Polymer Journal (2016) 48, 229–237 & 2016 The Society of Polymer Science, Japan (SPSJ) All rights reserved 0032-3896/16 www.nature.com/pj INVITED REVIEW Development of glycosaminoglycan mimetics using glycopolymers Yoshiko Miura1, Tomohiro Fukuda2, Hirokazu Seto1 and Yu Hoshino1 Glycosaminoglycans (GAGs) are polysaccharides found in living systems that have key biological roles and function as polyelectrolytes owing to their large number of sulfate groups. There have been many reports describing the syntheses of GAGs and the development of GAG mimetics and analogs. The preparation of such GAG mimics has utilized versatile methods ranging from total syntheses to synthetic polymer chemistry approaches. The core of GAG mimetic production is the fusion of complex chemical structures with polymeric properties. Multivalent interactions of the saccharides with specific biological targets, such as proteins, are an essential function of GAGs and other multivalent saccharides. In this review, methods for generating GAGs from glycopolymers are presented and research reports describing the functional characterization of the synthesized GAGs are outlined. Polymer Journal (2016) 48, 229–237; doi:10.1038/pj.2015.110; published online 11 November 2015 INTRODUCTION chondroitin sulfate is a sulfated alternating copolymer of GalNAc and Saccharides on cell surfaces have important roles in living systems1 GlcA or IdoA. The complex sulfation patterns of the saccharides, and exist as glycolipids, glycoproteins and polysaccharides. Glycans saccharide structures, chiral (epimeric) centers and stereo-selective have various functions that include serving as an energy source, glycoside bonds result in rich structural diversity. increasing the water solubility of proteins and chemicals, forming Because GAGs cover the cell surface as an extracellular matrix, they components of cell walls and acting as biological ligands for molecular have numerous roles in living systems, including cell growth, cell recognition. In particular, their role as biological ligands has received differentiation and defense against pathogen infection. Some GAGs significant attention in the fields of biochemistry and biomaterials. have been used in practical settings. The antithrombogenic activity of Saccharides on the cell surface mediate biological signals through heparin has received significant attention for use as a therapeutic and molecular recognition. Among the numerous types of saccharides, biomaterial.3 Heparin, which is used clinically, binds antithrombin III glycosaminoglycans (GAGs) and proteoglycans are important (ATIII) to control the activity of thrombin and inhibits thrombus biological ligands, because they mediate an abundance of biological formation. The application of heparin-immobilized materials for phenomena and control living systems. GAGs and proteoglycans exist antithrombogenic applications has been previously investigated, for universally on the cell surface. Proteoglycans are glycoproteins that example, for use in artificial vessels.4,5 The cell growth factor of have GAGs and core proteins, making their functions dependent on b-fibroblast growth factor (FGF) has been reported to bind heparin; GAGs (Figure 1). such heparin binding facilitates dimerization, activation and cell GAGs have an alternating copolymer structure composed of amino- proliferation.6 Other growth factors, such as vascular endothelial sugars (N-acetyl glucosamine (GlcNAc) and N-acetyl galactosamine growth factor and epidermal growth factor, have been reported to (GalNAc)) and uronic acids (iduronic acid (IdoA) and glucuronic acid bind GAGs for activation and stabilization of these proteins.7,8 GAGs (GlcA)).2 Most GAGs are highly sulfated, with the exception of also protect cells from pathogen infection by bacteria, viruses and hyaluronic acid, and their sulfation patterns and saccharide combina- other toxic proteins. Although GAGs protect cells from infectious tions are used to distinguish the different types of GAGs. GAGs are pathogens,2 some viruses specifically target GAGs for cell invasion.9–11 natural polymers and polyelectrolytes with molecular weights reaching By contrast, the infection of some viruses is inhibited by the presence 50 kDa, in the case of heparan sulfate. GAGs are divided into different of GAGs.12,13 GAGs also regulate inflammation by interacting with types of polysaccharides based on their unique saccharide structures, selectins.14–16 sulfation patterns and molecular weights (Figure 2). For example, Studies of GAGs hold a prominent position in the fields of hyaluronic acid is an unsulfated alternating copolymer of GlcNAc and glycoscience, biochemistry and biomaterials. GAGs are challenging GlcA. Heparin and heparan sulfate are highly sulfated alternating to study because of difficulties associated with their availability, copolymers of GlcNAc and GlcA or IdoA, respectively. Similarly, whereby their synthesis or production is a limiting factor. Research 1Department of Chemical Engineering, Faculty of Engineering, Kyushu University, Fukuoka, Japan