Novel trans-Stilbene-based Fluorophores as Probes for Spectral

Research Article pubs.acs.org/chemneuro

Novel trans-Stilbene-based Fluorophores as Probes for Spectral Discrimination of Native and Protofibrillar Transthyretin Raúl I Campos, Xiongyu Wu, Mathias Elgland, Peter Konradsson, and Per Hammarström* IFM−Department of Chemistry, Linköping University, Linköping 581 83, Sweden S Supporting Information *

ABSTRACT: Accumulation of misfolded transthyretin (TTR) as amyloid fibrils causes various human disorders. Native transthyretin is a neurotrophic protein and is a putative extracellular molecular chaperone. Several fluorophores have been shown in vitro to bind selectively to native TTR. Other compounds, such as thioflavin T, bind TTR amyloid fibrils. The probe 1-anilinonaphthalene-8-sulfonate (ANS) binds to both native and fibrillar TTR, becoming highly fluorescent, but with indistinguishable emission spectra for native and fibrillar TTR. Herein we report our efforts to develop a fluorescent small molecule capable of binding both native and misfolded protofibrillar TTR, providing distinguishable emission spectra. We used microwave synthesis for efficient production of a small library of trans-stilbenes and fluorescence spectral screening of their binding properties. We synthesized and tested 22 trans-stilbenes displaying a variety of functional groups. We successfully developed two naphthyl-based trans-stilbenes probes that detect both TTR states at physiological concentrations. The compounds bound with nanomolar to micromolar affinities and displayed distinct emission maxima upon binding native or misfolded protofibrillar TTR (>100 nm difference). The probes were mainly responsive to environment polarity providing evidence for the divergent hydrophobic structure of the binding sites of these protein conformational states. Furthermore, we were able to successfully use one of these probes to quantify the relative amounts of native and protofibrillar TTR in a dynamic equilibrium. In conclusion, we identified two trans-stilbene-based fluorescent probes, (E)-4-(2-(naphthalen-1-yl)vinyl)benzene-1,2-diol (11) and (E)-4-(2-(naphthalen-2-yl)vinyl)benzene-1,2-diol (14), that bind native and protofibrillar TTR, providing a wide difference in emission maxima allowing conformational discrimination by fluorescence spectroscopy. We expect these novel molecules to serve as important chemical biology research tools in studies of TTR folding and misfolding. KEYWORDS: transthyretin, amyloid, stilbene, fluorescence, probe, spectrum

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downhill polymerization process where, in contrast to most other amyloid proteins, a first step of dissociation of the native homotetramer is necessary.7,8 This step is strongly inhibited by hydrophobic molecules able to bind to the active site.9 This is believed to be a reason why TTR rarely deposits in the CNS, where a large population is bound to T4,10 while TTR is systemically deposited in heart and peripheral nerves. Currently, the best available treatments for this disease are liver transplantation and the use of small molecule misfolding inhibitors. Liver transplantation, the organ where plasma TTR is synthesized, is only useful in the cases of TTR amyloidosis caused by one of the >130 different mutant variants found today. Recently, an aggregation inhibitor that binds to the hydrophobic pocket and prevents native tetramer dissociation, named tafamidis, was developed.11 This is the only small molecule drug approved today to treat the cause of an amyloid disease. In addition, two more repurposed drug molecules, diflunisal12 and tocalpone,13 show great promise through the same mechanism.

ransthyretin (TTR) or prealbumin is a 55 kDa homotetrameric protein that circulates in human plasma1 and in the cerebrospinal fluid2 in approximate concentrations of 0.2 mg/mL (3.6 μM tetramer) and 0.02 mg/mL (0.36 μM tetramer), respectively. Approximately 15−20% and 80% of the protein circulates bound to thyroxine (T4) in plasma and in the cerebrospinal fluid, respectively.3 This molecule (T4), composed of two aromatic rings, is known to bind to TTR’s active site. This structural region is comprised by two hydrophobic pockets formed by hydrophobic interactions between two TTR dimers, and the entrance to the active site is flanked by opposing Lys15 residues. Only one T4 molecule is able to bind there at physiological concentrations of both protein and small molecule, believed to be due to negative cooperativity.4 Native TTR is believed to function as an extracellular molecular chaperone mitigating Aβ aggregation in Alzheimer’s disease.5 However, one of the main features of TTR is that, together with other ∼30 human proteins, both WT and mutants of the protein in an alternate misfolded conformation are implicated in the development of archetypal amyloid diseases. These diseases are characterized by the deposition of filamentous protein aggregates called amyloid fibrils.6 In the case of TTR, fibril formation is a © XXXX American Chemical Society

