Skip to main content
NCBI home page
ACS Omega logoLink to ACS Omega
. 2019 Nov 26;4(24):20690–20696. doi: 10.1021/acsomega.9b02877

Screening of a Glycopolymer Library of GM1 Mimics Containing Hydrophobic Units Using Surface Plasmon Resonance Imaging

Yuri Kimoto 1, Yuhei Terada 1, Yu Hoshino 1, Yoshiko Miura 1,*
PMCID: PMC6906939  PMID: 31858054

Abstract

graphic file with name ao9b02877_0007.jpg

Effective screening methods for the development of glycopolymers as molecular recognition materials are desirable for the discovery of novel biofunctional materials. A glycopolymer library was prepared to obtain guidelines for the design of glycopolymers for the recognition of cholera toxin B subunits (CTB). Glycopolymers with varying ratios of hydrophobic and sugar units were synthesized by reversible addition fragmentation chain transfer polymerization. N-tert-Butylacrylamide, N-phenylacrylamide, and N-cyclohexylacrylamide as hydrophobic units were copolymerized in the polymer backbone, and galactose, which contributes to CTB recognition, was introduced into the side chains by “post-click” chemistry. The thiol-terminated glycopolymers were immobilized on a gold surface. The polymer immobilization substrate was analyzed in terms of interaction with galactose recognition proteins (CTB, peanut agglutinin, and Ricinus communis agglutinin I) using surface plasmon resonance imaging. The polymers with high ratios of sugar and hydrophobic units had the strongest interactions with the CTB, which was different from the trend with peanut agglutinin and Ricinus communis agglutinin I. The binding constant of the CTB with the glycopolymer with hydrophobic units was 4.1 × 106 M–1, which was approximately eight times larger than that of the polymer without hydrophobic units. A correlation was observed between the log P value and the binding constant, indicating that the hydrophobic interaction played an important role in binding. New guidelines for the design of recognition materials were obtained by our screening method.

Introduction

Carbohydrates are used as the energy sources for living organisms, and the carbohydrates on cell surfaces also play important roles in living systems.1 Carbohydrates mediate various biological phenomena including bacterial and viral infections and cancer metastasis by interaction with proteins. Generally, bioactive carbohydrates composed of oligosaccharides are expensive to produce because they are difficult to synthesize and extract.2,3 Thus, it is difficult commercially use oligosaccharides as biomaterials. Therefore, there is a demand for substitutes that can be obtained inexpensively and in large quantities.

Glycopolymers, which have sugar units on the side chains, have been used as scaffolds for the development of compounds mimicking oligosaccharides.4−6 Monovalent carbohydrates only bind weakly to carbohydrate recognition proteins (lectins); however, the interaction between multivalent carbohydrates and lectins can be amplified, which is termed as “the cluster glycoside effect”.7,8 This effect is essential to provide glycan capability artificially and can be exhibited by glycopolymers. Glycopolymers that have high affinity and specificity to various target proteins have been developed, and this development provides guidelines for the design of biofunctional materials.

Many groups have investigated the interaction of glycopolymers with viruses, bacteria, and lectins, and various glycopolymers with different sugar densities and distances between sugars and linker lengths have been studied.9−13 However, the optimal design of glycopolymers for a specific target is difficult because the structures of the target proteins are complex and diverse. Recently, screening methods using glycopolymer libraries with various parameters have attracted attention. The Bradley group has reported the preparation of a glycopolymer library using a substrate and high-throughput analyses with lectins, showing the utility of the library method.14

Vibrio cholerae can cause serious diseases. The natural ligand of the cholera toxin secreted by V. cholerae is the oligosaccharide GM1,15,16 which is too expensive to produce for use in inhibitors and diagnostic medicine. To obtain a more readily available material for such applications, many groups have developed inexpensive GM1 functional mimics that strongly bind to cholera toxin, using glycopolymer screening methods.17−20 Our group has shown that a glycopolymer with galactose (Gal) and neuraminic acid (Neu5Ac) is advantageous for CTB recognition in an analysis using a glycopolymer library incorporating various sugar units.21 The Gibson group has prepared a glycopolymer library containing monomers incorporating hydrophobic groups that could facilitate improvements in the CTB recognition ability.22 However, both the synthesis and screening methods for these libraries were tedious and ineffective, requiring multistep synthesis, and the development of inexpensive GM1 mimics with sufficient affinity has not yet been achieved.

