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Synthetic models of quasi-stable amyloid #40 oligomers with significant neurotoxicity Yumi Irie, Kazuma Murakami, Mizuho Hanaki, Yusuke Hanaki, Takashi Suzuki, Yoko Monobe, Tomoyo Takai, Ken-ichi Akagi, Taiji Kawase, Kenji Hirose, and Kazuhiro Irie ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00390 • Publication Date (Web): 27 Dec 2016 Downloaded from http://pubs.acs.org on December 28, 2016

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ACS Chemical Neuroscience

Synthetic models of quasi-stable amyloid β40 oligomers with significant neurotoxicity

Yumi Irie,† Kazuma Murakami,† Mizuho Hanaki,† Yusuke Hanaki,† Takashi Suzuki,† Yoko Monobe,‡ Tomoyo Takai,‡ Ken-ichi Akagi,‡ Taiji Kawase,§ Kenji Hirose,§ and Kazuhiro Irie*,†

†

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University,

Kyoto 606-8502, Japan ‡

National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085, Japan

§

Nihon Waters K. K., Tokyo 140-0001, Japan

*Corresponding author. Tel. +81-75-753-6281, fax +81-75-753-6284 E-mail address: [email protected] (K. Irie)

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ABSTRACT: The formation of soluble oligomers of amyloid β42 and 40 (Aβ42, Aβ40) is the initial event in the pathogenesis of Alzheimer's disease (AD). Based on previous systematic proline replacement and solid-state NMR, we proposed a toxic dimer structure of Aβ42, a highly aggregative alloform, with a turn at positions 22 and 23, and a hydrophobic core in the Cterminal region.

However, in addition to Aβ42, Aβ40 dimers can also contribute to AD

progression because of the latter's 10-fold greater abundance. Here, we describe the synthesis and characterization of thee dimer models of the toxic-conformation constrained E22P-Aβ40 using L,L-2,6-diaminopimeric acid (DAP) or L,L-2,8-diaminoazelaic acid (DAZ) linker at position 30, which is incorporated into the intermolecular parallel β-sheet region, and DAP at position 38 in the C-terminal hydrophobic core. E22P-A30DAP-Aβ40 dimer (1) and E22PA30DAZ-Aβ40 dimer (2) existed mainly in oligomeric states even after 2 weeks incubation without forming fibrils, unlike the corresponding monomer. Their neurotoxicity towards SHSY5Y neuroblastoma cells was very weak. In contrast, E22P-G38DAP-Aβ40 dimer (3) formed β-sheet-rich oligomeric aggregates, and exhibited more potent neurotoxicity than the corresponding monomer. Ion mobility–mass spectrometry suggested that high molecular-weight oligomers (12–24-mer) of 3 form, but not for 1 and 2 after 4 h incubation. These findings indicate that formation of the hydrophobic core at the C-terminus, rather than intermolecular parallel β-sheet, triggers the formation of toxic Aβ oligomers. Compound 3 may be a suitable model for studying the etiology of Alzheimer's disease.

KEYWORDS: amyloid β, β-sheet, dimer, ion mobility–mass spectrometry, neurotoxicity, oligomer


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INTRODUCTION Amyloid β proteins (Aβ) are causative agents in Alzheimer's disease (AD).1,2 They consist mainly of 40- and 42-mer species. The latter is more aggregative and neurotoxic, while the former is more abundant.3 Mounting evidence suggests that quasi-stable Aβ oligomers and protofibrils rather than mature Aβ fibrils could be more pathological, and that oligomeric assemblies of Aβ including protofibrils cause cognitive dysfunction and synaptotoxicity during AD progression.4,5 Given their varying sizes and shapes, these oligomers (ADDL,6 Aβ*56,7 amylospheroid8) are thought to form through two pathways (2 × n-mer and 3 × n-mer) based on each dimer and trimer unit.9 For example, a hexameric β-barrel structure has been identified in Aβ42 oligomers.10 In particular, Aβ dimers were detected in cerebrospinal fluid and isolated from AD brains.11,12 However, structural information concerning the dimer and trimer is limited and how the oligomerization of Aβ is initiated has not been fully elucidated since these oligomers are too unstable or aggregative to be identified. “Aggregation” in this paper is defined as the change from Aβ monomers into amyloid fibrils via oligomers and protofibrils. Chemical modifications of Aβ that introduce conformational and structural constraints to stabilize the oligomeric species are indispensable for studying them. We previously identified a toxic conformer of Aβ42 with a turn at Glu22 and Asp23 using systematic proline replacement13 and solid-phase NMR.14 Recently, atomic resolution structures of monomorphic Aβ42 fibrils have been reported by three independent groups using solid-state NMR.15-17 Their reports support our initial observations13,18 that Glu22 and Asp23 are located in a loop region, and that both the carboxylic acid side chains are on the same side exposed to the surrounding water (Figure 1A). Given that mostly trimers and tetramers were detected in the toxic conformation–constrained Aβ42 analogs by SDS-PAGE,14,19 the toxic conformer appears to have a propensity to form toxic oligomers, in which the residues at positions 15–21 and 24–32 are incorporated into intermolecular parallel β-sheets as shown in Figure 1A.13,20 The C-terminal hydrophobic core is thought to be derived from another turn at positions 38 and 39, together with an intramolecular β-sheet at positions 35–37 and 40–42 in Aβ42.20 3 ACS Paragon Plus Environment


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The most abundant alloform, Aβ40, could adopt the toxic turn at positions 22 and 23 quite similarly to Aβ42, whereas the C-terminal hydrophobic core is not as predominant in Aβ40 aggregates based on Wetzel's model (Figure 1B).21 Hitherto, several covalently linked Aβ40 dimers have been prepared.22-26 Since generic dityrosine levels are elevated in the brains of AD patients, Kok et al. synthesized two dimer models for Aβ40 in which either racemic 2,6diaminopimeric acid (DAP, Figure 2A)22 or dityrosine23 was incorporated at position 10 instead of Tyr10.