Received: March 1, 2016 Accepted: May 4, 2016

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DOI: 10.1021/acschemneuro.6b00062 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience

two (SB 11 and 14) that have distinguishable fluorescence spectra when bound to either native or protofibrillar TTR. Our data supports that the structure of fibrillar forms of TTR entails a very different conformation of the constituent monomeric building block than that within the native TTR tetramer.

However encouraging this is, the results suggest that deeper understanding of disease progression is needed to generate a treatment capable of stalling or reversing disease progression. It is also urgent to find early diagnostic methods. The development of a small fluorescent molecule able to bind to both native and fibrillar conformations of an amyloidogenic protein is highly desirable for in vitro and in vivo studies of these conformational transitions. Several fluorophore heterocyclic scaffolds, for example, dibenzofuran, have been tested in vitro and bind selectively to native tetrameric TTR.14,15 Other similar compounds bind TTR amyloid fibrils, such as thioflavin T and Nile red.16 Furthermore, 1-anilinonaphthalene-8-sulfonate (ANS) and to some extent 4,4-bis-1-phenylamino-8-naphthalenesulfonate (Bis-ANS) and Nile red bind to both native and fibrillar TTR, becoming highly fluorescent but with indistinguishable emission spectra.16,17 To our knowledge, there are no reports of a fluorophore that binds to both native and fibrillar TTR that distinguishes these different conformational states. Herein we report our efforts to develop a fluorescent small molecule capable of binding native TTR and misfolded TTR aggregated into protofibrils. We synthesized and used fluorescence spectral screening as our main methods. We chose the transstilbene (SB)-based fluorophore resveratrol as a starting scaffold since it binds native TTR18 and becomes fluorescent.19 Moreover, other compounds having a trans-stilbenoid structure (11C-BF-227 and 18F-florbetapir) have successfully been used as positron emission tomography ligands to trace cardiac TTR amyloid.20,21 Thereafter, we synthesized and tested 22 analogs, and selected

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RESULTS AND DISCUSSION Rationale for Selection of trans-Stilbene Fluorophores to Transthyretin. We screened different trans-stilbene based molecules using resveratrol as starting scaffold (Figure 1).19 As initial target molecules, we incorporated fluorine in the structure because it provides the potential for imaging properties (18F-PET) as well as for 19F-NMR experiments, which has become an increasingly popular method to probe protein aggregation and misfolding processes.22 and has previously been shown to make good binders toward native TTR as substituents on biophenyls23 and anthranilic acid derivatives.24,25 Furthermore, the fluorinated stilbenes feature a benzimidazole motif, which is a widely used pharmacophore in therapeutic agents that elicit antimicrobial, antiviral, anticancer, and anti-inflammatory properties.26 To determine binding response to native TTR and TTR protofibrils, we used fluorescence spectroscopy using excitation at 330 nm and monitored emission in the 350−600 nm range. All our experiments using native TTR were carried out in PBS (phosphate buffered saline) buffer at pH 7.5, whereas TTR protofibrils were assayed in their original NaAc (sodium acetate) buffer at pH 3.0. We selected a 0.1 mg/mL native TTR and TTR protofibril concentration

Figure 1. Chemical structures of resveratrol and synthesized trans-stilbenes. B


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ACS Chemical Neuroscience Table 1. Selection of Trans-Stilbene Probes for Discrimination of Native TTR (N) and TTR Protofibrils (PF).

