Glycodendrimers of G1 and G2 (Figure 1) were synthesized using click chemistry according to the divergent method depicted in Scheme 1, Scheme 2, Scheme 3, Scheme 4, Scheme 5, Scheme 6 and Scheme 7.

The trimer of 6-sulfo-GlcNAc (G0) was synthesized via the connection of azide-terminated 6-sulfo-GlcNAc to 1,3,5-triethynylbenzene, using click chemistry [19,20] (Scheme 1 and Scheme 2). p-Aminophenyl-6-sulfo-D-GlcNAc was synthesized according to a method previously reported in the literature [14,15] and subsequently connected to N-phenylhex-5-ynamide using 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl uranium hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIEA) (Scheme 3). Starting from 5-hydroxyisophthalic acid, the dendron of 1,3-bis(chloromethyl)-5-(prop-2-yn-1-yloxy)benzene was synthesized by sequential alkylation, hydrogenation and chlorination reactions, with the resulting dendron being attained in high yield (G1-N₃: 93%, G2-N₃: 56%) (Scheme 4 and Scheme 5).

Glycodendrimers of G1 and G2 were obtained by click chemistry in a modest yield (G1: 28%, G2: 21%) (Scheme 6 and Scheme 7). 1,3,5-Tris(azidomethyl)benzene was used as the core of the dendrimer and was itself derived from 1,3,5-trimesic acid via sequential hydrogenation, chlorination and azidation reactions (Scheme 3). The structures of the compounds were confirmed by ¹H and ¹³C-NMR, electrospray ionization mass spectroscopy (ESI-MS) and matrix-assisted laser desorption ionization and time of flight mass spectroscopy (MALDI-TOF-MS). Detailed syntheses and characterizations are provided in the Experimental Section.

Although the interaction mechanism of WGA is different from that of Aβ, the synthesized glycodendrimers were evaluated in a lectin recognition assay to estimate their biological ability (Figure 2) [19].

Fluorescence quenching of the fluorescein isothiocyanate (FITC)-WGA complex occurred upon the addition of the glycodendrimers [21]. The quenching behavior was analyzed using a Scatchard plot. The binding constants (K_a) of G0, G1 and G2 were found to be 6.56 × 10⁵, 5.55 × 10⁶ and 2.12 × 10⁶ (M⁻¹), respectively, with the affinity constants therefore being of the order G1 > G2 >> G0. In contrast to WGA, the monomeric sulfonated GlcNAc did not show the same detectable affinity for lectin, and the addition of the glycodendrimers to FITC-bovine serum albumin did not induce fluorescence quenching. The results showed that the glycodendrimer of G1 and G2 had the ability to amplify the protein-saccharide interactions, indicating that the protein-saccharide interactions were specific to a certain extent. G0 did not show a high level of affinity for WGA, suggesting the potential for weak interactions to proteins including Aβ.

The hydrophobic domains of the dendrimes of G1 and G2 were analyzed by fluorescence of 8-anilino-1-naphthalene sulfonate (ANS) (Figure 3). The solution of G1 showed a larger blue shift in its emission spectra than that of G2, whereas the solution of G0 did not show any blue shift.

The hydrophobicity of dendrimer suggested that they would possess self-assembling properties in solution. Interestingly, the larger fluorescence of G1 versus that of G2 indicated the larger self-assembling properties of G1 in the aqueous solution. In addition, the glycodendrimers induced the formation of WGA aggregates. The diameters of WGA-glycodendrimer aggregates in the presence of WGA (1.00 μM) were 752, 496 and 384 nm, respectively, with G1, G2, and G0 (data not shown).

Multivalent GlcNAc generally showed a strong interaction based on the cluster glycoside effect [22], which was similar to the interaction observed for G1 and G2. The positive net charge of WGA also contributed to its interactions with G1 and G2 in an electrostatic manner. The sizes of the dendrimers by dynamic light scattering (DLS) became semi-micro in order in the presence of the WGA because of the cross-linking between the WGA and the dendrimer that occurred as a consequence of molecular recognition. The affinity of G1 was larger than that of G2, although the structure of G2 apparently exhibited a greater degree of multivalency. In addition, considering that the distance between the sugar binding sites was 1.5–5 nm in WGA [23,24], the sugar distance of G2 was better suited to accommodate WGA than that of G1. The affinities of dendrimers, however, showed an opposing trend. Taking the ANS results into account, G1 formed larger glycoside clusters than G2 through self-assembly, which resulted in the larger affinity for WGA. Given that the self-assembly plays an important role in the cluster glycoside effect of dendrimers, the affinity between WGA and dendrimer was considered to be dynamic including the cross linking and statistical re-binding aspects of the process. The results suggested that the glycodendrimers of G1 and G2 provided glycoside clusters suitable for binding WGA and Aβ.

The interactions of the glycodendrimers with Aβ were investigated. The aggregation properties of Aβ (1-42) were analyzed by fluorescence spectroscopy with thioflavin T (ThT) [14,15]. It is known that ThT has an affinity for amyloid fibril and that the fluorescence of ThT increases when ThT is attached to amyloid fibrils. In the current experiment, the degree of amyloid formation was monitored by the fluorescence of ThT [25]. The level of amyloidosis was examined over an 8 h period and the fluorescence intensity following this period was evaluated as 1.0. The aggregation behaviors with various sugar additives are shown in Figure 4.

The monomeric 6-sulfonated GlcNAc did not exhibit an inhibitory effect on Aβ aggregation, as we previously reported [14,15]. In contrast, the addition of sulfonated glycodendrimer modestly inhibited the aggregation of Aβ. The degree of inhibition was in the order G1 = G2 > G0 >> monomer. The addition of dendrimer inhibited the aggregation by approximately 50% relative to the level of inhibition observed in the absence of the additives.

