molecules Review Chemical Modification of Glycosaminoglycan Polysaccharides Lais C. G. F. Palhares 1 , James A. London 2 , Aleksandra M. Kozlowski 3, Emiliano Esposito 4, Suely F. Chavante 1, Minghong Ni 4 and Edwin A. Yates 2,* 1 Programa de Pós-graduação em Bioquímica e Biologia Molecular, Departamento de Bioquímica, Universidade do Rio Grande do Norte (UFRN), Natal 59012-570, Brazil; [email protected] (L.C.G.F.P.); [email protected] (S.F.C.) 2 Department of Biochemistry and Systems Biology, ISMIB, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK; [email protected] 3 Department of Chemistry and Chemical Engineering, Forest Products and Chemical Engineering, Chalmers University of Technology, Kemigården 4, 412 58 Göteborg, Sweden; [email protected] 4 Instituto di Richerche Chemiche e Biochemiche, ‘G.Ronzoni’, Via G Colombo 81, 20133 Milano, Italy; [email protected] (E.E.); [email protected] (M.N.) * Correspondence: [email protected] or [email protected]; Tel.: +44-151-795-4429 Abstract: The linear anionic class of polysaccharides, glycosaminoglycans (GAGs), are critical throughout the animal kingdom for developmental processes and the maintenance of healthy tissues. They are also of interest as a means of influencing biochemical processes. One member of the GAG family, heparin, is exploited globally as a major anticoagulant pharmaceutical and there is a growing interest in the potential of other GAGs for diverse applications ranging from skin care to the treatment of neurodegenerative conditions, and from the treatment and prevention of microbial infection to biotechnology. To realize the potential of GAGs, however, it is necessary to develop effective tools that are able to exploit the chemical manipulations to which GAGs are susceptible. Here, the current Citation: Palhares, L.C.G.F.; knowledge concerning the chemical modification of GAGs, one of the principal approaches for London, J.A.; Kozlowski, A.M.; the study of the structure-function relationships in these molecules, is reviewed. Some additional Esposito, E.; Chavante, S.F.; Ni, M.; methods that were applied successfully to the analysis and/or processing of other carbohydrates, Yates, E.A. Chemical Modification of but which could be suitable in GAG chemistry, are also discussed. Glycosaminoglycan Polysaccharides. Molecules 2021, 26, 5211. https:// Keywords: glycosaminoglycans; polysaccharides; sulfation; chemical modification doi.org/10.3390/molecules26175211 Academic Editor: Farid Chemat 1. Introduction Received: 20 July 2021 Among polysaccharides, the glycosaminoglycans (GAGs) are notable because of their Accepted: 21 August 2021 Published: 27 August 2021 participation in diverse biological events and their many biomedical applications. Their structural analysis can also depend on their chemical derivatization. The availability of methods for the chemical modification of GAGs enables the preparation of products with Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in improved therapeutic potential [1]. Several studies describing or utilizing the modification published maps and institutional affil- of GAGs for structural analysis or biotechnological purposes have been reported [2–7]. In iations. this review, we address chemical methods for the modification of GAG polysaccharides, concentrating largely on modifications within the polysaccharide chain that result in naturally-occurring residues being produced, albeit in potentially unnatural sequences, rather than modifying the structure through the addition of new chemical groups. The modifications at reducing ends or the non-reducing terminus, e.g., the formation of a Copyright: © 2021 by the authors. double bond by, for example, lyase enzyme action, with a few exceptions, have largely Licensee MDPI, Basel, Switzerland. This article is an open access article been excluded. We also discuss selected methods taken from the wider chemical literature distributed under the terms and that, in future, could be applied to modify GAGs. conditions of the Creative Commons 1.1. Structure and Classes of Glycosaminoglycans Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ GAGs represent a complex group of macromolecules found in the extracellular ma- 4.0/). trices and tissues of vertebrates and invertebrates [8], and can be localized inside and Molecules 2021, 26, 5211. https://doi.org/10.3390/molecules26175211 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 22 Molecules 2021, 26, 5211 1.1. Structure and Classes of Glycosaminoglycans 2 of 22 GAGs represent a complex group of macromolecules found in the extracellular ma- trices and tissues of vertebrates and invertebrates [8], and can be localized inside and on the surface of cells. These linear