a Different probes (1.8 μM) were tested with native (a) or protofibrillar TTR (b) (0.1 mg/mL). Ex. WL: 330 nm. Emission recorded 350−600 nm range. Intensities are given at the peak wavelength. Measurement error is ±5% (n = 2). bMeasurement error is ±2% (n = 2). From the intensities (a) and (b) we calculated the fold change upon addition of either native TTR or TTR protofibrils to each probe in their respective buffers. Fold changes: 0−2 are highlighted in black, 1−2 are highlighted in bold black, above 2 are highlighted in orange and over 4 are highlighted in bold orange. For those probes where 2 wavelengths gave an increased fluorescence, we calculated the ratio of the fold changes for native TTR and protofibrils in the ∼500 nm range of the spectra (N red/PF red) and similarly on the ∼400 nm range of the spectra for TTR protofibrils and native TTR (PF blue/ N blue). cEntries represent percent fibril formation and thus aggregation inhibition efficacy relative to TTR WT in the absence of probe (assigned to be 100%): complete inhibition is equivalent to 0% fibril formation. Average measurement error is ±7% (n = 3). Fibril formation of 0.2 mg/mL native TTR and 7.2 μM of each molecule (1:2 TTR tetramer to probe ratio) after 72 h incubation in stagnant conditions at pH = 4.4, using turbidity measurement at 400 nm.

(providing 1.8 μM on a tetramer basis) and 1.8 μM probe concentration. This renders a 1:1 native TTR to compound ratio. We failed to obtain satisfying fluorescence results from the fluorinated stilbenes (SB 1−3). Interestingly, replacing the trifluoromethyl substituents in SB 3 with methoxy groups (SB 4) increased the fluorescence performance for native TTR but not for protofibrils. Our data hence strongly indicates that fluorinated stilbenes are poor fluorescent probes. Then we decided to mimic the structure of the resveratrol molecule by moving one of the hydroxyl groups to the 4-position and forming two ortho-hydroxyl structural analogs of resveratrol (SB 5). This gave a slight red shift of the fluorescence emission compared with resveratrol. Removing the 4-hydroxy group (SB 6) provided an even larger shift, and in the presence of TTR protofibrils, it showed a higher fluorescence increase. Replacing the phenyl ring with a naphthalene moiety further improved the response to TTR protofibrils (SB 11). Then we synthesized the naphthalen-2-yl SB 14. To better understand the properties of SB 11 and 14, we synthesized their methylated derivatives (SB 12, 13, and 15). In addition to the highlighted probes discussed above, several diverse stilbene molecules were

included in the study to further screen the chemical space for TTR selective fluorescent probes (Figure 1). Fluorescence Screening of trans-Stilbenes. In general terms, most of our developed probes bound to native TTR and showed an emission peak at ∼400 nm, while some probes gave an additional signal at ∼500 nm (Table 1). This ease of binding to native TTR reflects the small size and hydrophobic nature of the probes, which hence can easily fit into the hydrophobic T4 pockets of native TTR,23,27 and underlines the promiscuity of TTR binding activity. A selection of molecules showed a positive increase in fluorescence intensity upon binding to protofibrils, namely, SBs resveratrol, 5, 7, 9 and 11−22. Most of them gave a peak at ∼400 nm. In order to more easily analyze their capacity to discriminate between the conformational states, we calculated the fold change of the signal upon binding native TTR or protofibrils at those wavelengths that had potential to be used for spectral discrimination. SB 11 and 14 were selected from the screening by using the following selection criteria: first, we wanted a probe that allowed spectral discrimination, thus giving a peak at different wavelengths (∼400 nm vs ∼500 nm) for the different conformational C


