Chapter 1 Introduction
4.2 Experimental section
4.2.6 Synthesis of sugar azide 4
Neuraminic acid azide (Neu5Ac-N3) was synthesized according to the previous method.42,43 To a solution of 100 mM N-Acetyl-2-chloro-2-deoxyneuraminic acid methyl ester 4,7,8,9-tetraacetate in 1:1 v/v CH2Cl2/aq.NaHCO3, TBAHS (1 eq) and NaN3 (5 eq) were dissolved and stirred vigorously for 2 h at r.t. The organic phase was washed with sat. NaHCO3 and sodium sulfate was added and incubated over 30 min then filtered. The filtrate was vacuumed, and the reaction progress was confirmed by ESI-MS (m/z calcd. For C20H28N4NaO12 [M + Na]+ 539.2 found 539.2).
The obtained compound was dissolved in MeOH and cooled in ice bath. Then 0.5 M NaOMe/MeOH was added dropwise and stirred for 3 h at r.t. Amberlyst 15 DRY (ORGANO) was added and stirred for 30 min to neutralize the mixture. Subsequently filtered and MeOH was removed by vacuum. The residue was dissolved in water and freeze dried. (Crude yield: 72%) The synthesis scheme is shown in Scheme 4-4.
Scheme 4-3 Synthesis of sugar azides (3’SALac-N3, 6’SALac-N3).
Scheme 4-4 Synthesis of Neu5Ac-N3.
4.2.7 Synthesis of polymer backbone by RAFT polymerization
Polymer backbone was synthesized according to the previous report.39 TMS BtnAAm or AAm, CPBTC as the RAFT agent, and a initiator AIBN were dissolved in DMSO.
The feed ratio of monomer : RAFT agent : initiator was 100 : 1 : 0.4, and the total monomer concentrateon was 1.0 M in all polymerization. The solution was brought out to freeze thaw cycle three times, and sealed under vacuum. Then the solution was incubated at 70oC for 18 h. Conversion of the polymerization was confimed by
1H-NMR (DMSO-d6). The polymers were purified by dialysis agains excess amount of acetone : methanol = 1 : 1 mixture for a day, and vacuum dried.
Secondly, TMS groups were deprotected. The polymer was stirred in TBAF/THF for 9 h, and purified by dialysis against excess amount of Acetone : MeOH = 1 : 1 mixture for 2 days, and vacuum dried. The deprotection of TMS groups in the polymer was confirmed by 1H-NMR (MeOH-d4). The synthesis scheme is shown in Scheme 4-5.
4.2.8 Synthesis of glycopolymer by click chemistry
Sugar units were incorporated into the synthesized polymer backbone referring the previous report.39 The synthesized polymer backbone, sugar azide (3 eq against alkyne units), CuSO4 (0.4 eq against alkyne units), TBTA (0.4 eq against alkyne units), and
Scheme 4-5 Synthesis of polymer backbone by RAFT polymerization.
L·Asc·Na (2 eq against alkyne units) were dissolved in 1:1 v/v solution of MilliQ and CH3CN. Then the mixture was kept at 60oC for 6 h, and N2 was bubbled through during the reaction. Then the mixture was purified by dialysis against excess amount of 1 mM HCl aqueous solution and MilliQ for a day, and freeze dried. Incorporation of sugar units into the polymer backbone was confirmed by 1H-NMR (D2O). The synthesis scheme is shown in Scheme 4-6. The entries of the synthesized glycopolymers are summarized in Table 4-2.
4.2.9 SPRI measurement of protein binding onto glycopolymer array
SPRI gold chips were prepared by deposition of Cr (1.0 nm) and Au (50 nm) onto S-TIM35 glass substrate. SPRI gold chip was rinsed with EtOH and MilliQ, then dried by air blow. Glycopolymers were immobilized onto the gold spots by incubation of 10 mg / mL glycopolymer aqueous solution for over 3 h (Figure 4-3).
Prior to the protein adsorption measurement, 10 mM Phosphate buffered saline (PBS) (pH 7.4, 137 mM NaCl, 2.68 mM KCl) was flew through (0.1 mL / min), and SPRI reflectivity change (defined as “SPRI signal”) was monitored until the SPRI signal was stable. Then, protein solution with a certain concentration was injected with
Scheme 4-6 Synthesis of glycopolymer by click chemistry.
flow rate of 0.1 mL / min in all experiments, and the SPRI signal was monitored.