These dimer models formed fibrils rapidly as well as oligomeric aggregates

responsible for neurotoxicity. The dityrosine-linked Aβ40 dimer was also studied by O’Malley et al. This dimer forms typical amyloid fibrils, but inhibits long-term potentiation in rats.26 Both the disulfide-linked Aβ40 dimer at position 225 and position 2624 formed protofibril-like assemblies, and the latter inhibited long-term potentiation. It is still unclear by using these conventional models whether oligomers or fibrils are more responsible for the pathogenesis of AD. To gain structural insights into the toxic Aβ oligomers, we recently reported a covalently linked E22P-Aβ40 dimer with an L,L-DAP linker at position 30 (Figure 2B, E22P-A30DAPAβ40 dimer, 1) based on a putative dimer structure of Aβ40 (Figure 1B)21 as a preliminary communication.27 Although 1 formed stable oligomeric species after 2 weeks of incubation, it did not show any neurotoxicity towards human SH-SY5Y neuroblastoma cell lines. However, it is likely that the length and/or position of the linker are not optimized to form toxic oligomeric Aβ40 species. According to the proposed dimer structure (Figure 1B), we synthesized two additional model dimers (Figure 2B) of Aβ40 with a longer linker (L,L-2,8-diaminoazelaic acid: DAZ, Figure 2A) at position 30 (E22P-A30DAZ-Aβ40 dimer, 2), and a DAP linker at position 38 (E22P-G38DAP-Aβ40 dimer, 3). Here, we report our characterization of the biological and biophysical properties of these dimers.


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RESULTS AND DISCUSSION Synthesis of 1–3 with a DAP or DAZ Linker at Position 30, and with a DAP Linker at Position 38. We synthesized three model dimers for E22P-Aβ40 (1–3) with the toxic turn at positions 22 and 23 to examine the effects of dimerization on their biological and biophysical properties. E22P-Aβ40 is neurotoxic towards PC12 cells, albeit 10-fold less than wild-type Aβ42, while wild-type Aβ40 is almost inactive.28 We chose the connecting position 30 based on our previous solid-state NMR studies using rotational resonance (R2).29 The Ala30 residues in aggregates of E22K-Aβ42 (Italian mutant) are proximal each other (98% 5 ACS Paragon Plus Environment


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by HPLC analysis, and their molecular weights and formulae were verified by ESI-qTOF-MS measurements (Figure S1, Supporting Information). The synthesis and characterization of various dimer models for Aβ are initial steps toward elucidating the toxic oligomeric structure. Establishment of pertinent dimer models is very difficult because of their synthetic complexity and transient nature. In fact, most dimer models are aggregative,22-26 and the difficulty is involved in synthesizing Aβ42 dimers.23 We also failed to synthesize an Aβ42 dimer with a DAP liker at position 30. In spite of its less aggregative and neurotoxic properties compared with Aβ42, we focused at first on Aβ40 because of its 10-fold abundance and propensity to form dimers.19,32

Neurotoxicity of 1–3 towards SH-SY5Y Neuroblastoma Cell Lines. The neurotoxicity of 1–3 towards SH-SY5Y cells was first measured with the MTT assay. SH-SY5Y, like PC12, is a neuronal cell model. After being incubated for either 16 or 48 h in the presence of 1–3 or E22PAβ40 as a positive control, cell viability was estimated by the ability to reduce 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). As shown in Figure 4A, E22PAβ40 at 2.5 µM concentration significantly decreased cell viability after a 48-h incubation period as reported previously for PC12 cells.28 In contrast, the dimers at position 30 (1, 2) were not as neurotoxic even at 10 µM, although 2 exhibited slightly greater neurotoxicity than 1. These results suggest that such dimers with intermolecular β-sheet around position 30 could not be critically involved in the pathogenesis of AD. It is noteworthy that the intermolecular β-sheet nearby position 30 exists in both fibrils of Aβ4033 and Aβ4215-17 as shown by studies using solidstate NMR.

This finding does not contradict the hypothesis that Aβ fibrils have low

neurotoxicity.3 Conversely, 3 with a DAP linker in the C-terminal core (position 38) was as neurotoxic as E22P-Aβ40 at all concentrations tested (2.5–10 µM) after a 48-h incubation period. E22P-Aβ42 dimer with a DAP linker at position 40 was more neurotoxic than the corresponding monomer after 16 h incubation.34 As expected, 3 exhibited potent neurotoxicity at 2.5 µM concentration 6 ACS Paragon Plus Environment

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after 16 h compared with E22P-Aβ40, but the other dimers did not (Figure 4B). Oligomerization is the earliest event during Aβ aggregation. These data indicate that the formation of the Cterminal hydrophobic core is important for the neurotoxicity of Aβ in the earlier phases during aggregation.

Since SH-SH5Y cells used in this study were not differentiated, the weak

concentration-dependency observed in Figure 4 may be mainly due to the intrinsic cell proliferation after 16 h or 48 h incubation. Indeed, the weaker concentration-dependency was observed after 48 h incubation than 16 h incubation. Our previous results using rat primary cultures32 also supported these observations.

Ability of 1–3 to Form Oligomers and Fibrils Estimated by Th-T and TEM Analyses. Given the neurotoxicity of 1–3, their aggregative ability was evaluated using thioflavin-T (Th-T), a reagent that fluoresces when bound to Aβ aggregates. Unlike wild-type Aβ40,28 the toxic conformer-constrained E22P-Aβ40 aggregated with a lag time of ~8 h to achieve maximum fluorescence after 48 h (Figure 5). However, the fluorescence of 1–3 was very weak, and remained almost unchanged after 2 weeks (Figure 5), suggesting that these dimers did not form fibrils, unlike E22P-Aβ40. Transmission electron microscopy (TEM) analysis supported these results (Figure 6). Although E22P-Aβ40 formed typical amyloid fibrils after 48 h incubation, globular aggregates were predominantly observed for 1 and 2. The mean diameter of the aggregates of 1 and 2 were 7.57 nm (SD = 0.78) and 7.88 nm (SD = 0.75), respectively, meaning that the oligomer size of 2 was similar to that of 1. The oligomer size of 1 after 48 h was estimated to be 6–8-mer using size exclusion chromatography as reported previously.27 These properties of 1 and 2 are similar to those of ADDL.6 On the other hand, the morphology of the aggregates of 3 was different from those of 1 and 2. The aggregates of 3 contained not only the globular forms like 1 and 2 but also protofibrillar ones, whose mean length and width were 143 nm (SD = 44) and 9.0 nm (SD = 1.4), respectively.

The characteristics of the protofibrils were in good agreement with previous

studies.35 Comparing the mean length (200–400 nm) and width [11.8 nm (SD = 1.1)] of E22P7 ACS Paragon Plus Environment


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Aβ40 aggregates showing typical fibrils, the morphology of the aggregates of 3 was clearly different. These results imply that Aβ oligomers must be of a certain molecular size in order to exhibit potent neurotoxicity.