In the present study, hydrophobic monomers that are readily available were copolymerized into a polymer scaffold by reversible addition fragmentation chain transfer (RAFT) polymerization with the aim of further improving the affinity for CTB. Gal was attached to the polymer side chain by “post-click” chemistry because it has been reported that Gal is an essential structure for the GM1-recognition protein, CTB.23 In the high throughput screening of sugar–protein interactions, a sensitive screening method is essential, and optics are useful because a precise method is necessary. In this study, surface plasmon resonance imaging (SPRI), which is capable of rapidly screening large libraries with high sensitivity and does not require the labeling of proteins, was used.24,25 The glycopolymer was immobilized on a gold-deposited substrate using a terminal thiol.21,26 The sugar and hydrophobic unit ratios were analyzed for the effects on the ability of the glycopolymers to recognize the CTB and other Gal lectins, using SPRI of the prepared substrates.

Results

Preparation of Glycopolymers with Hydrophobic Groups

4-Trimethylsilyl-3-butynyl acrylamide (TMS BtnAAm) and acrylamide (AAm) were used as the alkyne monomer and spacer, respectively. N-tert-Butylacrylamide (TBAm), N-phenylacrylamide (PhAAm), and N-cyclohexylacrylamide (CyHex), which have different degrees of hydrophobicity, were used as the hydrophobic monomers. These monomers were incorporated to evaluate the contribution of hydrophobic groups to CTB recognition. The monomers were polymerized in different ratios by RAFT polymerization. Glycopolymers with 10, 30, and 50% sugar units were prepared to evaluate the interactions between the protein and the sugar groups in the glycopolymers. The ratios of the alkyne and functional groups in the obtained polymer backbones were confirmed by 1H NMR, and the values corresponded to the monomer feed ratios (Table 1, Figure 1). Size exclusion chromatography (SEC) analysis revealed that the Mw/Mn values of the polymer backbones were low and the relative molecular weights were close to the theoretical values (Table 1). The results suggested that living radical polymerization was achieved successfully, and the glycopolymers were synthesized as designed. The TMS groups were deprotected in a tetrahydrofuran (THF) solution with tetrabutylammonium fluoride (TBAF) or in aqueous solution with KOH for activation of the alkyne groups in the polymer backbones. The deprotection of the TMS groups was confirmed by the disappearance of the peak at 0.13 ppm in the 1H NMR spectra.

Table 1. Properties of the RAFT Polymerization of the Glycopolymersa.

  monomer feed ratio (%)
            
entry TMS TBAm PhAAm CyHex AAm conv.b (%) alkyn ratioc (%) hydro. ratioc (%) Mn, thd (g/mol) Mne (g/mol) Mw/Mne
T10 10 0 0 0 90 94 10 0 8500 6100f 1.20f
T10T20 10 20 0 0 70 80 9 21 9500 6100f 1.25f
T10P20 10 0 20 0 70 79 10 19 9700 15 000 1.44
T30 30 0 0 0 70 98 28 0 9900 15 000 1.41
T30T20 30 20 0 0 50 90 31 21 10 900 11 200 1.48
T30P20 30 0 20 0 50 86 28 21 11 700 13 100 1.43
T50 50 0 0 0 50 96 49 0 10 800 11 800 1.36
T50T20 50 20 0 0 30 93 53 25 12 600 13 900 1.47
T50P20 50 0 20 0 30 89 45 21 12 600 17 300 1.57
T50C20 50 0 0 20 30 64g 52 23 12 600g 13 600 1.49
Open in a new tab
a

The target degree of polymerization was set at 100 ([M]/[RAFT] = 100). The ratio of the initiator ([RAFT]/[2,2′-azobis isobutyronitrile (AIBN)]) was fixed at 250. [M] = 1.0 mol/L.

b

Monomer conversions were determined by 1H NMR.

c

Alkyne and hydrophobic unit ratios were determined by 1H NMR after purification.

d

Theoretical molecular weight was calculated by the following formula: Mn,th = (MWmonomer × each unit ratio/100) × 100 × Conv. + MWRAFT.

e

Relative molecular weight and polydispersity index were determined by SEC analysis calibrated with a polystyrene standard. The eluent was 10 mM LiBr DMF.

f

Relative molecular weight and polydispersity index were determined by SEC analysis calibrated with a pullulan standard. The eluent was 10 mM LiBr DMSO because of poor solubility in water.

g

Monomer conversions and theoretical molecular weight were calculated from the yield because of the overlap of monomer and CyHex peaks.

Figure 1.

Figure 1

Open in a new tab

Synthetic scheme of the glycopolymer library and the compound list used in the study.