For the kinetic analysis of CTB binding onto the glycopolymer immobilized gold surface, the data fitting was performed by the single exponential curve described in Equation (1), and the binding rate constant kon and dissociation rate constants koff were calculated by relaxation method.
Δ𝑅 = Δ𝑅6HI 1 − 𝑒LNM (1) where 𝜏LP= 𝑘QR 𝐶𝑇𝐵 + 𝑘QVV (2)
Here, ΔR and ΔRmax are SPRI signal change and SPRI singal value at the point of binding saturation, respectively.
4.3 Results & Discussion
4.3.1 Synthesis of glycopolymers by post-click chemistry
The results for synthesis of polymer backbone and polyacrylamide (PAAm) by RAFT polymerization were summarized in Table 4-1. The conversions of PAAm and poly(TMS BtnAAm) were calculated from 1H-NMR and were 96% and 62%, respectively. The molecular weight and PDI of the polymers were obtained using SEC.
Figure 4-3 Immobilization of glycopolymers onto SPRI gold spot.
The molecular weight of PAAm and poly(TMS BtnAAm) were 1.20 × 104 and 1.28 × 104 , respectively. The PDI of the polymers were lower than 1.5 in both polymers.
The polymers were synthesized according to the previous report, and the obtained results resembled the previous results.39 The molecular weight of the polymer backbone was close to the theoretical value, and PDI was narrow (< 1.5). Thus, it is indicated that the well-defined polymer backbone was synthesized successfully. The TMS groups in the polymer was deprotected by reaction in TBAF/THF solution, and the deprotection was confirmed by disappearance of TMS peak in 1H-NMR (data shown in appendix).
Furthermore, UV spectra before and after the deprotection step were measured, and confirmed that the peak corresponding to the trithiocarbonate at 310 nm disappeared after the deprotection step. This indicate that the trithiocarbonate groups were cleaved in the deprotection step.
Synthesized glycopolymer entries were summarized in Table 4-2. The sugar units were incorporated in to the polymer backbone poly(BtnAAm) by copper-catalyzed azide-alkyne cyclo addition as the previous report.39 Each sugar azide compounds were synthesized according to the previous method,41 and was used in the reaction with the ratio shown in Table 4-2.
Incorporation of sugar units into the polymer backbones were basically confirmed
Table 4-1 Synthesis of the polymer backbone and polyacrylamide.
MnTH describes the theoretical molecular weight of 100 mer polymer times conv.(%)/100.
by 1H-NMR (data shown in appendix). The ratio of proton peak corresponding to a proton of triazole ring and the proton of alkyl carbon next to the triazole ring was 1:2, indicating the complete incorporation of sugar units into the alkyne groups of poly(BtnAAm). Same backbone was used for all of the polymers.
4.3.2 Surface analysis of polymer immobilized gold substrate using XPS
XPS Au(4f) and C(1s) spectra of the gold substrates were shown in Figure 4-4 and Figure 4-5, respectively. For Au(4f) spectra, peaks corresponding to Au at 84.0 eV and 87.5 eV were observed in all gold substrates. For C(1s) spectra of unmodified gold substrate (Figure 4-5a), no specific peak was observed. For C(1s) spectra of PAAm immobilized gold substrate (Figure 4-5b), peaks corresponding to C-C bonds and C=O bonds were observed at 285.0 eV and 288.0 eV, respectively. For C(1s) spectra of P1 and P10 immobilized gold substrate (Figure 4-5c and 4-5d), peaks corresponding to C-C bonds, C-O or C-N bonds, and C=O bonds were observed at 285.0 eV, 286.5 eV and 288.0 eV, respectively.
The intensity of the peak corresponding to Au decreased in polymer immobilized
Table 4-2 Synthesized glycopolymers and ratio of sugar azides added against alkyne in click reaction.
gold substrates compared to unmodified gold substrates. This indicated that the surface of gold was covered with materials. Peaks corresponding to carbon were observed only in polymer immobilized film, and the peak corresponding to the bonds included in each polymers were observed. The peak corresponding to C-O bonds were only observed in glycopolymer immobilized gold substrate (Figure 4-5c and 4-5d), and not in PAAm immobilized gold substrate. This is due to the hydroxyl groups in the sugar units. From these results, it is confirmed that the polymers were immobilized onto the gold surface successfully.
Figure 4-4 XPS spectra for Au(4f). (a) Blank, (b) PAAm immobilized, (c) P1 immobilized, and (d) P10 immobilized gold substrate.
