Glycoglycan Mimic by Synthetic Polymers - American Chemical Society

Chapter 3

Glycoglycan Mimic by Synthetic Polymers

Downloaded by UNIV OF FLORIDA on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1253.ch003

Yoshiko Miura,*,1 Tomohiro Fukuda,2 and Yu Hoshino1 1Department

of Chemical Engineering, Graduate School of Engineering, Kyushu University, 744 Motooka, Nisi-ku, Fukuoka 819-0395, Japan 2National Institute of Technology, Toyama College, 13 Hongo-machi, Toyama city, Toyama 939-8630, Japan *E-mail: [email protected]

Glycosaminoglycans (GAGs) are important polysaccharides in the living system, but the availability of GAGs is limited due to the complicated structures. In this investigation, GAGs were re-organized by synthetic glycopolymers. GAGs mimic glycopolymers were synthesized by polymerization of acrylamide sugar derivatives carrying sulfated N-acetyl glucosamine (GlcNAc). The interaction with proteins were investigated in view of inhibition of Alzheimer disease. The glycopolymer were prepared as molecular library with changing molecular weight, sugar structure and sugar ratios. The inhibitory activity of Alzheimer disease was studied by inhibitions of protein amyloidosis of amyloid beta (Aβ) peptides. The biological activities depended on the chemical structure of glycopolymers. We found the correlation of glycopolymer activities to the native GAGs.

1. Introduction The saccharides exist as glycolipids, glycoproteins and polysaccharides, and play important roles in the living system (1). Specially, polysaccharides, glycosaminoglycans (GAGs), the saccharide moiety of proteoglycans, are paid much attention due to the biological function such as cell adhesion, growth factor activation and anti-thrombogenicity (2, 3). GAGs are polysaccharides having alternating saccharides of amino-sugars (N-acetyl glucosamine (GlcNAc) or N-acetyl galgactosamine) and uronic acids (iduronic acid (IdoA) or glucuronic acid (GlcA). Most of the GAGs are highly sulfated except hyaluronic acid, and © 2017 American Chemical Society Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


the sulfation patters and saccharide structures are specific to each GAG. There are several famous GAGs such as heparin, heparan and chondroithin sulfate. Since GAGs have important in biology, many researchers have investigated the preparation of GAGs by organic and biological synthesis (5). Actually, total organic synthesis of GAGs has enthusiastically been studied for various synthetic chemists (4–7). The synthesis of oligosaccharides of GAGs have been attained. For example, the synthesis of disaccharide and of heparin like αIdoA(2S)αGlcNS(6S) have been reported (8). The disaccharide showed the function of GAGs, but the biological activity of anti-thrombogenicity was much weaker than the original GAGs (9). It is considered that the macromolecular structure of GAGs is important to exhibit strong biological activities. The biological synthesis of GAGs with enzymes are another solution for GAGs preparation. However, enzymes and sugar nucleic acids are expensive and difficult to control, and so the biological synthesis was also difficult. Considering the saccharide-protein interaction, the key of the interaction is multivalency. We have reported the sugar multivalent compounds with polymer, so-called glycopolymers (10). We have reported the various glycopolymers, which exhibit large biological interaction. Suda group have reported glyco-dendrimer (dendritic polymer) with GAGs oligosaccharides (11). Hsieh-Wilson group has reported the glycopolymer with GAGs oligosaccharide. Those glycopolymers with GAGs oligosaccharides showed the strong biological activities like the original GAGs (12, 13). We took a hint to re-organize GAGs with glycopolymers. GAGs structure was divided into the functional saccharide component with polymerizable sugars. The sugar monomers were polymerized by radical initiator. The glycopolymers can exhibit the strong interaction to the target proteins. The advantage of this polymer method is the facile preparation of GAGs mimic molecules (Figure 1).

2. Glycopolymer with Sulfonated GlcNAc The specific feature of GAGs is sulfated sugars in the polysaccharides. We focused on the sulfated GlcNAc in GAGs. In order to simplify the synthetic procedure, p-nitrophenyl glucosides were used for the starting material. Acrylamide phenyl 6-sulfo-GlcNAc was prepared as the monomer for GAGs mimic, because 6-sulfo-GlcNAc was reported to play important roles in the living systems (4). GAGs mimic polymers were prepared with radical initiator (14). The sugar content was changed from 10% to 60%. The biological activities of glycopolymer with sulfated GlcNAc was investigated by interaction with proteins. Since GAGs have related the protein amyloidosis, the glycopolymers with sulfo-GlcNAc was incubated with amyloid β (Aβ) peptide. Aβ peptides ((1-42) and (1-40)) are known as the pathogen of Alzheimer disease, and spontaneously formed protein amyloid. The aggregation of Aβ(1-42) spontaneously induced the aggregation which was monitored by fluorescence intensity of thioflavin T(ThT) (Figure 2). 70 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 1. Concept of GAGs mimic glycopolymers. Original GAGs was re-constructed with GAGs derived sugar and multivalency.