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Selected trans-Stilbenes Allow for Native versus Protofibril Spectral Discrimination. The best probes for native TTR and protofibril discrimination in our fluorescence spectroscopy screening were SB 11 and 14 (Figure 2A−D). SB 15 was left out because of expected low solubility and poor fibril formation inhibition. Both probes offered a different spectral signature in binding either native or protofibrillar TTR. They displayed an array of emission peaks, presumably from different excited states. SB 11 (Figure 2A), upon binding to TTR protofibrils, had a blue-shifted emission peak at 395 nm and a three additional peaks at 410, 425, and 475 nm. Upon binding to native TTR, there was an emitting species that can be useful for discrimination, because it is very red-shifted, having a peak fluorescence intensity at 505 nm. It is still possible to observe the three more blue-shifted emitting species at 395, 410, and 425 nm; however, their intensity is comparatively much weaker. SB 14 (Figure 2B) showed a similar spectral profile to SB 11. A difference is that the 395 nm peak is more prominent compared with the 410 and 425 components. Also, the emitting species that can be useful for native TTR discrimination appeared slightly more blue-shifted, with a peak at 490 nm. SB 14 had the added advantage of an apparent lower background fluorescence, because there was very little fluorescence given by the free probe in either buffer. To visualize the different response these probes provide upon binding either native or protofibrillar TTR, we show fluorescence of SB 11 under UV table excitation (303 nm). The probe offers very little background fluorescence (Figure 2C). It increases considerably its fluorescence intensity upon native TTR binding, giving a cyan emission by eye. It is not easy to distinguish SB 11 emission bound to TTR protofibrils by naked eye, because the emission peak is very blue-shifted toward the UV region, where the coloring is seen as a silver blue haze mixed from light scattering protofibrils (Figure 2D). TTR protofibrils are 4-fold change intensities for native TTR and protofibrils at the different wavelengths. Then, we calculated the ratio of the fold changes for native TTR and protofibrils in the ∼500 nm part of the spectra (N red/PF red) and similarly on the ∼400 nm part of the spectra for TTR protofibrils and native TTR (PF blue/N blue). Our third discrimination criterion is that both ratios had to be ≥1.5. Three SBs passed these three criteria, namely, SBs 11, 14, and 15. Notably all three SBs were naphthalene conjugated hydroxyl trans-stilbenes (Figure 1). Some other SBs are worth highlighting. SB 9, even though it did not pass the selection criteria, may represent a very useful native TTR fluorescent probe; it gives a considerably higher increase in fluorescence compared with the commonly used resveratrol molecule. SB 21 gave a 2.2-fold intensity increase upon binding of protofibrils, while not giving an intensity change upon native TTR binding at the selected wavelength. It was the only probe with this quality. Moreover, the spectral signatures of several of the trans-stilbenes displayed the characteristics of species originated from intramolecular proton transfer in the excited (ESIPT) state. This phenomena has been previously characterized using 3-hydroxyflavone and 5-hydroxyflavone28 and opens a variety of possible applications for these molecules as biological and inorganic detection probes.29 As a future step in the search for valuable SBs, several analogues to the amyloid probe X-3430 were desired. We hence synthesized bistilbene compounds 16, 18, 20, and 22 with larger conjugated systems. We expected some to show a fluorescence increase with TTR protofibrils at longer wavelengths. These were included in the current screening series but will be developed further for amyloid probe applications and will not be further elaborated upon in this report. Screening of Fibril Formation Inhibition by transStilbenes. Some of our SBs bound native TTR with a higher increase in fluorescence than the already effective aggregationinhibitor resveratrol.18,31 Hence, we wanted to test them sideby-side with resveratrol and, using the absence of probe as a reference of 100% fibril formation, to find other strong misfolding inhibitors, which reflect native TTR binding. We used the standard turbidity assay at pH 4.4 (37 °C), which is the optimal pH for TTR WT aggregate formation in these experimental conditions.32 Resveratrol and SBs 8, 11−14, and 22 were all deemed good fibril inhibitors ( 10 μM. We have recently shown that TTR protofibrils are in continuous exchange with unfolded monomers.33 Possibly these lower affinity probe binding sites are screened by surface remodeling due to high local concentration of exchanging unfolded monomeric TTR.33 The binding affinities determined for native TTR and SB 11 or 14 interactions were similar to those expected for small aromatic compounds. This binding event was weaker than that previously obtained for T4, which falls in the low nanomolar range.3 The binding strength of the first binding event resembles more closely that of the nonsteroidal anti-inflammatory drug (NSAID) diflunisal, which was determined by isothermal titration calorimetry (Kd1 = 75 nM; Kd2 = 1.1 μM).12 Spectral Differences of Bound trans-Stilbenes Can Partially Be Explained by Binding Site Polarity Effects. Given the profound spectral differences provided by SB 11 (Figure 3A,B) and 14 (Figure 4A,B) bound to either native TTR or protofibrils, we went further into characterizing the origin of these spectral differences. We tested SB 11 and 14 using a variety of solvents. These solvents offered a range of dielectric constants (ε) from 2.39 to 80.1 and viscosities (η) from 0.59 to 1 mPa (Supporting Table 1). There were no evident spectral differences