4.3.3 SPRI measurement of CTB binding to glycopolymer array
SPRI screening of CTB interaction to glycopolymer array of P1-P8 was performed. The results were summarized in Figure 4-6. Time dependent SPRI signal change by addition of 1 µM CTB was monitored (Figure 4-6a). Large SPRI signal change was observed in P1 immobilized gold spot, and no large signal intensity change was observed in gold spots immobilized with other polymers. The SPRI signal value at the signal saturated point was summarized in Figure 4-6b. P1 immobilized gold spot showed the largest SPRI signal intensity, and was more than 6 times larger than spots immobilized with other polymers.
Figure 4-5 XPS spectra for C(1s). (a) Blank, (b) PAAm immobilized, (c) P1 immobilized, and (d) P10 immobilized gold substrate.
Only P1 immobilized gold spot showed large SPRI singal change, indicating that CTB bound specifically with P1. Considering the chemical structure of GM1, it is consisted of four types of sugars, Gal, Neu5Ac, GalNAc, and Glc. Furthermore, it is indicated in the previous reports that Gal and Neu5Ac are mainly involved in the interaction between CTB and GM1, and also that GalNAc contribute to the interaction.44 Hence, we expected that SPRI signal change would also be observed in P4, P6, and Neu5Ac containing P7 and P8. However, CTB interaction to these polymers were not observed in this investigation. Hence, we considered that Gal is the main component for the interaction with CTB.
4.3.4 Investigation of CTB interaction with glycopolymers synthesized by glyco-module method
Considering that the interaction of GM1 is stronger than that of Gal, and also that GalNAc and Neu5Ac are also involved in the interaction between GM1 and CTB,44 I
Figure 4-6 SPRI signal change by addition of CTB (1 µM) to glycopolymer array of P1-P8. (a) Time dependent signal change, and (b) SPRI signal value at the saturated point.
synthesized glycopolymers baring multiple sugar units (P9-P12). Here, SPRI screening of CTB (200 nM) adsorption against glycopolymer arrays of P1 and P9-P11 was performed (Figure 4-7). P10 immobilized gold spot showed a large SPRI signal change by addition of CTB, and P1 also showed SPRI signal change. In contrast, no large SPRI signal change was observed in P9 and P11 immobilized gold spots.
P9 immobilized gold spot did not show SPRI signal change, which indicate that GalNAc units do not contribute to the interaction to CTB, rather disturb the interaction because the density of Gal units decreased and resulted in the loss of glyco-cluster effect. In contrast, P10 showed larger SPRI signal change than P1, indicating that P10 have better binding ability than P1. Although Neu5Ac containing glycopolymer P6 did not show large SPRI signal change (Figure4-6), glycopolymer containing Gal and Neu5Ac showed larger signal change than P1. It is considered that Neu5Ac units bound subsequently to Gal and contributed to the enhanced interaction. This is also indicated in other report.44 P11 showed larger signal change compared to P9, however, it did not
Figure 4-7 SPRI signal change by addition of CTB (200 nM) to glycopolymer array of P1, and P9-P11.
show large SPRI signal change than P1 although P11 contains both Gal and Neu5Ac. I considered that important point is the placement of Gal unit and Neu5Ac unit. For the enhanced interaction, Neu5Ac unit has to be close to Gal unit in molecular level. Hence, low signal for P11 is because the distance between Gal unit and Neu5Ac unit in the glycopolymer is stochastically far due to the incorporation of GalNAc unit.
4.3.5 Binding analysis of CTB binding to glycopolymer baring Gal and Neu5Ac It is indicated that incorporation of Gal and Neu5Ac units into the polymer enables enhanced interaction against CTB. I previously noted that GM1 is consisted of four types of sugars, however, it can be also assumed that GM1 is consisted of Gal, GalNAc and 3’SALac. Hence, glycopolymer incorporating Gal and 3’SALac was synthesized (P12). It can be expected that P12 also show enhanced interaction to CTB because 3’SALac contains Neu5Ac at the tip of its structure.
Figure 4-8 SPRI signal change by addition of CTB (1 µM) to glycopolymer array of P10 and P12.
The SPRI measurement for CTB binding onto P10 and P12 was shown in Figure 4-8.
P12 did not show large SPRI signal change although it contains both Gal units and Neu5Ac units. We considered that Gal binding to CTB was inhibited due to the steric hindrance of 3’SALac. Hence, we concluded that P10 interacts the most with CTB.