The dendrimers of G1 and G2 were effective inhibitors of Aβ aggregation, whereas the inhibitory effect of G0 was insufficient. The observed order of the inhibitory effect was similar to that observed for the lectin recognition. The results suggested that G1 and G2 displayed saccharides in a favorable manner for effective protein affinity. The molecular recognition of the saccharides by lectin was affected by the cluster glycoside effect, which was controlled by the spatial arrangement of the saccharides in the dendrimers or the self-assembled dendrimers. The appropriate glycoside clusters for WGA could also become a better inhibitor for Alzheimer amyloid aggregation.

It has been reported that GAGs bind to Aβ via electrostatic interactions with the basic segment 13HHQK16 [26]. Glycopolymers containing sulfonated saccharides were considered to interact with Aβ in the same way via electrostatic interactions [14,15]. Glycodendrimers are also considered to bind to Aβ via the same electrostatic interactions. Although the glycodendrimer G1 showed the largest binding properties, its binding to Aβ was almost identical to that of G2. Although the structures of WGA and Aβ are totally different, it was considered that the larger cluster of the glycosides would show high levels of affinity that were similar to those observed in their interaction with WGA.

Multivalent sulfonated groups have been reported to interact with Aβ with GAGs [26], 6-sulfoGlcNAc polymer [14,15], sulfonated Glc polymer [27], and the polymer with sulfonic acid [28]. It is obvious that the sulfonic acid interact with Aβ via electrostatic interaction. The structural specificity and the mechanism of Aβ interactions are under investigation in our group.

The conformation of Aβ was investigated in the presence of the sulfonated glycodendrimer (Figure 5). In the absence of the sugar additives, Aβ showed the negative cotton effect around 220 nm, suggesting β-sheet conformation [29]. Aβ also showed β-sheet structure in the presence of the monomeric sulfonated saccharide and G1. In contrast, the additions of G1 and G2 led to significant changes in the conformation, with the negative cotton effect observed around 220 nm almost disappearing entirely. The addition of G0 did not lead to a decrease in the negative cotton effect, suggesting that the interaction with Aβ was weak.

CD spectra recorded in the presence of the sulfonated glycodendrimer indicated that its addition led to the inhibition of the aggregation and conformation change of Aβ. Since the amyloidosis relates to changes in the conformation and aggregation, these results were in good agreement with the Th-T experiments. The inhibition of Aβ aggregation was also evaluated with the sulfonated glycopolymer. The results revealed that the sulfonated glycoside clusters inhibited oligomer formation during the early stages of the amyloidosis. It was also considered that the sulfonated glycodendrimer interacted with Aβ and inhibited the formation of the Aβ oligomer [14].

To develop an understanding of the amyloidosis and the properties of the inhibitors, it was necessary to develop an understanding of the Aβ morphology, because the results from the ThT fluorescence and CD spectra only provided an indirect reflection of the protein amyloidosis. The properties of the amyloid, including its cytotoxicity, were strongly related to the size and shape of the aggregates, and so the AFM measurements were indispensable in evaluating these properties [30]. Aβ spontaneously formed nanofiber upon incubation, with widths of 45-70 nm and length of only a few micrometers (Figure 6a). Aβ also formed nanofibers in the presence of the monomeric saccharide with widths of 35–70 nm, although the fibers were shorter in length at less than 1 μm (Figure 6b). In contrast, the additions of G1 and G2 led to significant changes in the morphology of Aβ. Nanofibers of the Aβ amyloid were not observed at all in the presence of G1 and G2 (Figure 6d,e). Interestingly, the addition of the sulfonated trimer of G0 induced the largest aggregates (Figure 6c).

The morphology of Aβ correlated well with the results of the ThT and CD analyses. Aβ formed nanofibers in the absence of any additives. AFM analysis revealed that the monomeric 6-sulfonated GlcNAc showed the lowest level of Aβ aggregation inhibition, and this result was consistent with the results of the ThT fluorescence and CD spectra. In contrast, the AFM results indicated that the dendrimers of G1 and G2 showed the highest levels of Aβ aggregation inhibition, which were in good agreement with the results of the ThT and CD spectra. The formation of large aggregates was inhibited by the strong interaction with the glycodendrimer.

For G0 (trimer of 6-sulfonated GlcNAc), the AFM results indicated that the material only exerted a minor impact on the inhibition of Aβ aggregation, with the result being consistent with the ThT and CD results. Furthermore, the aggregates in this case were much larger than those formed in the absence of the additives. The shape of the aggregates was different from the nanofiber of the amyloid. The morphology of G0 was measured in the absence of Aβ and the results indicated that G0 formed small aggregates (Figure 7) even though the hydrophobicity by ANS fluorescence was small.

The morphology of Figure 6c was considered to be representative of a mixture of Aβ and G0 aggregates. Since the interaction of Aβ with G0 was weaker than its interactions with G1 and G2, the nanofiber formation of Aβ aggregates was not hindered. G0 was envisaged to have behaved as a glue, effectively bringing Aβ units together to yield the observed large objects. The morphology in this particular experiment was determined to be mixture of Aβ, Aβ-dendrimer and dendrimer aggregates. In spite of this, it was expected that the cytotoxicity of Aβ would be reduced by the addition of G0 because of the resulting reduction in the number of toxic Aβ aggregates.