generated from the different conformational states. We can see that the spectral shape as we titrated SB 14 to native TTR provided three main peaks (395, 435, and 490 nm) where the 435 nm peak is from free probe (Figure 4A,B), while only one (395 nm) peak was observed when 14 was titrated on protofibrils. To characterize the binding of SB 11 and 14 to native TTR and protofibrils, we took the non-normalized intensities and fit them to single or double exponential binding functions (solid lines, Figures 3 E,F and 4,E,F and Table 2). SB titrations on fixed concentration of native TTR could not be fitted to two binding sites as we expect native TTR to have two available binding pockets. The determined dissociation constant (Kd) for a single binding site was 2.9 ± 0.2 μM for SB 11 and 2.0 ± 0.3 μM for SB 14. For TTR protofibrils, we obtained a slightly stronger dissociation constant for SB 14 (0.9 ± 0.3 μM), while for SB 11 the data could not be fitted to a fixed protofibril concentration. To obtain additional affinity data, we calculated Kd for SB−protein binding, where we carried out a reverse titration of native TTR or protofibrils, having SB 11 or 14 as fixed titrand at 1.8 μM (dashed lines, Figures 3E,F and 4E,F). These titrations using native TTR could, as expected from negative cooperativity of TTR, be fitted to a two binding-site equation. The affinities of either molecule were quite similar both for the strong binding site and for the weak binding site (Kd1 = 40 nM and Kd2 = 4.0 μM for SB 11 and Kd1= 60 nM and Kd2 = 3.3 μM for SB 14). The titrations using variable protofibril concentrations showed a peculiar behavior. At low concentrations of protofibrils, we E


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Figure 3. Spectral determination of binding affinity of native TTR and TTR protofibrils versus stilbene 11. (A) Normalized spectra of native TTR titration on SB 11. (B) Normalized spectra of TTR protofibril titration on SB 11. Color coding from lowest to highest concentrations yellow to red. Concentrations in micromolar from 0.0 to 16.2 μM. (C) SB 11 titration on native TTR, background corrected for free probe. (D) SB 11 titration on TTR protofibrils, background corrected for free probe. The titrand was kept at 1.8 μM in all cases (tetramer concentration for native TTR). Color coding from lowest to highest concentrations yellow to red. Concentrations in micromolar from 0.015 to 12 μM in panels C and D. (E) Binding curves generated using the non-normalized spectra of the above titrations. Curves show fits to single or double exponential binding functions, depending on which gave the best correlation (Table 2). Inset in panel F shows a low amplitude high affinity binding event in the nanomolar range.

originating from viscosity differences. However, in the nonnormalized spectra (not shown), one could appreciate an expected increase in intensity as the viscosity increased. Moreover, there was a slight red shift of peak wavelength of less than 5 nm as viscosity decreased when comparing solvents offering ε ≈ 40. It is expected that the lower the viscosity the more pronounced is solvent relaxation of the excited state and the more pronounced is energy loss leading to species emitting from a lower energy level (red-shifted wavelengths). For both probes, we could observe a strong solvent polarity effect causing change of the spectral signature from blue-shifted to red-shifted excited-species along with increasing ε from

toluene (nonpolar) to water based PBS buffer (polar protic) (Figure 5A,B). For the probes bound to TTR reference spectra to compare with the solvent data, we here used a protein concentration of 46 μM to ensure saturation and subtracted a small contribution from the free probe. For SB 11 (Figure 5A) incubated with native TTR, we observed a signature that resembled a very polar environment closest to SB 11 dissolved in PBS buffer judging from the high ∼500 nm peak. Therefore, we expect the ε of the binding site to be >80. For protofibrils, the signature on the contrary indicated that the binding site has a polarity close to that of methylene chloride, suggesting the binding site to have ε close to 9.14. This is in good agreement F