Secondary Structure of 1–3 Based on CD Spectra.

To obtain secondary structural

information for 1–3, we carried out circular dichroism (CD) measurements. As a positive control, E22P-Aβ40 exhibited a positive peak at ~200 nm and a negative peak at ~220 nm after 4 h incubation, and reached a maximum after 48 h (Figure 7).

This finding indicates the

transformation of random structure into β-sheet structure during incubation. The structure of 1, however, remained mostly random even after 48 h in a manner similar to wild-type Aβ40.32 The dimers 2 and 3 formed β-sheet-rich structure within 4 h after dissolution, and their curves remained constant, suggesting that they might adopt a quasi-stable oligomeric structure. The CD data of 3 was almost time-independent compared with E22P-Aβ40 that formed fibrils with cross β-sheet. This suggests that the observed toxicity of 3 could be unrelated to cross β-sheet formation. Furthermore, the considerably weaker intensity of 2 and 3 compared to that of E22PAβ40 supported the results that 2 and 3 formed no fibrils even after 48 h (Figure 5). It should be noted that 1 with the shorter linker (DAP) exhibited mainly random structure unlike 2 with the longer linker (DAZ). Thus, the DAZ linker would be suitable for forming stable intermolecular β-sheet in the midsection nearby position 30, while the DAP linker is likely to be sufficient to tether the C-terminal region of each monomer.

Oligomerization of 1–3 Based on IM-MS. Which Aβ oligomers have the greatest causative effect on AD pathogenesis has been the subject of extended debate. Although much effort using conventional techniques such as SDS-PAGE, size-exclusion chromatography, and photo-induced cross-linking have been devoted to the characterization of toxic oligomers, the strong, inherent tendency of native oligomers to form the structurally heterogeneous assemblies has hindered further studies. In particular, the usage of SDS could cause the overestimation of the amounts of 8 ACS Paragon Plus Environment

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oligomer because SDS tends to induce Aβ dimerization.36 Size-exclusion chromatography also cannot avoid a partial uncertainty in calculated molecular weights since the calibration curve is usually based on dextran for denatured proteins.27 Recently, ion mobility–mass spectrometry (IM-MS) combined with native ionization techniques have made it possible to separate various oligomers and clarify their distribution without using organic solvents that disrupt non-covalent interactions within Aβ oligomers.37,38 To estimate precisely the molecular weights of oligomers, IM-MS measurement of 1–3 was carried out after 4 h incubation according to the CD spectra (Figure 7). The two-dimensional heat map composed of the domain of m/z and drift time is shown in Figure 8 (Figure S2 in Supporting Information represents projection of 2D spectra of 1-3 in Figure 8 on the mass spectra axis).

After deconvolution based on the observed mass (Table S1, Supporting

Information), peaks corresponding to oligomeric orders were assigned to the series of multivalent ions [n = 1, 2, 3… of a dimer model denotes dimer (Dim), tetramer (Tet), hexamer (Hex)…, respectively] depending on their drift time. As exemplified by the red rectangle on the spectrum of 2 in Figure 8, the signals (z/n = 3: z denotes charge) for Hex9– and Tet6– overlapping in m/z 2850–2920 region were clearly resolved into two signal groups with different drift times (9.3 and 11.1 ms, respectively). The spectrum of 1 shows a distribution of oligomers (n = 1–6), indicating the formation of dimers to dodecamers, especially the predominance of hexamers (n = 3). This is in good coincidence with the previous data using size exclusion chromatography.27

Notably, the

oligomer distribution of 2, which is linked with a longer linker than 1, was similar (n = 1–6) to that of 1, whereas the oligomer intensity of 2 was more visible than that of 1. Furthermore, 3 produced higher molecular-weight oligomers (n = 6–12: dodecamers to tetracosamers) compared with 1 and 2. It is interesting that the distribution of 3 converged between dodecamers and ocatadecamers. These findings are basically consistent with the report on the importance of tetramers and dodecamers of Aβ40 monomer by Bernstein et al.37 In contrast, ionization signals necessary to the analysis of oligomer distribution were not detected in the case of E22P-Aβ40 9 ACS Paragon Plus Environment


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after 4 h incubation (data not shown), possibly because of its high aggregation velocity in PBS buffer. After 4 h incubation, considerable amounts of E22P-Aβ40 would have aggregated, as shown in Figure 5. Furthermore, we observed the time-dependency of oligomerization of 3, the most neurotoxic dimer, during 0~5 h incubation in 25 mM ammonium acetate (pH 7.4) at 37 °C (Figure 9). Although the 2D spectrum after 1 h incubation was almost the same as that after 0 h (data not shown), it is likely that at most 4 h incubation was required for the definitive formation of higher molecular-weight oligomers (n = 6–12) under this condition.

CONCLUSIONS The dimer models for Aβ40 (1, 2) with a linker at position 30 in the intermolecular β-sheet region produced mainly stable oligomers after 48 h incubation (Figures 5 and 6). Their CD spectra (Figure 7) suggested that 1 did not effectively form β-sheet structure, and that 2 formed β-sheet-rich structure even after 4 h.

However, both compounds demonstrated very weak

neurotoxicity towards SH-SY5Y neuroblastoma cells (Figure 3). These indicate that such dimers, the minimum structure of Aβ40 fibrils,33 are not involved in the pathogenesis of AD. Recent solid-state NMR studies on Aβ42 fibrils15-17 suggested that the tertiary structure about the turn between Glu22 and Asp23 is qualitatively similar to that in our previous paper.13,14 However, there is a difference in the C-terminal core structure between them. The S-shaped structure comprising short β-strand segments originating from the salt bridge between the amino group of Lys28 and the carboxyl group of Ala42(Cα) is characteristic to the recently proposed structure,15-17 while our model of the C-terminal hydrophobic core consists of intramolecular anti-parallel β-sheet among Met35-Ala42.39

This discrepancy may reflect the structural

difference between less toxic fibrils and toxic oligomers. These observations led us to synthesize Aβ dimer models connected within the C-terminal core. The dimer 3 as well as 1 and 2 had a propensity to form oligomers and protofibrils after 48 h incubation (Figures 5 and 6). However, only 3 was more neurotoxic towards SH-SY5Y cells than E22P-Aβ40 after 16 h incubation; its potency was almost equal to that of E22P-Aβ42 dimer at position 40 with a DAP linker that we 10 ACS Paragon Plus Environment