The following reagents were used as received: N-acetylglucosamine, copper(0) powder, copper(II) sulfate, copper(I)iodide, N,N-dimethylformamide (DMF), chloroform (CHCl₃), ethyl acetate (EtOAc), n-hexane, magnesium sulfate (MgSO₄), methanol (MeOH), methylene chloride (CH₂Cl₂), potassium bromide (KBr), potassium carbonate (K₂CO₃), 2-propanol, sodium L-ascorbate, sodium azide (NaN₃), sodium bicarbonate (NaHCO₃), sodium carbonate (Na₂CO₃) sodium hydride (NaH), sodium hydroxide (NaOH), tetrahydrofuran (THF), thioflavin-T(ThT), thionyl chloride (SOCl₂), (Kanto Chemical Co., Inc., Tokyo, Japan), 2-[2-(2-chloroethoxy)ethoxy]ethanol, DIEA, 5-hexynoic acid, propargyl bromide, 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), trichloroisocyanuric acid, 1,3,5-triethynylbenzene, trimesic acid (TCI Co., Tokyo, Japan), amyloid β protein (human, 1-42) (Aβ (1-42)), HATU (Peptide Institute Inc., Osaka, Japan), palladium/charcoal (Pd/C) (Merck & Co., Inc., Whitehouse Station, NJ, USA), and deuterated solvents (CDCl₃, CD₃OD, DMF-d₇ and D₂O), dimethyl 5-hydroxyisophtalate, and lithium alminium hydride (LiAlH₄) (Sigma-Aldrich, St. Louis, MO, USA). 6-sodium sulfo-N-acetyl-β-D-glucosamine (6) was synthesized according to the previous literature [5,14].

The identities of the compounds were confirmed by the following methods. Both ¹H-NMR (300 MHz) and ¹³C-NMR (75 MHz) spectra were recorded using a Varian Gemini 2000 spectrometer and ¹H-NMR (500 MHz) spectra was recorded using a Varian UNITY 500plus spectrometer, equipped with a Sun workstation, respectively. The spectra were measured in CDCl₃, CD₃OD, DMF-d₇ and D₂O solvents at room temperature. Mass spectra were measured on a MALDI-TOF-MS (Voyager, Applied Biosystems, Foster City, CA, USA) and ESI-MS (LCQ Deca xp, Thermo Fisher Scientific, Waltham, MA, USA). CD spectra were recorded using a JASCO J-720 spectrometer (JASCO, Tokyo, Japan). AFM measurement was carried out using SPA400 instrument (Seiko Instruments Inc., Tokyo, Japan) with a 40 N cantilever. Fluorescence spectra were measured on a JASCO FP-6500 (JASCO) at 25 °C. DLS for the GNPs was determined using a Zetasizer 3000 (Sysmex, Kobe, Japan).

[2-(2-Chloroethoxy)ethoxy]acetic acid (2). [2-(2-Chloroethoxy)ethoxy]ethanol (1, 2.20 g, 13.0 mmol) was dissolved in acetone (30 mL), and 15% NaHCO₃ aq. was added to the solution at 0 °C. KBr (0.312 g, 2.60 mmol) and TEMPO (0.040 g, 0.040 mmol) were added to the solution, and trichloroisocyanuric acid (6.10 g, 26.2 mmol) was added dropwise. The solution was allowed to warm up to room temperature, and stirred for 8 h. The reaction was confirmed by TLC (EtOAc–n-hexane = 3:1), and 2-propanol (10 mL) was added to quench the reaction. The reaction mixture was filtrated with Celite, and neutralized with sat. Na₂CO₃ aq. The solution was acidified with 1 N HCl, and extracted with CHCl₃. The organic phase was dried over MgSO₄, and MgSO₄ was removed by filtration. The solution was evaporated to yield yellow oil; Yield 1.94 g, 88.6%. ¹H-NMR (300 MHz, r.t., CDCl₃): δ/ppm 3.58–3.60 (m, 2H, CH₂), 3.64–3.68 (m, 2H, CH₂), 3.69–3.70 (m, 2H, CH₂), 3.71–3.76 (m, 2H, CH₂), 4.18 (s, 2H, CH₂). IR wavenumber [cm⁻¹]: 3470 (OH) 1734 (C=O) 1112 (C-O). ESI-MS (negative): 181.2 [M−H]⁻.

Methyl 2-[2-(2-chloroethoxy)ethoxy]acetate (3). Compound 2 (0.312 g, 1.78 mmol) was dissolved in DMF (2 mL), and NaH (0.530 g, 2.22 mmol) was added to the solution at 0 °C. After stirring for 30 min, MeI (0.649 g, 3.33 mmol) was added to the solution. The reaction mixture was stirred for 22.5 h at room temperature. The reaction was confirmed by TLC (EtOAc–n-hexane = 1:3). The solution was evaporated, and the residue was dissolved in CHCl₃. The solution was washed with 1 N HCl, sat. NaHCO₃ aq. and brine. The solution was dried over MgSO₄, and MgSO₄ was removed by filtration. The residue was purified by column chromatography (EtOAc–n-hexane = 1:3) to yield colorless oil; Yield 0.242 g, 74.4%. ¹H-NMR (300 MHz, r.t., CDCl₃): δ/ppm 3.62 (t, 2H, J = 6 Hz, J = 6 Hz, CH₂), 3.71–3.72 (m, 2H, CH₂), 3.72–3.74 (m, 2H, CH₂), 3.73 (s, 3H, CH₃), 3.74–3.76 (m, 2H, CH₂), 4.16 (s, 2H, CH₂). ESI-MS (positive): 219.1 [M+Na]⁺.