The glycopolymers were obtained by postmodification of azide-terminated Gal into the polymer backbones. The progress of the copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction was confirmed by 1H NMR: the ratio of the integral values of the anomer peak at approximately 5.6 ppm and the peak from the alkyl group next to the triazole at approximately 8.0 ppm reached 1:2. The relative molecular weights of the glycopolymers were evaluated by SEC analysis (Table 2). The glycopolymers with PhAAm or CyHex (G10P20, G30P20, G50P20, and G50C20) were dissolved in dimethyl sulfoxide (DMSO) because they were not soluble in water. The relative molecular weights of the glycopolymers with TBAm (G10T20, G30T20, and G50T20) were smaller than those of the glycopolymers without hydrophobic units (G10, G30, and G50). For example, the molecular weight of G50T20 with TBAm was smaller than that of G50 without hydrophobic groups. The results suggested that the glycopolymers incorporating hydrophobic groups were slightly aggregated in water.

Table 2. Glycopolymers Prepared by the CuAAC Reactiona.

entry sugar unit ratiob (%) Mn,thc (g/mol) Mnd (g/mol) Mw/Mnd
G10 8 9500 24 700 1.22
G10T20 7 10 400 8700 1.34
G10P20 10 10 800 18 500e 1.25e
G30 27 13 100 21 700 1.55
G30T20 28 14 200 8600 1.39
G30P20 28 14 800 15 600e 1.23e
G50 49 15 000 8000e 1.40e
G50T20 49 17 700 10 300 1.41
G50P20 46 18 900 20 000e 1.14e
G50C20 53 19 400f 15 500e 1.36e
Open in a new tab
a

The target degree of polymerization was set at 100 ([M]/[RAFT] = 50). The ratio of the initiator ([RAFT]/[AIBN]) was fixed at 250.

b

Alkyne and hydrophobic unit ratios were determined by 1H NMR after purification.

c

Theoretical molecular weight was calculated by theoretical molecular weight-based conversion.

d

Relative molecular weight and polydispersity index were determined by SEC analysis calibrated with a pullulan standard. The eluent was 100 mM NaNO3 aq.

e

Relative molecular weight and polydispersity index were determined by SEC analysis calibrated with a pullulan standard. The eluent was 10 mM LiBr DMSO because of the poor solubility in water.

f

The theoretical molecular weight was determined assuming the yield to be equal to the conversion because the conversion could not be calculated.

Analysis of the Interaction between the Glycopolymers and the Proteins

The load concentrations of CTB, peanut agglutinin (PNA), and ricinus communis agglutinin I (RCA120) were 1 μM, 500 nM, and 50 nM, respectively (Figure 2). In Figure 3, the x- and y-axis represent the hydrophobic units incorporated into the glycopolymers and the ratio of the incorporated sugar units, respectively. The z-axis shows the SPR reflection intensity change (SPRI signal) on the glycopolymer-immobilized surface after injection of the protein solution for 1 h.

Figure 2.

Figure 2

Open in a new tab

Schematic illustration of the current investigation.

Figure 3.

Figure 3

Open in a new tab

SPRI signals of the glycopolymer library with (a) CTB (1 μM), (b) PNA (500 nM) and (c) RCA 120 (50 nM).

The SPRI signal changes after the addition of the proteins are shown in Figure 3. All the glycopolymers showed a SPRI signal change after the addition of the proteins. The SPRI intensities differed according to the ratios of Gal and the hydrophobic units incorporated in the polymers. The CTB binding was shown to be dependent on the Gal ratio. The glycopolymers with higher Gal ratios showed the largest SPRI signals. The glycopolymers with 50% Gal tended to show the highest adsorption of CTB. PNA and RCA120 showed a different tendency regarding the Gal incorporation ratio compared with CTB. The polymer with 30% Gal showed the highest amount of adsorption after the addition of PNA or RCA120. The effect of hydrophobicity was also investigated by comparing the signals for the polymers with the same sugar ratios (Figure 3). CTB binding was amplified by the addition of hydrophobic groups as the SPRI signals of the glycopolymers with TBAm and PhAAm (blue and green bars, Figure 3) were larger than those of the polymers without hydrophobic units (red bars, Figure 3). In contrast, the SPRI signals of the polymers, with and without hydrophobic units, were approximately the same for PNA and RCA120 binding.

Calculation of the Binding Constants for the Proteins

The binding constants for each glycopolymer were calculated from the SPRI screening results. The curves formed by the SPRI signal plots for the protein solutions for each concentration were analyzed by fitting based on eq 1. For the CTB solution, the binding constants for the surface immobilized G50, G50T20, and G50P20 were 0.39, 2.7, and 4.1 × 106 M–1, respectively (Figure 4). The binding constant of the glycopolymer incorporating hydrophobic groups (G50P20) was eight times larger than that of the glycopolymer without hydrophobic groups (G50). The binding constant of G50P20 with PhAAm was larger than that of G50T20 with TBAm. The binding constants of PNA for the surface-immobilized G50, G50T20, and G50P20 were 8.8, 9.5, and 11.7 × 106 M–1, respectively. The binding constant of G50P20 with hydrophobic units was slightly larger than that of G50 without hydrophobic groups, but the increase in binding between G50P20 and G50 was only 1.3 times. In the case of RCA120, the binding constants for the surface-immobilized G50, G50T20, and G50P20 were 3.1, 2.5, and 2.5 × 108 M–1, respectively. The binding constants of G50T20 and G50P20 with hydrophobic units were slightly smaller than that of G50 without hydrophobic groups.

