iduronic acid and the C3 and C6 positions of the glucosamine residues. A combinatorial study of multi-enzyme, one-pot, in vitro biocatalytic synthesis, carried out in tandem with sensitive analytical techniques, reveals controlled structural changes leading to heparin products similar to animal- derived heparin active pharmaceutical ingredients. Liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy analysis confirms an abundance of heparin’s characteristic trisulfated disaccharide, as well as 3-O-sulfo containing residues critical for heparin binding to antithrombin III and its anticoagulant activity. The bioengineered heparins prepared using this simplified one-pot chemoenzymatic synthesis also show in vitro anticoagulant activity.

Edible snails have been widely used as a health food and medicine in many countries. A unique glycosaminoglycan (AF-GAG) was purified from Achatina fulica. Its structure was analyzed and characterized by chemical and instrumental methods, such as Fourier transform infrared spectroscopy, analysis of monosaccharide composition, and 1D/2D nuclear magnetic resonance spectroscopy. Chemical composition analysis indicated that AF-GAG is composed of iduronic acid (IdoA) and N-acetyl-glucosamine (GlcNAc) and its average molecular weight is 118kDa. Structural analysis clarified that the uronic acid unit in glycosaminoglycan (GAG) is the fully epimerized and the sequence of AF-GAG is →4)-α-GlcNAc (1→4)-α-IdoA2S (1→. Although its structure with a uniform repeating disaccharide is similar to those of heparin and heparan sulfate, this GAG is structurally highly regular and homogeneous. Anticoagulant activity assays indicated that AF-GAG exhibits no anticoagulant activities, but considering its structural characteristic, other bioactivities such as heparanase inhibition may be worthy of further study.

about the influence of the extraction process on composition of the extracts and in ulvan itself. In this context, the two main objectives of the present work are (1) the establishment of an efficient extraction process for ulvan and (2) development of an accurate characterization methodology to evaluate the extract composition and ulvan content. Three ulvan-rich extracts obtained by different schemes of extraction were studied. The methodology for the analysis was improved and a detailed analysis of extracted ulvan was provided. The polysaccharide is rich in ulvanobiuronic acid 3-sulfate type A [→4)- -d-GlcAp-(1 → 4)- -l-Rhap 3S-(1→], with minor amounts of ulvanobiuronic acid 3-sulfate type B [→4)- -l-IdoAp-(1 → 4)- -l-Rhap 3S-(1→]. The extract with the higher degree of purification is a high molecular weight polysaccharide (790 kDa) composed of rhamnose (22.4%), glucuronic acid (22.5%), xylose (3.7%), iduronic acid (3.1%) and glucose (1.0%). It is highly sulfated (32.2%) and contains 1.3% of proteins and 10.3% of inorganic material. Applying simple extraction scheme it was possible to obtain an extract from green algae with high content of ulvan without affecting the overall chemical structure of the polysaccharide., This work was supported by the project IBEROMARE ('Centro Multipolar de Valorizacao de Recursos e ResiduosMarinhos'), approved by Operational Programme for Cross-border Cooperation: Spain - Portugal, 2007-2013 (POCTEP), with funding contribution through the European Regional Development Fund (ERDF co-funding) and POCTEP.Anabela Alves is grateful for financial support from Fundacaopara a Ciencia e Tecnologia (FCT) through the SFRH/BD/39359/2007 grant.The authors thank Novozymes for kindly providing the enzymatic mixture. Authors are also grateful to Dra. Mariana Andrade and Materials Center of the University of Porto (CEMUP) for the acquisition of 13C NMR spectra (NMR spectrometer is part of the National NMR Network and was purchased in the framework of the National Program for Scientific Re-equipment, contract REDE/1517/RMN/2005, with funds from POCI 2010 (FEDER) and FCT).

Controlled physical depolymerization of heparin was performed in aqueous solution in the presence of isopropanol by γ-irradiation. Isopropanol both makes selective the radical-induced scission of the glycosidic linkage involved in the depolymerization and acts as hydrogen donor scavenger of heparin intermediate radicals that usually result in UV-absorbing by-products. Several preparations of heparin-derived oligosaccharides (HDO) were reproducibly obtained from different unfractionated heparins (UFH). From each preparation, an intermediate fraction with an average molecular weight of about 2200 Da (γ-HDO) was isolated by gel permeation chromatography and its sulfation pattern was characterized by nuclear magnetic resonance spectroscopy. The comparison of the sulfation pattern of HDO with that of parent UFHs revealed a significant decrease in the relative content of d -glucuronic acid with respect to the total amount of uronic acid. Further γ-ray treatments of heparin chains that survived the first depolymerization procedure led to a further decrease of d -glucuronic acid and also slightly reduced the content of N-acetyl-glucosamine residues, thus enriching oligosaccharide chains in the highly sulfated sequences. MALDI mass spectrometric analysis of oligomeric components indicated that γ-HDO prevalently contains highly sulfated chains (tetra to decasaccharides, including both odd and even number of monosaccharidic units) chiefly constituted of repeating sequences of the trisulfated disaccharide l -iduronic acid 2-sulfate (1→4) d -glucosamine-N,6 disulphate. γ-Irradiation in the presence of isopropanol thus generated heparin fragments largely preserving the prevalent structure of the parent heparin and involved glucuronic acid as the major site of cleavage.

A method for the isolation and purification of peptidoglycan heparan sulfate (HS) from bovine liver is described. Peptidoglycan HS was purified from bovine liver and its structure was characterized. The purity and identity of this HS was determined following treatment with heparin lyases by using polyacrylamide gel electrophoresis. The disaccharide compositional analysis on the peptidoglycan samples, determined by treatment with a mixture of heparin lyases followed by high-resolution capillary electrophoresis, showed that bovine liver HS was more highly sulfated than the standard porcine intestinal mucosal HS, but it contained no measurable amount of trisulfated disaccharide. One-dimensional 1H-NMR spectroscopy was used to calculate the relative amounts of glucuronic and iduronic acid residues that were present. The presence of peptide chain in this HS was confirmed by amino acid composition analysis and its selective biotinylation and conjugation with streptavidin.

The present study describes the isolation, purification and structural characterization of heparan sulfate (HS) from marine scallop. Enzymatic depolymerization of the isolated HS, followed by polyacrylamide gradient gel electrophoresis (PAGE), confirmed its presence of HS is by metachromatic activity and agarose gel electrophoresis. The purity of HS is determined by the following treatment with heparin lyases though PAGE. 1 H NMR spectroscopy revealed that the marine scallop HS is enriched in both glucuronic and sulfated iduronic acid residues. The result of this analysis clearly demonstrates that the isolated glycosaminoglycan (GAG) is HS. Marine scallop HS has contents of carbon, hydrogen, nitrogen and sulfated disaccharide and an elevated average sulfation level. The activated partial thromboblastin time (APTT) and prothrombin time (PT) of marine scallop HS, are lower than standard bovine intestinal HS. Moreover, marine scallop HS shows equal degree of sulfation to HS standard.