The glycopolymer was incubated with Aβ(1-42). The increase of ThT fluorescence was effectively inhibited in the presence of glycopolymers. The inhibitory effect of protein aggregation was depended on the sugar structure of glycopolymer. When Aβ was incubated with glycopolymer without sulfo-GlcNAc, the aggregation behavior of Aβ was similar to that without additives. The inhibitory effect of Aβ aggregation was depended on the sugar content. The glycopolymer with higher sugar content (100% and 65 %) didn’t show the inhibitory effect on protein aggregation. On the other hand, sulfo-GlcNAc glycopolymer with lower sugar content (20% and 10%) showed the good inhibitory effect. Glycopolymer having 6-sulfo-GlcNAc electrostatically interacted with Aβ via cationic residues such as His13. However, at the same time, the net charge of Aβ was negative. Not only glycopolymer can electrostatically interact with Aβ with the cationic residues, but the polymer induced the electron repulsion due to the negative Aβ net charge. The balance of interaction and repulsion determined the degree of inhibitory effect, and so the glycopolymer partially substituted with 6-sulfo-GlcNAc showed the strong inhibitory effect. The morphology of Aβ was measured by atomic force microscope (AFM). Aβ without additive formed nanofiber after incubation. The addition of glycopolymer with low sugar content (10-20%) changed the morphology of Aβ. It showed the interaction of glycopolymer with Aβ induced the different morphology from nanofibril. 71 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 2. The inhibitory effect on glycopolymer on Aβ(1-42) aggregation. (a) Time course of the fluorescence change in ThT with Aβ(1-42) and polymer additives. (b) Chemical structure of glycopolymers with 6-sulfo-GlcNAc (1) and GlcNAc (2) and polyacrylamide (3) (14). The neutralization of Aβ cytotoxicity was also investigated with Hela cell. The addition of Aβ induced the cytotoxicity, and the cell survival rate was decreased. The addition of glycopolymer with 6-sulfo-GlcNAc recovered the cell survival rate due to the neutralization of Aβ. The glycopolymer with 6-sulfo-GlcNAc itself did not induced cytotoxicity. These investigations showed that the glycopolymer with 6-sulfo-GlcNAc could be a polymer nanomedicine based on GAGs mimetics.

3. GAGs Mimic Polymers with Controlled Molecular Weight The biological ability of GAGs depends on the molecular weight due to the physical property and cell permeability (15). It has been reported that heparin with low molecular weight and high molecular weight show the different biological activities. It is difficult to prepare and investigate the synthetic GAGs with different molecular weight, but it is easy to prepare GAGs mimic polymers with different molecular weight. Controlled polymerization provides the facile method to prepare the molecules with different defined molecular weight. We tried to prepare the glycopolymer with different molecular weight using living radical polymerization with reversible addition fragmentation chain transfer (RAFT) reagent (Figure 72 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


3) (16), because RAFT living radical polymerization is advantageous of bulky monomer like sugar. The glycopolymers were prepared with different molecular weights, 105, 104 and 103 order (17). Sugars used were 6-sulfo-GlcNAc and GlcA from GAGs structure.

Figure 3. Chemical structure of GAGs mimic polymers with different molecular weights via RAFT living radical polymerization (17).

The function of glycopolymer with different molecular weights were investigated with Aβ(1-40). The kinetics of amyloidosis was investigated with the polymer additives using ThT fluorescence. The glycopolymer with 6-sulfo-GlcNAc showed the inhibitory effect on Aβ, but the glycopolymer with GlcA did not. Interestingly, the glycopolymer having both 6-sulfo-GlcNAc and GlcA showed the strongest inhibitory effect. The molecular weight effect of glycopolymer on Aβ protein aggregation was clear. Though the monomers didn’t not show the inhibitory effect on protein aggregation, the glycopolymers with low molecular weight showed the better activity than that with high molecular weight. Since the sugar density in the polymer was same, the multivalent effect of glycopolymer to Aβ exhibited in a similar degree. The mobility of glycopolymer in solution was dependent on the molecular weight, and the glycopolymer with low molecular weight. The kinetics of protein aggregation was studied by time course of ThT fluorescence. The glycopolymer with 6-sulfo-GlcNAc inhibited the nucleation of protein aggregation, and the glycopolymer with lower molecular weight exhibited the stronger inhibitory effect on nucleation (Figure 4). The glycopolymer 73 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


with GlcA showed the weak inhibitory effect on protein aggregation, but the glycopolymer with GlcA inhibited the elongation of protein fibril. The elongation inhibitory effect was related to molecular weight, and the glycopolymer with GlcA with low molecular weight showed the better inhibitory effect on elongation.