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Figure 4. Spectral determination of binding affinity of native TTR and TTR protofibrils versus stilbene 14. (A) Normalized background corrected spectra of native TTR titration on SB 14. (B) Normalized spectra of TTR protofibril titration on SB 14. Color coding from lowest to highest concentrations yellow to red. Concentrations in micromolar from 0.0 to 16.2 μM. (C) SB 14 titration on native TTR, background corrected for free probe. (D) SB 14 titration on TTR protofibrils, background corrected for free probe. The titrand was kept at 1.8 μM in all cases (tetramer concentration for native TTR). Color coding from lowest to highest concentrations yellow to red. Concentrations in micromolar from 0.015 to 12 μM in panels C and D. (E) Binding curves generated using the non-normalized spectra of the above titrations. Curves show fits to single or double exponential functions, depending on which gave the best correlation (Table 2). Inset in panel F shows a low magnitude high affinity binding event in the nanomolar range.

with previous data for Nile red binding to TTR fibrils.16 The binding site characteristics for native TTR and protofibrils binding to SB 14 appeared similar to those for SB 11 (Figure 5B), confirming the previous analysis of assigning expected ε for each binding site. One can therefore predict that the binding sites are similar for both probes being dictated by the protein conformational state. In comparison of the two probes while showing similar binding affinities, it appears that SB 11 shows preferential binding to native TTR while SB 14 shows preferential binding to protofibrils (Table 2). From the expected structures of the different TTR conformational states, it is intuitive that the compressed molecular structure of SB 11 fits better to the native

T4 binding site (see below) compared with fibrillar TTR whereas the elongated structure of SB 14 fits better to an elongated groove formed from β-strands within the amyloid fibril fold. Protofibrils of TTR generated under these conditions (pH 3.0) are structurally very different from the native TTR fold (see ref 33 for discussion and references). Ultrastructural analysis of TTR protofibrils shows 10

Figure 5. Solvent polarity effects on emission spectra of selected stilbenes. (A) SB 11 and (B) 14 (1.8 μM) dissolved in a variety of solvents offering a range of dielectric constants and viscosities (see Supporting Table 1). The graph shows the change of the spectral signature from blue-shifted to red-shifted excited-species along with increasing dielectric constant from toluene (nonpolar, light blue) to PBS (polar protic, red). Reference spectra of probes bound to native TTR and TTR protofibrils (46 μM) are also shown (thicker dashed black and red lines, respectively) in their respective buffers. Free probe was subtracted from native TTR and protofibril spectra. H


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Figure 7. pH effects on emission spectra of selected stilbenes free and in complex with native TTR. Stilbenes (1.8 μM) tested in PBS buffer pH = 5−10 with (straight lines) and without (dashed lines) native TTR (N in figure legend). (A) Normalized spectra of SB 11 in a range of solvent pH. (B) Normalized spectra of SB 14 in a range of solvent pH. Spectra are the average of two independent measurements.

native TTR binding (Figure 7B). The apparent decrease in this normalized signal of the native TTR-SB 14 complex is due to an increase in ∼500 nm signal and was hence an effect of normalization. Upon binding native TTR, SB 14 seemed highly responsive to buffer pH in the 6−9 pH range especially at the 500 nm emission peak with an apparent pKa of 8. We therefore concluded that binding site pKa does not explain the 25 nm red shift observed upon native TTR binding to SB 11 compared with SB 11 in PBS buffer at pH 7.5 but supported SB 11 being exposed to a polar environment, likely with the naphthalene moiety at the outer part of the T4 binding cavity. Importantly, pH does affect the protein environment of bound SB 14. Hence SB 11 and SB 14 behave differently. The fibril inhibition results (at pH 4.4) as discussed above showed that SBs 11 and 14 were both good inhibitors (Table 1). However, only the para-methoxy derivative of SB 11 (SB 12) was a good inhibitor, while the corresponding derivative of 14 (SB 15) was very poor (Table 1). This supports different binding modes of the α versus β naphthalene conjugates of SB 11 and SB 14. Hence it is possible that the elongated structure of SB 14 compared with SB 11 could reverse the binding mode in the T4 binding pocket. The crystal structure of resveratrol bound to TTR was published in 200018 (Figure 8A). Interestingly the TTR binding mode of reseveratrol and its analogues appears ambivalent.37 Compared with the crystal structure of a resveratrol−TTR complex generated by soaking,18 the opposite binding mode of resveratrol has recently been revealed based on the crystal structure of the TTR−resveratrol complex obtained upon cocrystallization of TTR with resveratrol37 (Figure 8B). As deduced from our fluorescence data, SB 14 could bind with the naphthalene moiety inward and the diphenol ring facing the outer part of the binding pocket. Here Lys15 and Lys15′ are located at the entrance and are known to be accessible for molecules with similar structures14,37,38 (Figure 8B,C). This result would be compatible with pH dependent H-bonding of Lys15 and the phenolic −OH groups, providing the observed of pKa ≈ 8. The reversed binding modes to native TTR are also supported by the amplitudes of the fluorescence spectral peaks (∼400 nm vs ∼500 nm) for SB 11 (dominating ∼500 nm peak) versus SB 14 (dominating ∼400 nm peak) respectively (Figures 2−4). SB 11 as a Tool to Quantify the Relative Amounts of Native TTR and TTR Protofibrils in Dynamic Equilibria. We sought an opportunity to apply our new probes to solve an important scientific issue in TTR misfolding research. We needed to determine the relative amount of unfolded TTR and misfolded TTR protofibrils present in mixtures. As described above, protofibrils were formed and stored in NaAc buffer, pH 3.0. We recently