Methyl 2-[2-(2-azidoethoxy)ethoxy]acetate (4). Compound 3 (0.242 g, 1.31 mmol) was dissolved in DMF (15 mL). NaN₃ (0.511 g, 7.86 mmol) was added to the solution, and stirred for 14 h at 70 °C. The reaction was confirmed by TLC (EtOAc–n-hexane = 1:3). NaN₃ was removed by filtration, and the solution was evaporated. The residue was dissolved in CHCl₃, and the solution was washed with 1 N HCl, sat. NaHCO₃ aq. and brine. The organic phase was dried over MgSO₄, and MgSO₄ was removed by filtration. The solution was evaporated to yield colorless oil; Yield 0.220 g, 87.8%. ¹H-NMR (300 MHz, r.t., CDCl₃): δ/ppm 3.38 (t, 2H, J = 5 Hz, J = 5 Hz, CH₂), 3.65–3.67 (m, 2H, CH₂), 3.67–3.67 (m, 2H, CH₂), 3.70–3.72 (m, 2H, CH₂), 3.73 (s, 3H, CH₃), 4.16 (s, 2H, CH₂). ESI-MS (positive) 226.1 [M+Na]⁺.

[2-(2-Azidoethoxy)ethoxy]acetic acid (5). Compound 4 (0.220 g, 1.15 mmol) was dissolved in 1 N NaOHaq (15 mL), and the solution was stirred for 16 h. The reaction was confirmed by TLC (EtOAc–n-hexane = 1:3). The solution was acidified with 1 N HCl, and extracted with CHCl₃. The solution was dried over MgSO₄, and MgSO₄ was removed by filtration. The solution was evaporated to yield colorless oil; Yield 0.172g, 84.4%. ¹H-NMR (300 MHz, r.t., CDCl₃): δ/ppm 3.41 (t, 2H, J = 4.8 Hz, J = 5.1 Hz, CH₂), 3.67–3.69 (m, 2H, CH₂), 3.70–3.71 (m, 2H, CH₂), 3.75–3.78 (m, 2H, CH₂), 4.16 (s, 2H, CH₂). ESI-MS (negative): 188.6 [M−H]⁻.

p-N-[2-(2-Azidoethoxy)ethoxy]amidophenyl-2-acetamido-2-deoxy-6-sulfo-β-D-glucopyranoside (7). Compound 6 was synthesized by following the previous method [5,14]. Next 6 (0.142 g, 0.320 mmol) was dissolved in MeOH–H₂O = 2:1 (10 mL). Pd/C (20 mg) was added to the solution, and the solution was stirred overnight under H₂. Pd/C was removed by filtration, and the solution was evaporated. The residue was dissolved in DMF (5 mL), and 5 (86.2 mg, 0.456 mmol) was added to the solution. HATU (185 mg, 0.487 mmol) and DIEA (83 μL, 0.487 mmol) were added to the solution, and the solution was stirred overnight. The solution was evaporated, and the residue was purified by column chromatography (H₂O–MeOH = 5:1) to yield a brown solid; Yield 0.160 g, 83.2%. ¹H-NMR (500 MHz, r.t., D₂O): δ/ppm 2.09 (s, 3H, OCOCH₃), 3.36–3.39 (m, 2H, CH₂), 3.48 (t, 1H, J_H3-H4 = 9.0 Hz, J_H4-H5 = 9.5 Hz, H4), 3.54 (t, 1H, J_H2-H3 = 10.0 Hz, J_H3-H4 = 9.5 Hz, H3), 3.57–3.62 (m, 2H, CH₂), 3.66–3.68 (m, 2H, CH₂), 3.71–3.73 (m, 2H, CH₂), 3.74–3.75 (m, 1H, H5), 3.88 (t, 1H, J_H1-H2 = 9.5 Hz, J_H2-H3 = 9.5 Hz, H2), 4.11 (s, 2H, CH₂), 4.13 (dd, 1H, J_H5-H6proR = 4.0 Hz, J_{H6proR-H6proS} = 6.0 Hz, H6proR), 4.28 (d, 1H, J = 10 Hz H6proS), 5.01 (d, J = 8.5 Hz, H1), 6.98(d, 2H, J_o-m=7.0 Hz, Ph-o), 7.27 (d, 2H, J_o-m = 9.0 Hz, Ph-m). ESI-MS (negative): 421.1 [M−Na]⁻.

pNP 6-Sulfo-GlcNAc trimer (G0:9). Compound 7 (91.8 mg, 0.157 mmol) and 1,3,5-triethylbenzene (8) (5.12 mg, 0.0341 mmol) were dissolved in DMF (20 mL). To the solution, sodium ascorbate (12.2 mg, 0.6 eq.) and CuSO₄ (4.91 mg, 0.3 eq.) was added. The solution was stirred for 2 days, and the reaction was confirmed by reversed phase TLC (H₂O–MeOH = 5:1). Copper was removed by centrifugation, and the residue was purified by column chromatography (H₂O–MeOH = 5:1) to yield colorless solid; Yield 0.0320g, 49.2%. ¹H-NMR (300 MHz, r.t., D₂O):δ/ppm 1.86(s, 9H, CH₃), 3.41–3.47 (overlap, 6H, CH₂), 3.41–3.47 (overlap, 3H, H4), 3.52–3.57 (overlap, 3H, H3), 3.52–3.57 (overlap, 12H, CH₂ × 2), 3.66 (broad, 3H, 5H), 3.66 (broad, 6H, CH₂), 3.75–3.77 (overlap, 3H, H2), 3.77 (s, 6H, CH₂), 3.98 (s, 6H, NH), 4.05–4.07 (m, 3H, H6proR), 4.16 (d, 3H, J = 11.5 Hz H6proS), 4.61–4.62 (overlap, 3H, H1), 6.47 (d, 6H, J_o-m = 9.0 Hz, Ph-o), 6.77 (d, 6H, J_o-m = 8.5 Hz, Ph-m), 7.53 (s, 3H, Ar), 8.18 (s, 3H, C=CH). ¹³C-NMR (75 MHz, r.t., D₂O): δ/ppm 21.6, 49.5, 53.7, 54.8, 66.2, 67.8, 68.7, 68.8, 69.2, 72.9, 73.3, 99.3, 116.1, 121.2, 121.7, 122.6, 130.0, 130.6, 153.2, 169.2, 174.0. ESI-MS: m/z 976.1 [M+2Na]²⁺, (LCMS, negative) m/z 822.9 [M−3Na]³⁻.