Figure 4.

Figure 4

Open in a new tab

Binding constants of glycopolymers with Gal recognition proteins: (a) SPRI signals with varying CTB concentrations, (b) Ka values with CTB, (c) Ka values with PNA, and (d) Ka values with RCA120.

The binding constants calculated in the previous section were evaluated using the partition coefficient of the log P value, which is a calculated value representing the hydrophobicity of chemical substances. The x- and y-axis represent the log P value of the hydrophobic group monomer (AAm, TBAm, PhAAm, and CyHex) in each glycopolymer and the binding constant for each immobilized glycopolymer, respectively (Figure 5). Glycopolymers incorporating highly hydrophobic monomers had large binding constants.

Figure 5.

Figure 5

Open in a new tab

Relationship between the binding constant and the log P value of various monomers AAm, TBAm, CyHex, and PhAAm. log P was estimated using a software.

Inhibition Assay with Addition of the Natural Ligand GM1

An inhibition assay was performed to investigate the site of CTB that interacts with the glycopolymers (Figure 6). The vertical axis of the graph represents the SPRI signal after flowing the protein solution for 1 h. The left and right bars are the results for the CTB solution (250 nM) and a mixed solution with CTB (250 nM) and GM1 (10 μM), respectively. A SPRI signal appeared in all the polymer-immobilized substrates after the addition of the CTB solution. In contrast, no SPRI signal change was observed after the addition of the mixed solution.

Figure 6.

Figure 6

Open in a new tab

SPRI signals of the glycopolymers in the presence and absence of GM1. The polymers used were G50, G50T20, and G50P20.

Discussion

Hydrophobic units have been frequently added to the Gal units in glycopolymers to enhance the molecular recognition with CTB.27 The Bundle group reported that the ligand library conjugated with multivalent polymer scaffolds assisted in identifying hydrophobic units to complement the affinity of Gal for CTB.19 The Gibson group has reported that the affinity and selectivity of glycopolymers toward CTB and PNA were changed by incorporation of monomers with a secondary hydrophobic motif for Neu5Ac recognition sites next to Gal.22 In this study, an amplification effect on CTB binding was observed with glycopolymers having hydrophobic units and Gal residues. Inhibition assays indicated that the hydrophobic group performed a similar function to that of Neu5Ac in GM1, and that the hydrophobic unit and Gal interacted cooperatively with carbohydrate recognition domains.16 The results of the present study differ from Gibson’s report in that the interaction with the proteins had no correlation with the log P value of the hydrophobic groups in the glycopolymers, and the hydrophobic group might interfere with the interaction with PNA.22 The ligands on the side chains might have been able to reach the carbohydrate recognition sites because the polymers used in the present study are flexible compared with the polymers used by other groups. Although there are still some issues to consider, the introduction of Gal and hydrophobic units to a glycopolymer has the potential to be effective for enhancing CTB recognition.

The contribution of the hydrophobic groups to the CTB (PDB-ID: 3CHB(28)) binding was investigated based on the crystal structure from the PDB. CTB was found to have hydrophilic amino acids on the surface, but PNA (PDB-ID: 2PEL(29)) and RCA120 (PDB-ID: 1RZO) had hydrophobic groups scattered on the surface. CTB interacted strongly with the glycopolymers with hydrophobic units, and PNA and RCA120 did not interact with the hydrophobic glycopolymers, suggesting that the hydrophobic amino acid residues in Gal lectins contribute to the interaction only with the CTB. Unexpectedly, hydrophobic residues were found not only in CTB but also in PNA and RCA120, though a hydrophobic interaction was not observed with PNA or RCA120. One possibility is that some of the hydrophobic residues in CTB contribute to the recognition of Neu5Ac in GM1 or the hydrophobic unit in the glycopolymers and that the hydrophobic residues in PNA and RCA120 contribute to the protein folding. At the carbohydrate recognition sites in the CTB, Trp88, a hydrophobic amino acid, interact with Gal of GM1. The acetyl group of Neu5Ac in GM1 induced hydrophobic interaction with Glu11 and Tyr12, and Tyr12 form a hydrophobic cavity in the vicinity of Neu5Ac residue. The enhanced binding of CTB with molecules containing hydrophobic groups has been reported.19,22,27 Further studies are needed to determine whether hydrophobic groups acted on this hydrophobic pocket. Elucidation of hydrophobic units interacting on the cavity requires further study.