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recently synthesized.34 These results strongly suggest that the covalent cross-linkage in the Cterminal core is appropriate for the synthesis of model toxic Aβ oligomers, and that the formation of the hydrophobic core at the C-terminus, rather than intermolecular parallel β-sheet, triggers the formation of toxic Aβ oligomers. IM-MS analyses also supported the importance of the C-terminal hydrophobic core to oligomer formation (Figure 8). The cytotoxicity of E22PAβ40 incubated for 16 h and for 48 h may be originated from protofibrils coexisting in the fibrils because the concentration used in MTT assay (2.5, 5, and 10 µM) is lower than that used in several aggregation tests (25 µM). Formation of the hydrophobic core at the C-terminus is postulated to be an initial event for Aβ42 to exhibit neurotoxicity through radicalization according to the toxic conformation theory.9,20 Such a hydrophobic core is not easily formed in Aβ40 lacking the two C-terminal hydrophobic residues Ile41 and Ala42. Considering the fact that a shorter DAP linker was able to successfully tether Aβ molecules at the C-terminus, the C-terminal tail of Aβ40 should have a considerable flexibility. In other words, the C-terminus of Aβ40 may not be structured very well unlike Aβ42. This is one of the most important differences between Aβ40 and Aβ42, which would exhibit very different properties in aggregation and cytotoxicity. These implications are consistent with our previous studies20 regarding the significance of the formation of stable Cterminal core in Aβ42 in the etiology of Alzheimer's disease. However, introducing a DAP linker into position 38 of Aβ40 molecules could substitute for the hydrophobic residues at positions 41 and 42 of Aβ42.

The C-terminal hydrophobic

interaction might lead to oligomers like trimers19 and hexamers10 that do not result directly in fibrils. Such a route is called an “off-pathway”, while a route where Aβ directly aggregates into fibrils is called an “on-pathway”. Since 3 formed protofibrillar aggregates (12–24-mer) with significant neurotoxicity (Figures 4 and 8), 3 could be one of the most practical models for toxic dimers of Aβ that could aggregate to yield 3 × 2n oligomers (12–24-mer) for drug development of AD therapeutics with fewer side effects.



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METHODS General Remarks. The following spectroscopic and analytical instruments were used: digital polarimeter, model P-2200 (Jasco, Tokyo, Japan); NMR spectrometers, Avance III 400 and 500 (Bruker, Rheinstetten, Germany), tetramethylsilane was used as reference unless otherwise described; HR-ESI-qTOF-MS, Waters Xevo G2-S qTOF (Waters, Tokyo, Japan); peptide synthesizer, PioneerTM (Applied Biosystems, Foster City, CA); transmission electron microscope, H-7650 electron microscope (Hitachi, Ibaraki, Japan); micro plate readers, MultiScan JX and Fluoroskan Ascent (Thermo Fisher Scientific, Waltham, MA); CD, J-805 (Jasco, Tokyo, Japan); HPLC, Waters model 600E with a model 2487 UV detector. HPLC was carried out on a YMC PROTEIN-RP column (20 mm i.d. × 150 mm, Yamamura Chemical Research Institute, Kyoto, Japan) and a YMC ODS-A column (20 mm i.d. × 150 mm, Yamamura Chemical Research Institute). Wakogel C-200 (silica gel, Wako, Osaka, Japan) and YMC A60-350/250 gel (ODS, Yamamura Chemical Research Institute) were used for column chromatography. HATU,40 Fmoc amino acids, Fmoc-L-Val-polyethylene glycol-polystyrene support (PEG-PS) resin, and N,N-diisopropylethylamine (DIPEA) were purchased from Applied Biosystems (Foster City, CA). N,N-Dimethylformamide (DMF), trifluoroacetic acid (TFA), ethane-1,2dithiol, thioanisole, m-cresol, and diethyl ether (peroxide free) were purchased from Nacalai Tesque (Kyoto, Japan). Piperidine, MTT, and Th-T reagent were obtained from Sigma-Aldrich (St. Louis, MO).

Synthesis of Fmoc-L,L-DAZ. 1,4-bis[(R)-1'-phenylethyl]piperazine-2.5-dione

(4).

Intermediate

diketone

(4)

was

synthesized according to the protocol of L,L-DAP27 with slight modifications.30,31 In brief, (R)α-phenylethylamine (6.4 mL, 50 mmol) and K2CO3 (14 g, 100 mmol) were dissolved in acetonitrile (120 mL).30 After the solution was cooled to 0 °C, chloroacetyl chloride (4.0 mL, 50 mmol) was added and the solution was stirred at room temperature for 1.5 h. The reaction 12 ACS Paragon Plus Environment

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mixture was filtered through celite and concentrated in vacuo. The residues purified by column chromatography on Wakogel C-200 eluted with hexane containing increasing amounts of EtOAc (0, 40, and 50%) followed by recrystallization with EtOAc/hexane to give N-chloroacetyl-(R)-αphenylethylamine (4.6 g, 23 mmol, 47%) as a white solid. Sodium hydride (60% in mineral oil, 0.85 g, 21 mmol) was added to the solution of N-chloroacetyl-(R)-α-phenylethylamine (3.5 g, 18 mmol) in DMF (35 mL) at 0 °C, followed by stirring for 7 h at room temperature.31 The mixture was quenched with saturated aqueous NH4Cl (35 mL), and extracted with 50% EtOAc/hexane (40 mL × 4). The organic layer was washed with brine, dried over Na2SO4, filtered, and evaporated to dryness. The residue was purified by column chromatography on Wakogel C-200 eluted with hexane containing increasing amounts of EtOAc (0, 30, and 50%) to give 4 (1.1 g, 3.4 mmol, 39%) as a yellow solid. 1H NMR (500 MHz, CDCl3) δ 1.55 (6H, d, J = 7.1 Hz), 3.52 (2H, d, J =16.5 Hz), 3.86 (2H, d, J = 16.5 Hz), 5.95 (2H, q, J =7.1 Hz), 7.32 (10H, m) ppm. (3S)-1,4-bis[(R)-1'-phenylethyl]-3-(5-iodopentyl)piperazine-2.5-dione