4.3.6 Analyzing the cooperativity of Gal unit and Neu5Ac unit for the interaction with CTB
Furthermore, I prepared gold surfaces with Gal and Neu5Ac units by spotting P1 and P6 mixed solutions onto the gold spots. Polymer solution of total 10 g/L was prepared by mixing P1 and P6 in the weight ratio of 7:3 and 5:5. In addition, polymer solution f total 10 g/L was prepared by mixing P10 and PAAm in the weight ratio of 7:3 and 5:5. Then the solutions were spotted onto different gold spots, and CTB interaction onto the surface containing two different polymers were investigated (Figure 4-9a). The SPRI measurement result was shown in Figure 4-9b. The P10 immobilized spot showed the largest SPRI signal change, and the signal decreased with the decrease in ratio of P10 against PAAm. For the gold spots immobilized with both P1 and P6, no large SPRI signal change was observed.
The decrease in SPRI signal change by mixing P10 with PAAm suggested that the density of P10 decreased by co-adsorption of PAAm onto the gold surface. Hence, the polymer mixed surface was prepared successfully. The gold spot with both P1 and P6 did not show large SPRI signal change. Although the surface contains both Gal units and Neu5Ac units, there were not many points where Gal unit and Neu5Ac unit are placed closely. In my previous report, I investigated that the water soluble glycopolymers synthesized by RAFT polymerization has mushroom-like brush structure in water when immobilized onto a gold surface. Hence, there are little areas on gold
surface where Gal unit and Neu5Ac unit are placed closely in molecular level, and also resulted in the loss of glyco-cluster effect. From these results, I suggest that incorporation of Gal and Neu5Ac in the same polymer is necessary for the enhanced interaction with CTB.
4.3.7 Binding analysis of CTB binding to glycopolymer synthesized by glyco-module method
For quantitative comparison, the kinetic analysis of CTB binding onto P1 and P10 immobilized gold spots were performed. CTB with concentrations of 50, 75, 100, 150, 200, 500, and 1000 nM were added to the glycopolymer immobilized chip, and the data fitting of time dependent SPRI signal change was performed by the equation described in the experimental section. The reciprocal of the relaxation time (τ) obtained from the measurement was plotted against CTB concentration (Figure 4-10). The obtained binding/dissociation rate constant and binding constant were summarized in Table 4-3.
Figure 4-9 (a) Concept of the experiment, (b) SPRI signal change by addition of CTB (1 µM) to glycopolymer array of mixed surfaces.
The binding rate constant kon was an order higher in P10 than P1. The dissociation rate constant koff was three orders lower in P10 than P1. The binding constant Ka was three orders higher in P10 than P1.
The results shown that the affinity of P10 was significantly stronger that P1. This is due to the drastic decrease in dissociation rate constant. I consider that this is due to the subsequent bond of Neu5Ac unit to CTB after Gal unit bond which is also indicated in other report.44 Although there was an increase in binding rate constant, it was not as drastic as the dissociation rate constant. This may be because the Gal unit is the main component in the binding process. From these results I suggest that strong interaction of GM1 with CTB is due to the specific recognition of Gal and ‘locking’ of the interaction by subsequent bond of Neu5Ac. Hence, it can be said that the glycopolymer synthesized by glyco-module strategy is capable of mimicking the molecular recognition of physiologically active oligosaccharide.
Binding constant of CTB to GM1 is in order of 1011 M-1.45 Therefore, the affinity is stronger than P10. However, the interaction of the glycopolymer may improve by more precise design of the polymers. For instance, controlling the incorporation ratio, sequence, or polymer length. Gibson et al reported that longer linker for Gal containing glycopolymer results in better mimic of GM1.46 Hence greater potential of the glycopolymer can be expected.
I performed SPRI screening against glycopolymers synthesized by glyco-module method in multiple ways. This exhaustive analysis system is capable of extracting glycopolymers that interact with the target protein with high affinity, and furthermore might enable high-throughput cycle for developing useful molecular recognition materials. Therefore, the results are important in the field such as development of glycomaterials, and glyco-engineering.
4.4 Conclusion
Considering sugar units constructing oligosaccharide as a glyco-module, glycopolymer baring multiple kinds of sugars were synthesized successfully by post-click chemistry.