p-N-(5-Hexynoic)amidophenyl-2-acetoamido-2-deoxy-6-sulfo-β-D-glucopyranoside (10). Compound 6 was dissolved in MeOH–H₂O = 2:1. Pd/C was added to the solution, and the solution was stirred overnight under H₂. Pd/C was removed by filtration, and the solution was evaporated. The residue, p-aminophenyl-2-acetoamido-2-deoxy-6-sulfo-β-D-glucopyranoside (0.353 g, 0.853 mmol) was dissolved in DMF (20 mL), and 5-hexynoic acid (0.143 g, 1.28 mmol) was added to the solution at 0 °C. HATU (0.486 g, 1.28 mmol) and DIEA (217 μL, 1.28 mmol) was added to the solution, and the solution was stirred overnight. DMF was evaporated, and the residue was purified by reversed phase column chromatography (H₂O–MeOH = from 5:1 to 2:1) to yield white solid; Yield 0.366g, 81.6%. ¹H-NMR (500 MHz, r.t., D₂O): δ/ppm 1.74 (t, J = 7.0Hz, 2H, CH₂), 1.90(s, 3H, OCOCH₃), 2.15–2.18 (m, 2H, CH₂), 2.25 (t, J = 2.5Hz, 1H, C≡CH), 2.36–2.39 (m, 2H, CH₂), 3.47 (t, 1H, J_H3-H4 = 9.5 Hz, J_H4-H5 = 9.5 Hz, H4), 3.52–3.55 (m, 1H, H3), 3.70–3.73 (m, 1H, H5), 3.87 (t, 1H, J_H1-H2 = 8.5Hz, J_H2-H3 = 10 Hz, H2), 4.11 (dd, 1H, J_H5-H6proR = 5.5Hz, J_{H6proR-H6proS} = 5.0 Hz, H6proR), 4.25 (d, 1H, J = 11.5 Hz, H6proS), 4.99 (d, J = 8.5 Hz, H1), 6.93 (d, 2H, J_o-m = 9.5 Hz, Ph-o), 7.27 (d, 2H, J_o-m = 9.5 Hz, Ph-m).

Trimethyl 1,3,5-benzenetricarboxylate (12). Trimesic acid (11, 8.00 g, 38.1 mmol) was dissolved in MeOH (140 mL), and conc. H₂SO₄ (2 mL) was added to the reaction mixture. The solution was refluxed for 24 h, and the solution was evaporated. The residue was dissolved in CHCl₃, and washed with sat. Na₂CO₃aq. The solution was dried with MgSO₄, and removed by filtration. The solution was evaporated to obtain white solid; Yield 9.09 g, 94.7%. ¹H-NMR (300 MHz, r.t., CDCl₃): δ/ppm 3.96 (s, 9H, OMe), 8.83 (s, 3H, Ar).

1,3,5-Trihydroxymethylbenzene (13). Compound 12 (1.00 g, 3.97 mmol) was dissolved in THF (70 mL). LiAlH₄ (35.7 mmol) was added to the solution, and the solution was refluxed for 24 h. The reaction was confirmed by TLC (EtOAc–n-hexane=1:1), and water was added to the reaction in order to quench the reaction. The solution was dried over MgSO₄ and evaporated to yield white solid; Yield 0.571 g, 85.6%. ¹H-NMR (300 MHz, r.t., CDCl₃): δ/ppm 4.60 (s, 6H, CH₂), 7.25 (s, 3H, Ar).

1,3,5-Tribenzylchloride (14). Compound 13 (0.200 g, 1.19 mmol) was dissolved with SOCl₂ (5 mL) at 0 °C and to the solution the catalytic amount of DMF was added. The solution was stirred for 48 h at room temperature. The reaction was confirmed by TLC (EtOAc–n-hexane = 1:1). The reaction mixture was poured into ice-water, and extracted with CHCl₃. The organic phase was washed with water, and dried over MgSO₄. The solution was evaporated to yield white solid; Yield 0.241 g, 93.9%. ¹H-NMR (300 MHz, r.t., CDCl₃): δ/ppm 4.57 (s, 6H, CH₂), 7.36 (s, 3H, Ar).

1,3,5-Tribenzylazide (15). Compound 14 (0.214 g, 0.964 mmol) was dissolved in DMF (10 mL). NaN₃ (1.13 g, 17.4 mmol, 18 eq.) was added to the solution, and the solution was stirred at 70 °C for 21 h. The solution was evaporated, and the residue was dissolved with chloroform. The solution was washed with 1 N HCl aq., sat. NaHCO₃, and brine. The solution was dried over MgSO₄. The solution was evaporated to yield colorless oil; Yield 0.218 g, 93.0%. ¹H-NMR (300 MHz, r.t., CDCl₃): δ/ppm 4.38 (s, 6H, CH₂), 7.23 (s, 3H, Ar).