(5)

and

(3R)-1,4-

bis[(R)-1'-phenylethyl]-3-(5-iodopentyl)piperazine-2.5-dione (6). 1.0 M Hexamethyldisilazide (NaHMDS, 0.25 mmol) in THF (0.25 mL) was added to the solution of diketone 4 (0.081g, 0.25 mmol) in THF (0.8 mL) at –80 °C under an argon atmosphere. After stirring for 2 h at –80 °C, the reaction mixture was treated with 1,5-diiodopentane (0.37 mL, 2.5 mmol) and stirred for further 2.5 h. The temperature was subsequently allowed to rise to room temperature, and the mixture was stirred overnight. The mixture was quenched with 3 mL saturated aqueous NH4Cl and extracted with EtOAc (25 mL × 3). The organic layer was washed with 50% aqueous NaCl (3 mL × 2), followed by brine (3 mL × 2). The washed organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The reaction was repeated using 2.0 g (6.3 mmol) of 4. The crude products were combined and purified by column chromatography on Wakogel C-200 eluted with hexane containing increasing amounts of EtOAc (30, 50, and 80%), followed by recrystallization to give 5 (0.86 g, 1.7 mmol, 25%) as a colorless solid and 6 (0.55 g, 1.1 mmol, 16%) as a clear oil. 5: 1H NMR (500 MHz, ppm, CDCl3) δ 0.90 (3H, m), 1.04 (3H, m), 1.49 (2H, m), 1.55 (3H, d, J = 7.1 Hz), 1.58 (3H, d, J = 7.1 Hz), 3.00 (2H, dt, J = 1.7 Hz, 7.0 Hz), 13 ACS Paragon Plus Environment


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3.38 (1H, d, J = 7.4 Hz), 3.69 (1H, d, J = 7.3 Hz), 3.92 (1H, dd, J = 3.0, 8.9 Hz), 5.92 (2H, m), 7.22 (2H, m), 7.34 (8H, m) ppm; HR-ESI-qTOF-MS m/z 519.1473 [M + H]+ (calcd for C25H32N2O2I, 519.1508). 6: 1H NMR (500 MHz, CDCl3) δ 1.42 (4H, m), 1.51 (3H, d, J = 7.2 Hz), 1.62 (3H, d, J = 7.5 Hz), 1.83 (4H, m), 3.18 (2H, t, J = 6.9 Hz), 3.59 (1H, d, J = 17.2 Hz), 3.70 (1H, dd, J = 4.0, 9.2 Hz) 3.93 (1H, d, J = 17.2 Hz), 5.78 (1H, q, J = 7.2 Hz), 5.87 (1H, q, J = 7.1 Hz), 7.21 (4H, m), 7.34 (6H, m) ppm; HR-ESI-qTOF-MS m/z 519.1473 [M + H]+ (calcd for C25H32N2O2I, 519.1508). (1S,4S,1'R)-2,5-Bis-[N,N-(1'-phenylethyl)]-3,6-dioxo-2,5-diazabicyblo[5,2,2] undecane (7). 1.0 M NaHMDS (1.9 mmol) in THF (1.8 mL) was added to the solution of 5 (0.86 g, 1.7 mmol) in THF (65 mL) at –10 °C under argon atmosphere. After the mixture was stirred overnight at room temperature, saturated aqueous NH4Cl (60 mL) was added. The solution was extracted with EtOAc (50 mL × 3), washed with brine, and dried over Na2SO4. After filtration and concentration in vacuo, the residue was separated by column chromatography on Wakogel C-200 eluted with hexane, EtOAc, and increasing amounts of CHCl3 (Hexane:EtOAc:CHCl3 = 7:3:0, 7:3:0.6, and 7:3:2.8). Fractions containing 7 were combined and evaporated to dryness, followed by recrystallization with CH3CN and MeOH (1:1) to give 7 (0.21 g, 0.53 mmol, 32%) as a colorless solid. 1H NMR (500 MHz, CDCl3) δ 0.75 (2H, m), 0.93 (4H, m), 1.13 (2H, m), 1.41 (2H, m), 1.55 (6H, d, J = 7.1 Hz), 4.22 (2H, dd, J = 2.8, 7.6 Hz), 5.86 (2H, q, J = 7.0 Hz), 7.30 (6H, m), 7.41 (4H, m) ppm; HR-ESI-qTOF-MS m/z 391.2431 [M + H]+ (calcd for C25H31N2O2, 391.2386). L,L-Diaminoazelaic acid (DAZ). Bicyclic diketone 7 (0.023 g, 0.059 mmol) was refluxed in

57% hydriodic acid (5 mL) for 4 h. The solution was washed with EtOAc (5 mL × 3), and the water layer was concentrated in vacuo to 1–2 mL. The residue was charged on Amberlite 15 (Sigma-Aldrich), washed with water (10 mL), and eluted with 5 M NH4OH (30 mL). The eluates were evaporated with ethanol to dryness. The reaction was repeated using 7 (0.18 g, 0.47 mmol) to give L,L-DAZ (total 0.096 g, 0.44 mmol, 83%) as a colorless solid. [α]D21 + 30.3°(c = 0.19, 1 M HCl); 1H NMR (500 MHz, ref. DHO = 4.79 ppm, D2O) δ 1.39 (6H, m), 1.86 (4H, m), 14 ACS Paragon Plus Environment

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3.73 (2H, dd, J = 5.7, 6.5 Hz) ppm; HR-ESI-qTOF-MS m/z 217.1183 [M – H]– (calcd for C9H17N2O4, 217.1188). Fmoc-L,L-DAZ. 9-Fluorenyl methyl succinimidyl carbonate (0.33 g, 0.98 mmol) was added to the suspension of L,L-DAZ (0.094 g, 0.43 mmol) in acetone and water (1:1) (12 mL). Na2CO3 (0.12 g, 1.8 mmol) was added to the solution and the mixture was stirred at room temperature overnight. The mixture was adjusted to pH 1–2 using 1 M HCl, extracted with EtOAc (15 mL × 3), washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on Wakogel C-200 eluted with hexane containing increasing amounts of EtOAc (0, 50, and 100% EtOAc containing 0.1% AcOH) to give Fmoc-L,L-DAZ (0.17 g, 0.25 mmol, 58%) as a colorless solid. 1H-NMR (500 MHz, ref. CD2HOD = 3.31 ppm, CD3OD) δ 1.40 (6H, m), 1.69 (2H, m), 1.83 (2H, m), 4.14 (2H, br q, J = 4.6 Hz), 4.21 (2H, br t, J = 6.9 Hz), 4.35 (4H, br d, J = 7.1 Hz), 7.30 (4H, t, J = 7.4 Hz), 7.37 (4H, t, J = 7.4 Hz), 7.67 (4H, t, J = 7.8 Hz), 7.78 (4H, d, J = 7.6 Hz) ppm; HR-ESI-qTOF-MS m/z 661.2586 [M – H]– (calcd for C39H37N2O8, 661.2550).