The binding constant between G50P20 and CTB was 4.1 × 106 M–1, which was the highest value in this study, and this affinity is comparable to antigen–antibody interactions; this suggested that G50P20 may be applicable as a biofunctional material for molecular recognition. However, this affinity was still much weaker than the natural ligand for CTB, GM1 (Ka = 1010 to 1012 M–1). The strong binding of GM1 is achieved by multiple hydrogen bonding between the amino acids in the protein and the sugars in GM1, including Gal, GalNAc, and Neu5Ac.28 The binding constant of G50P20 is thought to be smaller than that of GM1 because only Gal in G50P20 functions as a sugar ligand with CTB. In addition, while the rigid structure of GM1 can suppress the entropy loss by binding with CTB, the flexible side chains of the glycopolymer G50P20 provide the potential advantage that the ligands can reach the carbohydrate recognition domains, but this advantage is canceled by an increased entropy loss.19

We have previously reported another GM1 polymer mimic, containing Gal and Neu5Ac, which had a binding constant of 2.7 × 109 M–1, which is much stronger binding than that observed for G50P20.21 A possible reason for the weaker binding of G50P20 is that G50P20 was composed of only 50% Gal, which is a lower amount than in the previous polymer; glycopolymers with higher sugar ratios might have better performance because of the cluster glycoside effect. The weak affinity of G50P20 indicated that the hydrophobic units could induce hydrophobic interactions but could not substitute for other interactions, such as electrical interactions and hydrogen bonds. Although hydrophobic groups have often been proposed to be substitutes for oligosaccharides and sialic acid in synthetic compounds,27 the design and scope of the application of hydrophobic groups require careful consideration.

Conclusions

The glycopolymers with high sugar ratios and strong hydrophobicity showed strong interactions with the CTB. For PNA and RCA120, the observed trends for glycopolymers that showed strong affinities were clearly different compared with the CTB; with these proteins, the hydrophobic units did not provide effective protein recognition. Quantitative binding constant analysis of the immobilized glycopolymers and CTB was performed to confirm the contribution of the hydrophobic units, and a correlation between the Ka values and the log P values of the introduced hydrophobic units was observed. Our analysis suggested that the hydrophobic units contributed to a hydrophobic interaction and functioned cooperatively with the Gal units. We were able to provide new design guidelines for glycopolymers for CTB recognition. This screening method using SPRI could be a useful approach for developing new molecular recognition materials in the future. Research is in progress in our group on new development methods for glycopolymers as molecular recognition materials that are comparable to current drug-screening methods.

Experimental Section

Preparation of Polymer Backbones30

All polymer backbones were prepared by RAFT polymerization (Figure 1). Briefly, AAm, TMS BtnAAm, and hydrophobic acrylamide derivatives (TBAm, PhAAm, and CyHex) were dissolved in DMSO with 2-cyanopropan-2-yl butyl trithiocarbonate (CPBTC) and AIBN. Polymer structures are shown in the Supporting Information. The monomer feed ratio and target degree of polymerization were varied (Table 1). The solution was degassed by freeze–thaw cycles (three times) and placed in an oil bath at 70 °C for 18 h. The polymer backbones were purified by dialysis against a solvent mixture of MeOH and acetone (MeOH/acetone = 1:1, MWCO = 3.5 kDa) or Milli-Q water (MWCO = 3.5 kDa).

Deprotection of TMS Groups from the Polymer Backbones30

For G10–G30, the polymer (100 mg) was dissolved in a mixture of Milli-Q water (10 mL): KOHaq (1 M, 3 mL) (Figure 1). The solution was kept stirring at room temperature for 3 h. The product was obtained by freeze-drying. For T50 and G30T20–G50C20, the polymer (100 mg) was dissolved in dry THF (5 mL), and TBAF solution (1 mL, 1.0 M in THF) was added into the solution. The solution was kept stirring at room temperature for 9 h. THF was removed under reduced pressure. The deprotected polymer was dialyzed against a mixture of MeOH and acetone (MeOH/acetone = 1:1, MWCO = 3.5 kDa).

Carbohydrate Addition by “Post-Click” Chemistry30

The CuAAC reaction was conducted between the alkyne groups in the polymer backbone and azide-terminated Gal, where the molar amounts of the alkyne units were calculated based on the monomer ratio in the polymer. The polymer (20 mg), azide-terminated Gal (3 equiv to alkyne unit), and CuSO4 (0.4 equiv to alkyne unit) were dissolved in Milli-Q water (1.5 mL). Tris(benzyltriazolylmethyl)amine (0.4 equiv to alkyne unit) in MeCN (1.5 mL) was added to the reaction mixture. Sodium ascorbate (l-Asc·Na) (2 equiv to alkyne unit) was added, and the reaction mixture was kept at 60 °C for 6 h with N2 bubbling. The products were purified by dialysis (Spectra/Por 7; MWCO 3500) against water with hydrochloric acid (pH = 4) for 24 h. The solution was changed subsequently to pure water (pH = 7) and kept for 24 h, and the product was obtained by freeze-drying.