1-(3-Acetyl-5-prop-2-ynyloxy-phenyl)ethanone (17). Dimethyl 5-hydroxyisophtalate (16, 5.01 g, 23.8 mmol) and K₂CO₃ (93 g, 35.7 mmol, 1.5 eq.) were dissolved in DMF (100 mL), and the solution was degassed with bubbling of N₂. The solution was heated up to 80 °C, and propargyl bromide (2.80 mL, 35.7 mmol, 1.5 eq.) was added under N_2. The solution was stirred for overnight. The solution was allowed to be cooled to room temperature. K₂CO₃ was removed by filtration, and the solution was evaporated. The residue was recrystallized in EtOH to yield white solid; Yield: 4.52 g, 76.5%. ¹H-NMR (300 MHz, r.t., CDCl₃): δ/ppm 2.53 (t, J = 2.1 Hz, J = 2.4 Hz, 1H, C≡CH), 3.92 (s, 6H, CH₃), 4.76 (d, J = 2.4 Hz, 2H, CH₂), 7.80 (d, J = 1.5 Hz, 2H, ArH), 8.30 (t, J = 1.5 Hz, J = 1.2 Hz, 1H, ArH).

(3-Hydroxymethyl-5-prop-2-ynyloxy-phenyl)methanol (18). Compound 17 (0.500 g, 2.01 mmol) was dissolved in THF (35.0 mL), and degassed. LiAlH₄ (12.1 mmol) was added to the solution. The reaction mixture was stirred for 6 h, and the reaction was confirmed by TLC (EtOAc–n-hexane = 1:1). Then, water was added to the solution in order to quench the reaction. The solution was dried over MgSO₄, and MgSO₄ was removed by filtration. The solution was evaporated to yield white solid; Yield 0.425 g, 99%. ¹H-NMR (300 MHz, r.t., CD₃OD): δ/ppm 2.91 (t, J = 2.7 Hz, J = 2.4 Hz, 1H, C≡CH), 4.57 (s, 4H, CH₂OH), 4.72 (d, J = 2.4 Hz, 2H, CH₂), 6.89 (s, 2H, ArH), 8.30 (d, J = 0.6 Hz, 1H, ArH).

1,3-Bischloromethyl-5-prop-2-ynyloxy-benzene (19). Compound 18 (0.425 g, 2.21 mmol) was dissolved in CH₂Cl₂, and SOCl₂ was added to the solution with catalytic amount of DMF at 0 °C. The reaction mixture was stirred for overnight, and the reaction was confirmed by TLC (EtOAc–n-hexane = 1:1). The reaction mixture was poured into ice-water, and extracted with CHCl₃. The organic phase was washed with water, and dried over MgSO₄. MgSO₄ was removed by filtration, and the solution was evaporated to yield white solid; Yield. 0.462 g, 91.2%. ¹H-NMR (300 MHz, r.t., CDCl₃): δ/ppm 2.52 (t, J = 2.4 Hz, J = 2.4 Hz, 1H, C≡CH), 4.53 (s, 4H, CH₂), 4.70 (d, J = 2.4 Hz, 2H, CH₂), 6.95 (d, J = 1.5 Hz, 2H, ArH), 7.02 (t, J = 0.6 Hz, J = 0.6 Hz, 1H, ArH).

G1-Cl (20). Compounds 15 (95.0 mg, 0.391 mmol) and 19 (0.420 g, 1.83 mmol) was dissolved in DMF (3 mL). To the reaction mixture, CuI (111 mg, 0.586 mmol) was added and suspended. DIEA (100 mL, 0.588 μmol) was added to the solution. The solution was stirred for 24 h. The reaction mixture was evaporated, and the residue was purified by column chromatography (CHCl₃–MeOH = 60:1) to yield white solid; Yield 0.268 g, 73.7%. ¹H-NMR (300 MHz, r.t., CDCl₃): δ/ppm 4.50 (s, 12H, CH₂), 5.20 (s, 6H, CH₂), 5.46 (s, 6H, CH₂), 6.94 (s, 6H, ArH), 6.99 (s, 3H, ArH), 7.10 (s, 3H, ArH), 7.54 (s, 3H, C=CH).

G1-N₃ (21). Compound 20 (0.268 g, 0.288 mmol) was dissolved in DMF (10 mL). NaN₃ (3.63 mmol) was added to the solution, and the solution was stirred at 50 °C for overnight. The solution was evaporated, and the residue was dissolved in CHCl₃. The organic phase was washed with water for three times. The solution was dried with MgSO₄, and MgSO₄ was removed by filtration. The residue was purified by column chromatography (CHCl₃–MeOH = 100:1) to yield white solid; Yield 0.260 g, 93.1%. ¹H-NMR (300 MHz, r.t., CDCl₃): δ/ppm 4.29 (s, 12H, CH₂), 5.21 (s, 6H, CH₂), 5.45 (s, 6H, CH₂), 6.85 (s, 3H, ArH), 6.88 (s, 6H, ArH), 7.13 (s, 3H, ArH), 7.55 (s, 3H, C=CH). ¹H-NMR (300 MHz, r.t., DMF-d₇): δ/ppm 4.50 (s, 12H, CH₂) 5.23 (s, 6H, CH₂), 5.71 (s, 6H, CH₂), 7.05 (s, 3H, ArH), 7.10 (s, 6H, ArH), 7.45 (s, 3H, ArH), 8.39 (s, 3H, C=CH). ¹³C-NMR (75 MHz, r.t., CDCl₃): δ/ppm 53.4, 54.4, 62.1, 114.3, 120.6, 122.9, 127.6, 136.8, 137.7, 144.4, 158.8. ESI-MS: m/z 969.9.0 [M+H]⁺, 992.0 [M+Na]⁺.