Synthesis of Dimers 1–3. The amino groups of L,L-DAP (Sigma-Aldrich) were protected with Fmoc for the solid-phase synthesis of the E22P-Aβ40 dimers (1 and 3); 1H NMR (500 MHz, ref. CD2HOD = 3.31 ppm, CD3OD): δ 1.54 (2H, m), 1.74 (2H, m), 1.88 (2H, m), 4.16 (4H, m), 4.30 (4H, m), 7.28 (4H, m), 7.34 (4H, t, J = 7.0 Hz), 7.63 (4H, m), 7.75 (4H, t, J = 7.4 Hz) ppm; HR-ESI-qTOF-MS: m/z 635.2410 [M + H]+ (calcd for C37H35N2O8, 635.2393). The E22P-Aβ40 dimers (Figure 2B) were synthesized in a stepwise fashion on 0.1 mmol of preloaded Fmoc-L-Val-PEG-PS resin by PioneerTM peptide synthesizer using the Fmoc method, as reported previously.28 Each coupling reaction was carried out using each Fmoc amino acid (0.4 mmol), HATU (0.4 mmol), and DIPEA (0.8 mmol) in 1.9 mL of DMF for 30 min. After each coupling reaction, the N-terminal Fmoc group was deblocked with 20% piperidine in DMF. Molar equivalent Fmoc-L,L-DAP or Fmoc-L,L-DAZ (0.05 mmol) was employed instead of



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Fmoc-L-Ala at position 30 or Fmoc-Gly at position 38 (0.4 mmol) in order to avoid formation of the mono-coupled peptide. After the completion of chain elongation followed by Fmoc deblocking, each peptide resin, washed with DMF and CH2Cl2, was shaken for 2 h at room temperature in a mixture containing TFA, m-cresol, ethanedithiol, and thioanisole for final deprotection and cleavage from the resin. Each crude peptide was precipitated by diethylether, followed by purification using HPLC on a YMC PROTEIN-RP column and elution at 8.0 mL/min by a 70-min linear gradient (curve 6) of 20–60% CH3CN containing 0.1% TFA for 1, by an 80-min exponential gradient (curve 7) of 30– 60% CH3CN containing 0.1% TFA for 2, or by an 80-min linear gradient (curve 6) of 20–60% CH3CN containing 0.1% TFA for 3. Subsequent purification was carried out using YMC ODSA and elution at 8.0 mL/min by an 80-min linear gradient (curve 6) of 20–60% CH3CN containing 0.1% TFA for 1, by an 80-min exponential gradient (curve 7) of 30–60% CH3CN containing 0.1% TFA for 2, or by a 60-min exponential gradient (curve 7) of 30–60% CH3CN containing 0.1% TFA for 3. Lyophilization gave each pure peptide, the purity of which was confirmed by HPLC (> 98%) (Figure S1, Supporting Information). The yield and molecular weight of each E22P-Aβ40 dimer were as follows: 1, 5.6% yield, m/z 8607.49 (calcd for [M (Av.)] 8607.80); 2, 3.1% yield, m/z 8636.26 (calcd for [M (Av.)] 8635.85); 3, 11.6% yield, m/z 8636.04 (calcd for [M (Av.)] 8635.85).

MTT Assay. Human neuroblastoma SH-SY5Y cell line (ATCC, Manassas, VA), which is used as a neuronal cell model to estimate the neurotoxicity of Aβ peptides, was maintained in a 1:1 mixture of Eagle's minimum essential medium and Ham's F12 medium (Wako) containing 10% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel). As reported previously,34 each Aβ was dissolved in 0.15% NH4OH to give a 110 µM stock solution. The resultant peptide solution (10 µL) was diluted with 0.15% NH4OH to the appropriate final concentrations (2.5, 5, and 10 µM) in the medium, and was subsequently added to 100 µL of the culture medium containing near-confluent cells (104 cells/well). After being treated at 37 °C for 16 ACS Paragon Plus Environment

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16 h or 48 h, 10 µL of 5 mg/mL MTT was added to the cells, followed by 4 h incubation at 37 °C. After removing the culture medium, cell lysis buffer (100 µL/well; 10% SDS, 0.01 M NH4Cl) was subsequently added to the cells. Next, the resultant cell lysate was incubated overnight in the dark at room temperature, and was subjected to absorbance measurements at 595 nm using a microplate reader (MultiScan JX). Absorbance obtained by the addition of vehicle (0.15% NH4OH) was taken as 100%.

Thioflavin-T (Th-T) Assay. The aggregative ability of each Aβ was evaluated by Th-T fluorescence assay.41 As reported previously,28 each Aβ was dissolved in 0.15% NH4OH at 250 (monomer) or 125 (dimer) µM, followed by 10-fold dilution using phosphate buffered saline [PBS (50 mM sodium phosphate and 100 mM NaCl, pH 7.4)] to a final concentration of 25 (monomer) or 12.5 (dimer) µM. After incubating at 37 °C for the desired duration, 2.5 µL of the reaction solution was added to 250 µL of 5.0 µM Th-T in 5.0 mM Gly-NaOH (pH 8.5). Fluorescence measurements (excitation at 430 nm and emission at 485 nm) were subsequently performed using a microplate reader (Fluoroskan Ascent).

Transmission Electron Microscopy (TEM). The aggregates of each Aβ solution (25 µM, monomer; 12.5 µM, dimer) in 50 mM PBS (pH 7.4) after 48 h incubation for the Th-T assay were examined under a H-7650 electron microscope.

The experimental procedure was

previously described34 with slight differences. After each Aβ aggregate was centrifuged (4 °C, 16,100 g, 10 min), the supernatant was removed from the pellet. The resultant pellet was gently resuspended in water (20 µL) using a vortex, and centrifuged at 2000 g for 1 min.