anionic polysaccharides are comprised of repeating di- on the surface of cells. These linear anionic polysaccharides are comprised of repeating saccharide units, consisting of a hexosamine (D-glucosamine or D-galactosamine) residue disaccharide units, consisting of a hexosamine (D-glucosamine or D-galactosamine) residue boundbound to to a non-nitrogenateda non-nitrogenated sugar sugar residue residue (D-glucoronate, (D-glucoronate, L-iduronate, L-iduronate, or D-galactose) or D-galactose) [9]. The[9]. identitiesThe identities of these of these residues, residues, the number the number and and position position of the of sulfatethe sulfate groups, groups, as well as well as theas the positions positions and and configurations configurations of glycosidic of glycosidic bonds, bonds, [10 ][10] distinguish distinguish the the different different classes clas- ofses GAGs, of GAGs, i.e., chondroitin-4 i.e. chondroitin- and4 -6 and sulfate -6 sulfate (CS, also (CS, known also asknown chondroitin as chondroitin sulfate-A sulfate-A and -C), dermatanand -C), dermatan sulfate (DS), sulfate heparan (DS), heparan sulfate (HS),sulfate heparin (HS), heparin (Hp), hyaluronic (Hp), hyaluronic acid (HA), acid (HA), and keratanand keratan sulfate sulfate (KS) [(KS)11]. [11]. 1.2.1.2. General General Properties Properties FromFrom a a structural structural point point of of view, view, GAGs GAGs are are linear linear anionic anionic polysaccharides polysaccharides composed composed ofof multiple multiple repeating repeating disaccharides disaccharides (Figure (Figure1 ),1), with with numerous numerous sequence sequence variants variants giving giving riserise to to heterogeneousheterogeneous populations. For For exampl example,e, octasaccharides octasaccharides from from HS HScould could have have over over1,000,000 1,000,000 hypothetical hypothetical sulfation sulfation arrangements arrangements [12] [and,12] and,if only if onlya small a small proportion proportion of these of thesepossibilities possibilities are observed, are observed, this still this represents still represents considerable considerable heteroge heterogeneity.neity. This wide This widerange rangeof structural of structural variations variations is accompanied is accompanied by extensive by extensive molecular molecular weight variation, weight variation, different differentelectronegativity electronegativity characteristics, characteristics, and sulfate and clustering sulfate clusteringto generate to a generatedistinct binding a distinct po- bindingtential and potential distinct and regions distinct within regions the withinchains. theWhile chains. the molecular While the weight molecular of purified weight Hp of purifiedis typically Hp around is typically 12–16 around kDa, HA 12–16 can kDa,reach HA 10,000 can kDa reach [13], 10,000 but, within kDa [13 a], particular but, within GAG a particularpopulation, GAG varying population, molecular varying weights molecular are usually weights observed are usually [14]. observed [14]. FigureFigure 1. 1.Typical Typical repeating repeating disaccharides disaccharides of chondroitinof chondroitin ((A ),((A upper)), upper) and and dermatan dermatan ((A), (( lower)A), lower) sul- sulfates ((A), lower) and repeating disaccharides of heparan sulfate ((B), upper) and heparin ((B), fates ((A), lower) and repeating disaccharides of heparan sulfate ((B), upper) and heparin ((B), lower). lower). The biological activities of GAGs arise predominantly from their interaction with proteins.The Thebiological binding activities forces involved of GAGs are arise usually predominantly dominated byfrom ionic their interactions, interaction espe- with ciallyproteins. between The binding the basic forces amino involved acids, lysine are usua andlly arginine dominated on the by proteinionic interactions, surface. These espe- interactionscially between are supportedthe basic amino by hydrogen acids, lysine bonding and involving arginine polaron the residues, protein whichsurface. are These cru- cial,interactions together are with supported a degree by of shapehydrogen complementarity, bonding involving to the polar formation residues, of GAG-proteinwhich are cru- complexescial, together between, with a for degree example, of shape growth complementarity, factors and their to receptors the formation [15]. High of GAG-protein molecular weightcomplexes HA canbetween, stimulate for cellexample, surface growth CD44 clustering,factors and while their smallreceptors oligosaccharides [15]. High molecular attenu- ateweight the clustering HA can stimulate effect [16 ].cell Besides surface the CD44 variation clustering, in molecular while small weight, oligosaccharides the polyelectrolyte atten- propertiesuate