pNP 6-Sulfo-GlcNAc hexamer(G1:22). Compounds 21 (6.15 mg, 6.34 μmol) and 10 were dissolved in DMF (400 μL), and an aqueous solution of sodium ascorbate (6.02 mg, 0.0304 mmol) and CuSO₄ (2.40 mg, 0.0150 mmol) was added to the solution. The reaction mixture was stirred for 3 days. The solvent was evaporated and the residue was purified by reversed phase chromatography (H₂O–MeOH = 2:1) to yield a white solid; Yield 7.70 mg, 28.2%. ¹H-NMR (300 MHz, r.t., DMF-d₇): δ/ppm 1.26–1.28 (m, 12H, CH₂), 1.88 (s, 18H, OCOCH₃), 1.96 (t, J = 7.5Hz, J = 7.5 Hz, 12H, CH₂), 2.41 (t, J = 7.5 Hz, J = 7.8 Hz, 12H, CH₂), 3.92 (dd, J_H1-H2 = 9.5Hz, J_H2-H3 = 9.0 Hz, 6H, H2), 4.03–4.06 (m, 6H, H5), 4.07–4.11 (m, 6H, H3), 4.19–4.26 (m, 6H, H4), 5.02 (d, J_H1-H2 = 9.0 Hz, 6H, H1), 5.16 (s, 6H, CH₂), 5.29 (broad, 6H, H6), 5.36 (broad, 6H, H6), 5.57 (s, 12H, CH₂), 5.66 (s, 6H, CH₂), 6.91 (s, 3H, ArH), 6.94 (d, 12H, J_o‑m = 9.0Hz, Ph-o), 7.03 (s, 6H, ArH), 7.44 (s, 3H, ArH), 7.58 (d, 12H, J_o-m = 9.0Hz, Ph-m), 7.94 (s, 6H, C=CH), 8.40 (s, 3H, C=CH). ¹³C-NMR (75 MHz, r.t., DMF-d₇): δ/ppm 23.5, 28.2, 30.6, 31.0, 47.9, 58.4, 59.8, 61.8, 67.1, 71.7, 76.8, 80.4, 81.2, 106.0, 115.2, 119.8, 122.5, 126.0, 130.6, 133.6, 140.1, 143.1, 144.2, 159.4, 164.6, 175.5, 176.4. ESI-MS: m/z 822.9 [M+H+4Na]⁵⁺.

G2-Cl (23). G1-N₃ (21, 0.0597 g, 0.0616 mmol) and 19 (0.114 g, 0.498 mmol) was dissolved in DMF (750 μL). CuI (21.0 mg, 0.110 mmol) was added to the solution and suspended. DIEA was added to the solution, and the solution was stirred for 24 h at room temperature. The reaction was confirmed by TLC (CHCl₃–MeOH = 10:1), and the residue was purified by column chromatography (CHCl₃–MeOH = 40:1) to yield a white solid; Yield 43.5 mg, 30.1%. ¹H-NMR (300 MHz, r.t., CDCl₃): δ/ppm 4.45 (s, 24H, CH₂), 4.99 (s, 6H, CH₂), 5.07 (s, 12H, CH₂), 5.12 (s, 6H, CH₂), 5.37 (s, 12H, CH₂), 6.73 (s, 3H, ArH), 6.75 (s, 6H, ArH), 6.88 (s, 12H, ArH), 6.94 (s, 6H, ArH), 7.05 (s, 3H, ArH), 7.55 (s, 3H, C=CH), 7.62 (s, 6H, C=CH).

G2-N₃ (24). 23 (0.0435 g, 0.0186 mmol) was dissolved in DMF (10 mL). NaN₃ (0.0201 g, 0.334 mmol) was added to the solution, and stirred at 50 °C for overnight. The solution was evaporated and the residue was dissolved in CHCl₃. The organic phase was washed with water three times. The organic phase was dried over MgSO₄, and MgSO₄ was filtrated. The residue was purified by column chromatography (CHCl₃–MeOH = 40:1) to yield a white solid; Yield 0.253 g, 56.3%. ¹H-NMR (300 MHz, r.t., CDCl₃): δ/ppm 4.27 (s, 24H, CH₂), 4.5.04 (s, 6H, CH₂), 5.16 (s, 12H, CH₂), 5.17 (s, 6H, CH₂), 5.40 (s, 12H, CH₂), 6.76 (s, 3H, ArH), 6.78 (s, 6H, ArH), 6.83 (s, 6H, ArH), 6.86 (s, 12H, ArH), 7.08 (s, 3H, ArH), 7.54 (s, 3H, C=CH), 7.61 (s, 6H, C=CH). ¹H-NMR (300 MHz, r.t., DMF-d₇): δ/ppm 4.48 (s, 24H, CH₂), 5.15 (s, 6H, CH₂), 5.24 (s, 12H, CH₂), 5.66 (s, 6H, CH₂), 5.69 (s, 12H, CH₂), 7.03 (s, 6H, ArH), 7.06 (s, 9H, ArH), 7.09 (s, 12H, ArH), 7.44 (s, 3H, ArH), 8.35 (s, 3H, C=CH), 8.41 (s, 6H, C=CH). ¹³C-NMR (75 MHz, r.t., CDCl₃): δ/ppm 53.3, 53.6, 54.3, 61.7, 62.0, 114.2, 114.6, 120.0, 120.6, 123.2, 123.5, 127.7, 136.7, 137.2, 137.7, 143.6, 144.1, 158.7, 159.0. MALDI-TOF-MS: m/z 2447.2 [M+Na]⁺.