The

suspension (5 µL) was applied to a 200 mesh carbon–coated copper grid (thickness: 20–25 nm; Veco, Eerbeek, Netherlands), and allowed to incubate for 5 min before being negatively stained for twice with 2% uranyl acetate (5 µL). Stained samples were subsequently subjected to TEM. The diameter, width, and length of the aggregates were calculated from at least three representative pictures using Hitachi EM viewer Ver03.01 software. 17 ACS Paragon Plus Environment


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Circular Dichroism (CD) Spectrometry. CD spectra were measured using a 0.1-mm quartz cell as described elsewhere.34 Each Aβ was dissolved in 0.1% NH4OH at 250 (monomer) or 125 (dimer) µM and diluted 10 times with PBS (pH 7.4) to a final concentration of 25 (monomer) or 12.5 (dimer) µM, and was incubated at 37 oC. After several intervals, an aliquot (200 µL) was loaded into a quartz cell, and the CD spectrum was recorded at 190–260 nm. The spectrum of each Aβ is shown after subtracting the spectrum of the vehicle alone.

Ion Mobility–Mass Spectrometry (IM-MS). Each Aβ-dimer (1, 2, or 3) was dissolved in 0.1% NH4OH at 125 µM, followed by 10-fold dilution with 25 mM ammonium acetate (pH 7.4) before incubating for 4 h at 37 °C. The incubated sample (12.5 µM) was centrifuged for 5 min at 13,000 g before infusion into MS apparatus using glass capillary (Nanoflow Probe Tip, Waters). Mass spectra and ion mobility experiments were accomplished on SYNAPT G2-Si HDMS (Waters) using a nano-electrospray as an ionization source. The instrument was operated in negative ion mode with a capillary voltage of 0.65–0.75 kV, a sample cone voltage of 100 or 150 V, a source temperature of 50 oC, and a desolvation temperature of 150 oC. For the ion mobility measurement, nitrogen gas was used in the ion mobility cell, and the cell pressure was maintained at approximately 2.95 mbar with a wave velocity of 1,000 m/s and a wave height of 40 V. Data acquisition and processing were performed with MassLynx (V4.1) and DriftScope (V2.8) software supplied with the instrument. The CsI cluster ions were used for m/z scale as a calibrator.

Statistical analysis. All data are presented as the mean ± SD. The differences were analyzed with one-way analysis of variance (ANOVA), followed by Bonferroni's test. These tests were implemented using GraphPad Prism software (version 5.0d). P values < 0.05 were considered significant.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxx. Figure S1. HPLC profile for purity check and ESI-MS data of the Aβ40 dimers (1–3). Figure S2. Projection of 2D spectra of 1–3 in Figure 8 in the main text on the mass spectra axis. Table S1. The calculated and observed masses of 1–3 in Figure 8 in the main text. Abbreviations AD, Alzheimer's disease; Aβ, amyloid β; CD, circular dichroism; DAZ, L,L-2,8-diaminoazelaic acid; DAP, L,L-2,6-diaminopimeric acid; IM-MS, ion mobility–mass spectrometry; NMR, nuclear magnetic resonance; PBS, phosphate buffered saline; TEM, transmission electron microscopy; Th-T, thioflavin-T.

AUTHOR INFORMATION Author Contributions Y.I., K.M., and K.I. designed the research. Y.I., K.M., M.H., Y.H., T.S., Y.M., T.T., T.K., and K.I. performed the experiments. Y.I., K.M., K.A., T.K., K.H., and K.I. analyzed data. Y.I., K.M., and K.I. wrote the paper. Funding This study was supported by JSPS KAKENHI Grant Number 26221202 to K.I. and K.M., and by funds for life science research 2013 from Takeda Science Foundation to K.I. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We thank Dr. Ryo C. Yanagita (Kagawa University) for molecular modeling and Mr. Harukuni Tokuda (Kyoto University) for assistance with cell culture. 19 ACS Paragon Plus Environment


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Watt, A. D., Perez, K. A., Rembach, A., Sherrat, N. A., Hung, L. W., Johanssen, T., McLean, C. A., Kok, W. M., Hutton, C. A., Fodero-Tavoletti, M., Masters, C. L., Villemagne, V. L., and Barnham, K. J. (2013) Oligomers, fact or artefact? SDS-PAGE induces dimerization of β-amyloid in human brain samples. Acta Neuropathol. 125, 549564.

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Bernstein, S. L., Dupuis, N. F., Lazo, N. D., Wyttenbach, T., Condron, M. M., Bitan, G., Teplow, D. B., Shea, J. E., Ruotolo, B. T., Robinson, C. V., and Bowers, M. T. (2009) Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer's disease. Nat. Chem. 1, 326-331. 24 ACS Paragon Plus Environment

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38.

Kloniecki, M., Jablonowska, A., Poznanski, J., Langridge, J., Hughes, C., Campuzano, I., Giles, K., and Dadlez, M. (2011) Ion mobility separation coupled with MS detects two structural states of Alzheimer's disease Aβ1-40 peptide oligomers. J. Mol. Biol. 407, 110124.

39.

Masuda, Y., Uemura, S., Nakanishi, A., Ohashi, R., Takegoshi, K., Shimizu, T., Shirasawa, T., and Irie, K. (2008) Verification of the C-terminal intramolecular β-sheet in Aβ42 aggregates using solid-state NMR: implications for potent neurotoxicity through the formation of radicals. Bioorg. Med. Chem. Lett. 18, 3206-3210.

40.

Carpino, L. A. (1993) 1-Hydroxy-7-azabenzotriazole. An efficient peptide coupling additive. J. Am. Chem. Soc. 115, 4397-4398.

41.

Naiki, H., and Gejyo, F. (1999) Kinetic analysis of amyloid fibril formation. Methods Enzymol. 309, 305-318.



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Legends to the Figures Figure 1. (A) Our proposed toxic dimer model for Aβ4213 in “the toxic conformation theory”. (B) A toxic dimer model for Aβ40 proposed by Wetzel's group.21 Both models were deduced from systematic proline replacements.

Figure 2. (A) Structures of L,L-DAP and L,L-DAZ as a molecular linker. (B) Structures of Aβ40 dimers (1–3) utilized in this study.

Figure 3. Synthesis of Fmoc-L,L-DAZ. (a) 1,5-diidopentane, NaHMDS, THF; 5, 25%; 6, 16%; (b) NaHMDS, THF, 32%; (c) HI, 83%; (d) Fmoc-OSu, Na2CO3, acetone / H2O, 58%.