and 2Department of Applied Chemistry and Chemical Engineering, National Institute of Technology, Toyama College, Toyama, Japan Correspondence: Professor Y Miura, Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan. E-mail: [email protected] Received 7 July 2015; revised 24 September 2015; accepted 25 September 2015; published online 11 November 2015 Practical approaches to synthesizing GAG mimics by polymerization chemistry YMiuraet al 230 Infection of Pathogens Antithrombogenic activity Tuning of Cell-Cell thrombin Enzyme Activitiy Interaction Anti-thrombin III Enzyme Virus Cell Growth factor Activation b-FGF Protein Amyloidosis Cell Figure 1 Schematic illustration of the functions of GAGs on the cell surface. Disaccharide Structure of Glycosaminoglycans has focused on preparing GAGs using chemical and biological Heparin and Heparan Sulfate syntheses, with a particular emphasis on oligosaccharide 17 α−IdoA-(1-4)-α-GlcNAc β-GlcA-(1-4)-α-GlcNAc production. However, GAGs are natural ‘polymers’ and function as polyelectrolytes; thus the production of new GAG mimetics using OR 1 - OOC OR polymer chemistry has considerable potential. In this review, we O O O 1 O O describe the preparation of glycopolymers that act as GAG mimetics. OH HO HO O - NHR2 OOC O OR1 HO O NHR2 SYNTHESIS OF GAGS USING SYNTHETIC AND BIOLOGICAL O OR O 1 METHODS Chondroitin Sulfate Dermatan Sulfate GAGs have been prepared using both chemical and biological β-GlcA-(1-3)-β-GalNAc α−IdoA-(1-3)-β-GalNAc methods. Because of their complex saccharide structures, they have - been a target of total synthesis within the field of organic chemistry. OR1 OSO3 OR1 - OH The total synthesis of GAGs enables the detailed investigation of the COO O O O O O various functions of GAGs; however, complicated glycosylation and O O HO NHAc OH OH -OOC O NHAc regioselective sulfonation procedures are required (Figure 3). The syntheses are frequently designed by retro-syntheses. An efficient Hyaluronic Acid O OH synthetic method that includes the use of protective groups β-GlcA-(1-3)-β-GlcNAc and glycosylation is necessary to successfully synthesize GAG oligo- - OH - saccharides. There have been many reports describing the synthesis OOC R1=SO3 or H, O HO O - 17,18 O O R2=COCH3 or SO3 of oligosaccharides of GAGs, with numerous research groups HO O OH NHAc reporting novel methods for synthesizing GAGs, including efforts to prepare longer saccharides using facile synthetic approaches. Figure 2 Chemical structure of GAG components. The synthesis and biological activity of heparin and heparan sulfate oligosaccharides have been previously studied. The biological functions of these saccharides have been investigated using various oligosaccharides. IdoA(2S)(α1-4)GlcNS(6S) is a major structure17 that Glycosylation has been reported to bind to ATIII,18 blood platelets,19,20 basic FGF (b-FGF)21 and the Alzheimer amyloid precursor protein.22 The Petitou group reported the synthesis of the disaccharide IdoA(2S) (α1-4)GlcNS(6S),23 whereas the Russo group reported the synthesis of a different GAG disaccharide.24 - Sulfonation O3SO The Kusumoto group reported the syntheses of heparin and O - O 20,25,26 OSO3 OH HO heparan sulfate oligosaccharides. This group reported the O - -O SHN synthesis of IdoA(2S)(α1-4)GlcNS(6S) and found that the disaccharide HO OOC O 3 OMe HO moiety inhibited heparin from binding to blood platelets.20 They also -O SHN - 3 - - O OSO3 α β COO O3SHN reported the synthesis of GlcNS(6S)( 1-4)GlcA( 1-4)GlcNS(3S, 6S) - O O O3SO (α1-4)IdoA(2S)(α1-4)GlcNS(6S), which efficiently inhibited heparin HO O O - binding to platelets. The Suda group reported the syntheses of a OSO3 - OSO3 glycoconjugate and multivalent compounds using a heparin oligo- saccharide, which is described in the next section.27 The Sinaÿ group Figure 3 Total syntheses of GAG pentasaccharides. The syntheses were 28 attained by conjugating functional disaccharides and trisaccharides and were reported the binding of an oligosaccharide to ATIII. By modifying a designed retro-synthetically. A full color version of this figure is available at heparin pentasaccharide, the authors found that this compound had a the Polymer Journal online. reduced affinity toward ATIII compared with heparin. This group also Polymer Journal Practical approaches to synthesizing GAG mimics by polymerization chemistry YMiuraet al 231 - - O3SO OH -O SO OOC -OOC 3 OH HO O O O O O O HO HO O OH O OH NHAc N n HO Enzymatic Polymerization -OOC OH HO O O with Hyaluronidase HO O OH O N - OH OH -O SO + OOC -OOC 3 OH O O O O O O HO O O -O SO HO -OOC 3 OH OH NHAc OH NHAc O O HO n HO O OH O N Figure 4 Preparation of chondroitin sulfate using chemoenzymatic synthesis based on the enzymatic polymerization of hyaluronidase. Functional Saccharides Multivalency OSO3Na HO O O HO O O HO OH NH CO CH2 CH2 NH CO CH2 NHCOCH2 NH CO CH NaO3SHN NaOOC OHHO NH 2 S HO OH S 2 2 OSO OSO OSO OSO OOC OOC OOC OSO OH O O O O O O SO HO O O O O HO O O HO O SHN HO O O SO O OH NHAc OH NHAc O 2 n Figure 5 Preparation of GAG mimetics with multivalent saccharides.