The glycopolymers used in this investigation are shown in Figure 1. We named glycopolymers as follows: the numbers following Gal show the incorporation ratio of Gal in the polymer, where G10 means 10% incorporation of Gal in the polymer. The letters and numbers represent the type and ratio of the hydrophobic group incorporated into the polymers, respectively.

SPRI Chips Preparation and SPRI Measurement21

In all the experiments, the glass substrate of S-TIM35 (Vidtec, Fukuoka, Japan) was exposed to UV/O3 light for 30 min. Sigmacote was put on the substrates for 1 min and rinsed with hexane, EtOH, and Milli-Q water. Cr (1 nm) and Au (50 nm) were deposited on the substrates and rinsed with EtOH and Milli-Q water. An aqueous solution of each polymer (0.5 μL, 10 g/L) was put onto each gold dot on the deposited substrate, and the glass substrate was incubated for 3 h. The chip was rinsed with Milli-Q water and blown with air to dry (Figure 2).

All of the SPR measurements were performed with the Kretschmann configuration. A 60° S-TIH11 prism (Sigmakoki Co., Ltd. Saitama, Japan) was used in all the measurements. A p-polarized helium–neon laser (λ = 632.8 nm) was used as the light source, and the reflected light at the interface was detected using a photodiode detector. The reflectivity of the light was measured as a function of the incidence angle (angle scan) and as a function of time at a fixed incidence angle (kinetic scan). Prepared chips were set in the SPRI chip cell. Before the protein solution in PBS was flowed into the cell, 10 mM phosphate buffered saline (PBS) (pH 7.4, 137 mM NaCl, 2.68 mM KCl) was flowed through (0.1 mL/min) until the SPR reflectivity became stable. The SPR reflectivity change (SPRI signal) was measured after injecting the protein solution (CTB, PNA, or RCA120) at a flow rate of 0.1 mL/min for 1 h (Figure 2).

Measurement of the Affinity between the Polymers and Proteins

SPRI chips were prepared in accordance with the method described above and set in the SPRI chip cell. Before the measurement, 10 mM PBS was flowed into the cell at 0.1 mL/min until the SPRI signal became stable. The protein solution (CTB, PNA, or RCA120) was injected with a flow rate of 0.1 mL/min, and the order of the injection was from the most diluted to the most concentrated solution. In the measurement, the SPRI signal was regarded as the amount of protein adsorption. The binding constants of each protein were calculated with the Langmuir isotherm using the SPRI signals

graphic file with name ao9b02877_m001.jpg 1

ΔR, ΔRmax, c, and Ka are the SPRI signal, the maximum SPRI signal, the protein concentration, and the binding constant, respectively. Based on eq 1, the plots of the SPRI signals were analyzed by nonlinear regression to derive the binding constants.

Inhibition Assay for the Carbohydrate Recognition Sites of CTB

SPRI chips were prepared in accordance with the method described above and set in the SPRI chip cell. Before the measurement, 10 mM PBS was flowed into the cell at 0.1 mL/min until the SPR reflectivity became stable. Protein solutions were injected with a flow rate of 0.1 mL/min for 1 h. A mixture of CTB (250 nM) and GM1 (10 μM) was used. GM1 used was whole lipid and not only the saccharide unit. GM1 was soluble in a monomeric state which was confirmed by dynamic light scattering (Figure S44). The concentration was under the critical micelle concentration of GM1.31

Acknowledgments

This work was supported by Grant-in-Aid for Scientific Research (B) (JP19H02766), Grant-in-Aid for Scientific Research on Innovative Areas (JP18H04420), Grant-in-Aid for challenging Exploratory Research (JP19K22971), and the Japan Agency for Medical Research and Development (JP18fk0108054). We appreciate for the measurement of XPS by Dr. Tatsuya Murakami and Ichiro Kimura in the Japan Advanced Institute of Science and Technology. We appreciate for the preparation of SPRI chips by Prof. Kaoru Tamada in Kyushu University. Victoria Muir, PhD, from the Edanz Group (www.edanzediting.com/ac) edited a draft of this manuscript.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.9b02877.