pNP 6-Sulfo-GlcNAc dodecamer (G2:25). Compounds 24 (8.00 mg, 3.30 μmol) and 10 were dissolved in DMF (350 μL). The aqueous solution (83 μL) of sodium ascorbate (6.28 mg, 31.7 mmol) and CuSO₄ (2.52 mg, 0.0158 mmol) was added to the solution. The reaction mixture was stirred for 3 days. The reaction was confirmed by TLC (H₂O–MeOH = 3:1). The solution was evaporated and the residue was purified by reversed phase column chromatography (H₂O–MeOH = 3:1) to yield a white solid; Yield 6.30 mg, 21.1%. ¹H-NMR (500 MHz, r.t., DMF-d₇): δ/ppm 1.26–1.28 (m, 24H, CH₂), 1.88 (s, 36H, OCOCH₃), 1.94–199 (m, 24H, CH₂), 2.40 (t, J = 7.5 Hz, J = 7.0 Hz, 24H, CH₂), 3.78 (broad, 12H, H5), 3.87–3.90 (m, 12H, H2), 4.06–4.10 (m, 12H, H3), 4.23–4.26 m, 12H, H4), 4.25 (m, 6H, H4), 5.01 (d, J_H1-H2 = 8.5 Hz, 12H, H1), 5.16–5.18 (m, 12H, H6), 5.22–5.24 (broad, 6H, CH₂), 5.31–5.34 (m, 6H, H6), 5.36 (broad, 6H, H6′), 5.53 (s, 24H, CH₂), 5.56 (s, 12H, CH₂), 5.58 (s, 12H, CH₂), 5.61–5.66 (broad, 6H, CH₂), 6.88 (s, 6H, ArH), 6.96–6.98 (overlap, 24H, Ph-o), 6.96–6.98 (overlap, 9H, ArH), 7.03(s, 12H, ArH), 7.44 (broad, 3H, ArH), 7.58 (d, 2H, J_o-m = 8.1 Hz, Ph-m), 7.94 (s, 12H, C=CH), 8.35 (s, 3H, C=CH), 8.35 (s, 6H, C=CH). ¹³C-NMR (75 MHz, r.t., DMF-d₇): δ/ppm 22.6, 28.2, 30.6, 31.1, 58.4, 61.3, 61.8, 67.1, 71.7, 76.8, 80.3, 81.2, 82.3, 91.5, 106.0, 115.4, 120.0, 122.5, 126.0, 127.8, 130.0, 140.1, 143.2, 143.4, 144.2, 148.7, 155.4, 156.6, 156.6, 164.6, 164.7, 176.4, 181.0. ESI-MS m/z 1238.0 [M+H+6Na]⁷⁺.

Each sugar dendrimer was dissolved in PBS (pH 7.4) at a concentration of 8.00 μM. The lectin (FITC-WGA) was also dissolved in PBS (pH 7.4) at a concentration of 0.100 μM. To a solution of WGA (300 μL) were sequentially added aliquots of the sugar dendrimer solutions (3, 3, 5, 5, 5, 5, 10, 10, 10, 10, 10, 20, 20 and 20 μL) at 25 °C. Fluorescent measurements were taken 5 min after the addition of each aliquot. Fluorescence spectra of the FITC were measured with an excitation wavelength at 490 nm and an emission wavelength of 510 nm. The PBS buffer without a sugar dendrimer was also added in the same way as a reference. Changes in the fluorescence of the sugar-protein interaction were calculated by subtracting two of fluorescence intensities (i.e., fluorescence change by sugar protein interaction = fluorescence change by addition of the sugar dendrimer solution–fluorescence change by the addition of the reference PBS solution). Changes in the fluorescence were plotted according to the following formula [19,20]:

where [Sugar], K_a, F_o, ΔF, and ΔF _max represent the sugar concentration of the dendrimer solution (M), the association constant (M⁻¹), the initial fluorescence intensity, the fluorescence change, and the maximum fluorescence change, respectively.

Aβ (1-42) was dissolved in a 0.02% ammonia solution at a concentration of 200 μM. Any aggregates formed were removed by centrifugation using a CS120FX centrifuge (Hitachi, Tokyo, Japan) for 3 h at 16,000 g and 4 °C. The supernatant was mixed with phosphate buffer (20 mM phosphate buffer, pH 7.4, 100 mM NaCl) to a final peptide concentration of 20 μM. The peptide solution was incubated with each sugar additive at 37 °C. The concentration of the sugar additives was 200 μM. Amyloid fibril formation was evaluated by the fluorescence emission of ThT. Following a period of incubation at 37 °C, a 5 μL solution of Aβ in buffer was added to 300 μL of the ThT solution (50 mM in the same phosphate buffer). Following a period of 10 s, the fluorescence intensity was measured at an excitation wavelength of 450 nm and an emission wavelength of 482 nm.

The Aβ peptide (1-42) was incubated in phosphate buffer (20 mM phosphate buffer, 100 mM NaCl) at 37 °C for 12 h, with the peptide and the sugar additives being added at a concentration of 20 μM. After the incubation of the sample, a 5 μL portion of the sample solution was placed on freshly cleaved mica and dried under N2. The mica substrates were then washed with 100 μL of MilliQ water.