Figure 4. The schematic image of inhibition of Aβ(1-40) with GAGs mimic polymer. AFM image of Aβ(1-40) (a) without glycopolymer and (b) in the presence of glycopolymer of poly(AAm/6S-GlcNAc/GlcA) (17). Reproduced with permission from reference (17). The results suggested that the sugar structure in GAGs relates the nucleation and elongation of Aβ. The addition of glycopolymer with 6-sulfo-GlcNAc and GlcA inhibited the nucleation and elongation of Aβ aggregates, respectively. The glyco-ter-polymer with 6-sulfo-GlcNAc and GlcA showed the strong inhibitory effect on Aβ aggregation. Among them, the glyco-ter-polymer with 6-sulfo-GlcNAc and GlcA showed the strongest inhibitory effects. The natural GAGs have various sugar structures which interacted with proteins and control activities. The Aβ kinetics experiments with glycopolymer can clarify the role of each saccharide in GAGs. The glycopolymer with lower molecular weight showed the better activities in this chapter’s experiment. Though the multivalent effect of polymer is essential, but the polymer with lower molecular weight was advantageous on inhibitory effect of protein aggregation due to the better mobility.

4. Other GAGs Mimic Polymers Preparation of the GAGs mimic polymer is a unique method to investigate the GAGs with different sugar structures and molecular weights. One problem of the synthetic polymer is inhomogeneous structure based on the polydispersity. Most of the polymer has polydispersity and inhomogeneous structure. On the other hand, dendrimers have uniform structure (18). Dendrimers with 6-sulfo-GlcNAc were synthesized (19). The dendrimers were prepared by click chemistry of Huisgen reaction, which enables facile synthesis without protective groups. The glycodendrimer was synthesized by divergent method. Glycodendrimers with three different generations (G0, G1 and G2) were synthesized (Figure 5). The inhibitory effect of glycodendrimer was studied with Aβ(1-42). The 74 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


glycodendrimer with 6-sulfo-GlcNAc in high generation (G2) showed the good inhibitory effect like GAGs mimic linear polymers.

Figure 5. Chemical structure of GAGs mimic dendrimers of G0, G1 and G2 (19). Reproduced with permission from reference (19). Copyright (2012) MDPI. The glycodendrimers were also utilized as microarray to analyze sugar-protein interaction in detail (20). Fan-type glycodendrimers with 6-sulfo-GlcNAc were synthesized by click chemistry as shown in Figure 6. The glycodendrimers were immobilized onto gold substrate by click chemistry. he interaction with Aβ was studied by SPR and AFM. The interaction with Aβ was amplified in trimer and dimer of 6-sulfo-GlcNAc. The multivalent structure of 6-sulfo-GlcNAc was indispensable to interaction with Aβ. But at the same time, multivalent 6-sulfo-GlcNAc induced the electrostatic repulsion due to the negatively charged protein and saccharides. The interaction and electric repulsion between Aβ and 6-sulfo-GlcNAc determined the morphology of Aβ aggregates. The weak 75 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


interaction induced the nano fibrils, and the strong interaction and repulsion of multivalent sugar induced the spherical objects that exhibited cytotoxicity.

Figure 6. A representative image of glycodendrimer array with 6-sulfo-GlcNAc (20). The dendrimer with 6-sulfo-GlcNAc provided the defined interaction to Aβ. The investigation with glycodendrimer and glycodendrimer array was useful to understand the mechanism of amyloidosis with GAGs.

5. Conclusion We investigated the preparation and investigation of GAGs mimic polymer using sulfonated saccharides. GAGs mimic polymer was facile way to prepare GAGs’ mimic libraries. The glycopolymer with 6-sulfo-GlcNAc interacted with Aβ based on GAGs function. The GAGs mimic polymer libraries can change the various factors such as the polymer structure, sugar structure, sugar content and molecular weight. The detailed study on Aβ with GAGs mimic polymer was useful to clarify the mechanism of Aβ aggregation with GAGs.

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77 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Glycoglycan Mimic by Synthetic Polymers - American Chemical Society

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