Immobilization of the synthesized glycopolymers onto a gold surface was confirmed using XPS. SPRI screening of CTB binding onto glycopolymer with single type of sugar unit suggested that Gal is the main component for the interaction with CTB.
Furthermore, SPRI screening was performed for glycopolymers baring multiple kinds of sugar units which were selected from the sugar units constructing GM1. Interestingly,
Figure 4-10 Plots for the invers of binding relaxation time (t) as a function of CTB concentration.
Table 4-3 Results for kinetic analysis of CTB biniding to glycopolymers.
glycopolymer incorporating Gal and Neu5Ac showed larger signal response compared to Gal containing glycopolymer. The kinetic analysis revealed that the affinity of the glycopolymer baring both Gal and Neu5Ac was markedly higher than that of glycopolymer containing only Gal. The results suggested the possibility of mimicking the molecular recognition ability of oligosaccharide by glyco-module strategy. This work and the performed system would contribute the progress in glyco-engineering.
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Appendix
UV spectra of polymer backbone before and after deprotection procedure and 1H-NMR spectra of the synthesized glycopolymers, and are shown.
Figure 4S-1 UV spectra of poly(TMS BtnAAm) and poly(BtnAAm)
Figure 4S-2 1H-NMR spectrum of poly(TMS BtnAAm).
Figure 4S-3 1H-NMR spectrum of poly(BtnAAm).
Figure 4S-4 1H-NMR spectrum of P1.
Figure 4S-5 1H-NMR spectrum of P2.
Figure 4S-6 1H-NMR spectrum of P3.
Figure 4S-7 1H-NMR spectrum of P4.
Figure 4S-8 1H-NMR spectrum of P5.
Figure 4S-9 1H-NMR spectrum of P6.
Figure 4S-10 1H-NMR spectrum of P7.
Figure 4S-11 1H-NMR spectrum of P8.
Figure 4S-12 1H-NMR spectrum of P9.
Figure 4S-13 1H-NMR spectrum of P10.
Figure 4S-14 1H-NMR spectrum of P11.
Figure 4S-15 1H-NMR spectrum of P12.
Chapter 5
Summary
Summary
In this thesis, molecular recognition ability of glycopolymers were investigated using optical biosensing methods, structural-color of photonic crystal and surface plasmon resonance techniques. Especially, difference in binding abilities of glycopolymers with different polymer conformation, sugar-incorporation ratio, and type of sugar units were determined. Furthermore, the relation between the polymer-brush structure and protein recognition ability, and detail of protein uptake into sugar-incorporating hydrogel nanoparticle were also investigated.
Chapter 2 presents development of biosensor by structural color produced from glycopolymer-immobilized two-dimensional photonic crystal (Figure 5-1). In this theme, two types of glycopolymers, sugar-incoporating hydrogel nanoparticle and sugar-homopolymer were synthesized and immobilized onto nano-imprinted photonic crystal films. The adsorption of target protein onto the glycopolymer-immobilized film was measured by the change in intensity of reflected light irradiated vertically to the film. Both type of glycopolymers showed strong and specific interaction with the target protein, and the hydrogel nanoparticle-immobilized film showed larger signal change compared to the homopolymer-immobilized film. The results revealed that
Figure 5-1 Schematic illustration of structural color biosensing using 2D-PhC.
sugar-incorporating hydrogel nanoparticle showed larger binding capacity because its three-dimensional polymer structure enabled three-dimensional adsorption of the proteins. It can be said that sugar-incoporating hydrogel nanoparticle is useful as the molecular recognition part of biomaterials.
Chapter 3 discuss about the polymer-brush structure of glycopolymers on gold surface and its relation with protein recognition (Figure 5-2). Glycopolymers were synthesized by RAFT polymerization of acrylamide and mannose monomer as the sugar unit. Two glycopolymers with different sugar-incorporation ratio (10% and 100%) were synthesized as designed, and immobilized onto gold surface. The glycopolymer-brush layer thickness in wet and dry condition, and protein binding analysis were measured using SPR technique. The SPR analysis revealed that the glycopolymer-brush form layer with thickness of 2 nm in air, and had larger thickness in water. This result suggested that the glycopolymer-brush was swollen muschroom-like structure in water, and had pancake-like structure in dry condition. From the binding analysis, the binding constant and binding rate constant of target protein to glycopolymer-brush layer was in the same order, although dissociation rate constant was lower in glycopolymer with
Figure 5-2 SPR analysis of glycopolymer interface on the gold surface in air and water.