  • 1H NMR spectra of the compounds and the detailed conditions for the polymerization (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao9b02877_si_001.pdf (5.1MB, pdf)

References

  1. Taylor M. E.; Drickamer K.. Introduction to Glycobiology, 2nd ed.; Oxford University Press: Oxford, 2002. [Google Scholar]
  2. Davis B. G. Synthesis of glycoproteins. Chem. Rev. 2002, 102, 579–602. 10.1021/cr0004310. [DOI] [PubMed] [Google Scholar]
  3. Bhattacharya S. K.; Danishefsky S. J. A total synthesis of the methyl glycoside of ganglioside GM1. J. Org. Chem. 2000, 65, 144–151. 10.1021/jo9912496. [DOI] [PubMed] [Google Scholar]
  4. Miura Y.; Hoshino Y.; Seto H. Glycopolymer nanobiotechnology. Chem. Rev. 2016, 116, 1673–1692. 10.1021/acs.chemrev.5b00247. [DOI] [PubMed] [Google Scholar]
  5. Geng J.; Mantovani G.; Tao L.; Nicolas J.; Chen G.; Wallis R.; Mitchell D. A.; Johnson B. R. G.; Evans S. D.; Haddleton D. M. Site-Directed Conjugation of ″Clicked″ Glycopolymers To Form Glycoprotein Mimics: Binding to Mammalian Lectin and Induction of Immunological Function. J. Am. Chem. Soc. 2007, 129, 15156–15163. 10.1021/ja072999x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Paluck S. J.; Nguyen T. H.; Maynard H. D. Heparin-mimicking polymers: synthesis and biological applications. Biomacromolecules 2016, 17, 3417–3440. 10.1021/acs.biomac.6b01147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Lundquist J. J.; Toone E. J. The cluster glycoside effect. Chem. Rev. 2002, 102, 555–578. 10.1021/cr000418f. [DOI] [PubMed] [Google Scholar]
  8. Cecioni S.; Imberty A.; Vidal S. Glycomimetics versus multivalent glycoconjugates for the design of high affinity lectin ligands. Chem. Rev. 2015, 115, 525–561. 10.1021/cr500303t. [DOI] [PubMed] [Google Scholar]
  9. Ladmiral V.; Mantovani G.; Clarkson G. J.; Cauet S.; Irwin J. L.; Haddleton D. M. Synthesis of Neoglycopolymers by a Combination of ″Click Chemistry″ and Living Radical Polymerization. J. Am. Chem. Soc. 2006, 128, 4823–4830. 10.1021/ja058364k. [DOI] [PubMed] [Google Scholar]
  10. Becer C. R. The Glycopolymer Code: Synthesis of Glycopolymers and Multivalent Carbohydrate-Lectin Interactions. Macromol. Rapid Commun. 2012, 33, 742–752. 10.1002/marc.201200055. [DOI] [PubMed] [Google Scholar]
  11. Miura Y. Design and synthesis of well-defined glycopolymers for the control of biological functionalities. Polym. J. 2012, 44, 679–689. 10.1038/pj.2012.4. [DOI] [Google Scholar]
  12. Jono K.; Nagao M.; Oh T.; Sonoda S.; Hoshino Y.; Miura Y. Controlling the lectin recognition of glycopolymersviadistance arrangement of sugar blocks. Chem. Commun. 2018, 54, 82–85. 10.1039/c7cc07107h. [DOI] [PubMed] [Google Scholar]
  13. Nagao M.; Matsubara T.; Hoshino Y.; Sato T.; Miura Y. Synthesis of various glycopolymers bearing sialyllactose and the effect of their molecular mobility on interaction with the influenza virus. Biomacromolecules 2019, 20, 2763–2769. 10.1021/acs.biomac.9b00515. [DOI] [PubMed] [Google Scholar]
  14. Neumann K.; Conde-González A.; Owens M.; Venturato A.; Zhang Y.; Geng J.; Bradley M. An Approach to the High-Throughput Fabrication of Glycopolymer Microarrays through Thiol-Ene Chemistry. Macromolecules 2017, 50, 6026–6031. 10.1021/acs.macromol.7b00952. [DOI] [Google Scholar]
  15. Merritt E. A.; Sarfaty S.; Akker F. V. D.; L’Hoir C.; Martial J. A.; Hol W. G. Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide. Protein Sci. 2008, 3, 166–175. 10.1002/pro.5560030202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Zuilhof H. Fighting cholera one-on-one: the development and efficacy of multivalent cholera-toxin-binding molecules. Acc. Chem. Res. 2016, 49, 274–285. 10.1021/acs.accounts.5b00480. [DOI] [PubMed] [Google Scholar]