Figure 4. Neurotoxicity towards SH-SY5Y of 1–3 and E22P-Aβ40 (2.5, 5, and 10 µM) (A) after 48 h incubation and (B) after 16 h incubation at 37 °C. ○, E22P-Aβ40; □, 1; ■, 2; ●, 3. Absorbance obtained after adding vehicle (Veh: 0.15% NH4OH) was taken as 100%. Viability of cells treated with 1 and 2 (2.5, 5, and 10 µM) after 16 h incubation did not significantly decrease compared with cells treated with vehicle only. *, p < 0.05; n.s., not significant. Data are expressed as mean ± SD (n = 3).

Figure 5. Th-T fluorescence of 1–3 (12.5 µM) and E22P-Aβ40 (25 µM) after incubation for the indicated duration at 37 °C. ○, E22P-Aβ40; □, 1; ■, 2; ●, 3. Data are expressed as mean ± SD (n = 8).

Figure 6. TEM analyses of the aggregates of 1–3 (12.5 µM) and E22P-Aβ40 (25 µM) after 48 h incubation at 37 °C. Mean diameter of the aggregates of 1 and 2, and mean width and length of the aggregates of 3 and E22P-Aβ40 were calculated from at least three representative pictures using Hitachi EM viewer Ver03.01 software (See text). Arrowheads indicate protofibrils. Scale bar = 50 nm. 26 ACS Paragon Plus Environment

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Figure 7. CD spectra of 1–3 (12.5 µM) and E22P-Aβ40 (25 µM) after incubation for the indicated duration at 37 °C.

Figure 8.

IM-MS of 1–3 (12.5 µM) after 4 h incubation at 37 °C. n denotes an integer corresponding to the number of units coexisting in the solution [n = 1, 2, 3… of a dimer model denotes dimer (Dim), tetramer (Tet), hexamer (Hex)…, respectively] depending on their drift time. The signal amplitude with linear scale is color-coded, increasing from purple (low intensity) to blue (high intensity). As exemplified by red rectangle in 2, signals (z/n = 3: z denotes charge) for Hex9– and Tet6– overlapping in m/z 2850–2920 region were clearly resolved into two signal groups with different drift times: 9.3 and 11.1 ms (see text).

Figure 9. Time-course experiments of IM-MS of 3, the most neurotoxic dimer (12.5 µM), during 0~5 h incubation in 25 mM ammonium acetate (pH 7.4) at 37 °C. The weak and diffused signals under m/z 2000 after 4 h and 5 h incubation could be originated from the background signals.



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Figure 1. Irie, Y. et al.　　

A

B

33!

D23! E22!

35! 42! 14! Ala42!

29! 30!

40!

38! 39!

1! 1!

D23!

E22!

10!

Our dimer model of Aβ42

Wetzel’s dimer model of Aβ40

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



A HO 2C ＊

＊ CO H 2

NH 2

HO 2C ＊

＊ CO H 2

NH 2

NH 2

NH 2

L,L-Diaminopimelic acid!

L,L-Diaminoazelaic acid!

(L,L-DAP)!

(L,L-DAZ)!

B 29!30!

29!30!

40!

1!

D23! E22P!

1

29!30!

40!

38! 40!

1! D23! E22P!

1! D23! E22P!

2


3


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O

O N

H 2N

Ph

Ph

18 % in 2 steps (R)-α-Phenylethylamine

a

Ph

N

Ph 1' N O

b

N 1' Ph 3

I

O 5 (3S,1'R)

4

O N Ph

Ph

N

I O 6 (3R,1'R)

c

NH 2 HO 2C 2

NH 2 8 CO 2H

L,L -Diaminoazelaic acid

NHFmoc

d HO 2C

NHFmoc CO 2H

Fmoc-L,L -Diaminoazelaic acid

(2S,8S)


O

R N

4 1 N R O 7 (1S,4S,1'R)

R=

1' Ph

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A

B

Normal incubation (48 h)

n.s.

80! 60!

20! 0!

n.s.

E22P-Aβ40

1 2 3

Veh!

Cell viability (%)!

100!

40!

Short incubation (16 h)

120!

120!

Cell viability (%)!

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


n.s.

100! 80! 60!

*

40! 20!

2.5!

5!

10!

0! Veh!

Concentration (µM)!


2.5!

5!

10!

Concentration (µM)!



Th-T fluorescence (× 103)!

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0.4! E22P-Aβ40

0.3!

1 2 3

0.2! 0.1! //

0! 0! 4! 8!

24!

48!

//

168!

Incubation time (h)!


336!

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E22P-Aβ40

1

2

3



0 h! 4 h! 8 h! 24 h! 48 h!

20! 15! 10! 5! 0! −5! −10! −15! 190!

E22P-Aβ40 210!

230!

250!

Wavelength (nm)!

5! 2.5! 0! −2.5! −5! −7.5! −10! 190!

2 210! 230! Wavelength (nm)!

250!

[θ] (kdeg・cm2 dmol-1 per residue)!

25!





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5! 2.5! 0! −2.5! −5! −7.5! −10! 190!


250!

5! 2.5! 0! −2.5! −5! −7.5! −10! 190!



250!

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Figure 8. Irie, Y. et al.　　 20!

Drift time (msec)!

2

3

4 5

6

n = 1 10!

1

Drift time (msec)!

0! 1000! 20!

m/z! 2000!

n = 1

3000!

4000!

2

4 6 5

3

5000!

6000!

7000!

10!

2 0! 1000! 20!

2000!

3000!

4000!

5000!

m/z! 7000!

6000!

9 10 11 12 n = 6 7 8

Drift time (msec)!

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10!

3 0! 1000!

2000!

3000!

4000!

5000!


6000!

m/z! 7000!



Drift time (msec)!

20!

10!

0 h 0!

2000!

4000! m/z!

6000!

2 h 2000!

4000! m/z!

6000!

20!

Drift time (msec)!

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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10!

4 h 0!

2000!

4000! m/z!

6000!

5 h 2000!


4000! m/z!

6000!

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TOC. Irie, Y. et al.　　

20!

E22P-G38DAP-Aβ40 dimer! (Toxic)

48 h!

Width! 9.0 nm ! (SD = 1.4) ! Length! 143 nm ! (SD = 44) !

Drift time (msec)!

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9 10 11 12 n = 6 7 8

10!

12 ~ 24-mer 4 h! 0! 4000!


5000! m/z!

6000!

Synthetic Models of Quasi-Stable Amyloid Î²40 ... - ACS Publications

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