  17. Kumar V.; Turnbull W. B. Carbohydrate inhibitors of cholera toxin. Beilstein J. Org. Chem. 2018, 14, 484–498. 10.3762/bjoc.14.34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Richards S.-J.; Jones M. W.; Hunaban M.; Haddleton D. M.; Gibson M. I. Probing Bacterial-Toxin Inhibition with Synthetic Glycopolymers Prepared by Tandem Post-Polymerization Modification: Role of Linker Length and Carbohydrate Density. Angew. Chem., Int. Ed. 2012, 51, 7812–7816. 10.1002/anie.201202945. [DOI] [PubMed] [Google Scholar]
  19. Tran H.-A.; Kitov P. I.; Paszkiewicz E.; Sadowska J. M.; Bundle D. R. Multifunctional multivalency: a focused library of polymeric cholera toxin antagonists. Org. Biomol. Chem. 2011, 9, 3658–3671. 10.1039/c0ob01089h. [DOI] [PubMed] [Google Scholar]
  20. Polizzotti B. D.; Kiick K. L. Effects of polymer structure on the inhibition of cholera toxin by linear polypeptide-based glycopolymers. Biomacromolecules 2006, 7, 483–490. 10.1021/bm050672n. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Terada Y.; Hoshino Y.; Miura Y. Glycopolymers mimicking GM1 gangliosides: cooperativity of galactose and neuraminic acid for cholera toxin recognition. Chem.–Asian J. 2019, 14, 1021–1027. 10.1002/asia.201900053. [DOI] [PubMed] [Google Scholar]
  22. Jones M. W.; Otten L.; Richards S.-J.; Lowery R.; Phillips D. J.; Haddleton D. M.; Gibson M. I. Glycopolymers with secondary binding motifs mimic glycan branching and display bacterial lectin selectivity in addition to affinity. Chem. Sci. 2014, 5, 1611–1616. 10.1039/c3sc52982g. [DOI] [Google Scholar]
  23. Turnbull W. B.; Precious B. L.; Homans S. W. Dissecting the Cholera Toxin–Ganglioside GM1 Interaction by Isothermal Titration Calorimetry. J. Am. Chem. Soc. 2004, 126, 1047–1054. 10.1021/ja0378207. [DOI] [PubMed] [Google Scholar]
  24. Fang S.; Lee H. J.; Wark A. W.; Corn R. M. Attomole microarray detection of microRNAs by nanoparticle-amplified SPR imaging measurements of surface polyadenylation reactions. J. Am. Chem. Soc. 2006, 128, 14044–14046. 10.1021/ja065223p. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Steiner G. Surface plasmon resonance imaging. Anal. Bioanal. Chem. 2004, 379, 328–331. 10.1007/s00216-004-2636-8. [DOI] [PubMed] [Google Scholar]
  26. Sumerlin B. S.; Lowe A. B.; Stroud P. A.; Zhang P.; Urban M. W.; McCormick C. L. Modification of Gold Surfaces with Water-Soluble (Co)polymers Prepared via Aqueous Reversible Addition–Fragmentation Chain Transfer (RAFT) Polymerization†. Langmuir 2003, 19, 5559–5562. 10.1021/la034459t. [DOI] [Google Scholar]
  27. Arosio D.; Baretti S.; Cattaldo S.; Potenza D.; Bernardi A. Ganglioside GM1 mimics: lipophilic substituents improve affinity for cholera toxin. Bioorg. Med. Chem. Lett. 2003, 13, 3831–3834. 10.1016/j.bmcl.2003.07.007. [DOI] [PubMed] [Google Scholar]
  28. Merritt E. A.; Kuhn P.; Sarfaty S.; Erbe J. L.; Holmes R. K.; Hol W. G. J. The 1.25 Å resolution refinement of the cholera toxin B-pentamer: evidence of peptide backbone strain at the receptor-binding site. J. Mol. Biol. 1998, 282, 1043–1059. 10.1006/jmbi.1998.2076. [DOI] [PubMed] [Google Scholar]
  29. Banerjee R.; Das K.; Ravishankar R.; Suguna K.; Surolia A.; Vijayan M. Conformation, protein-carbohydrate interactions and a novel subunit association in the refined structure of peanut lectin-lactose complex. J. Mol. Biol. 1996, 259, 281–296. 10.1006/jmbi.1996.0319. [DOI] [PubMed] [Google Scholar]
  30. Nagao M.; Fujiwara Y.; Matsubara T.; Hoshino Y.; Sato T.; Miura Y. Design of glycopolymers carrying sialyl oligosaccharides for controlling the interaction with the influenza virus. Biomacromolecules 2017, 18, 4385–4392. 10.1021/acs.biomac.7b01426. [DOI] [PubMed] [Google Scholar]
  31. Formisano S.; Johnson M. L.; Lee G.; Aloj S. M.; Edelhoch H. Critical micelle concentrations of gangliosides. Biochemistry 1979, 18, 1119–1124. 10.1021/bi00573a028. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao9b02877_si_001.pdf (5.1MB, pdf)

Articles from ACS Omega are provided here courtesy of American Chemical Society

RESOURCES