Figure 8. Binding modes of SB 11 and SB 14 compared with resveratrol in native tetrameric TTR. (A) The crystal structure of TTR soaked in complex with resveratrol.18 (B) (top) Zoom in (red square in panel A) of the two binding pockets and the directions of the resveratrol molecule in relation to Lys15 and Lys15′, PDB code 1DVS,18 and (bottom) cocrystal structure of single occupancy of resveratrol in complex with TTR and T4 C, PDB code 5CR1,37 showing reverse binding modes. (C) Schematic hypothetical binding modes of SB 11 and SB 14 in relation to the binding modes of resveratrol in panel B based on fluorescence spectroscopy data from solvent and pH titration experiments in Figure 7. Structure illustrations made with PyMol.

showed using small-angle X-ray scattering experiments and hydrogen−deuterium exchange mass spectrometry that there I


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Figure 9. Application of SB 11 to determine the relative amounts of native TTR and TTR protofibrils from a dynamic equilibrium. Application of SB 11 to determine the amount of TTR protofibrils that are maintained and how much unfolded monomer that refolds after diluting a concentrated stock (5 mg/mL) in fibrillation buffer (NaAc pH = 3.0) into PBS buffer pH = 7.5. (A) SB 11 (5 μM) was mixed with varying concentrations of native TTR (0−10 μM yellow to red gradient, monomer basis, shown in panel D legend) in PBS buffer, pH = 7.5. The inset shows the binding curve generated from these spectra. (B) SB 11 was mixed with varying concentrations of protofibrillar TTR (0−10 μM) in fibrillation buffer. The inset shows the binding curve generated from these spectra. (C) SB 11 mixed with unfolded TTR in 11% acetic acid (0−10 μM) showing no binding to unfolded TTR. (D) In order to assess (i) retained fibril and (ii) refolded tetramer (i.e., prior presence of unfolded monomer), protofibrils were subjected to refolding conditions in PBS. Both the tetramer peak at 525 nm and the fibril peak at 425 nm are clearly visible. (E) Semiquantitative assessment of the data from panel D based on the relative intensity at 425 and 525 nm of percent maintained fibril and percent refolded tetramer.

was constant presence of ∼15% unfolded monomer in equilibrium with protofibrils.33 We hence wanted to determine the amount of protofibrils that were maintained after diluting a concentrated stock (5 mg/mL) into PBS buffer, pH 7.5. We could foresee that the unfolded monomer population present around the protofibrils would refold into native tetrameric TTR in about 3 min.39 Hereby the use of our new probe would allow us to quantify the change in equilibrium using the refolded tetramer as a surrogate marker for unfolded monomers and concomitantly probe for the stability of protofibrils transferred to native conditions (pH 7.5). We chose SB 11, our best probe for spectral discrimination purposes, to quantify the relative amounts of protofibrils and

native TTR present after dilution in PBS, pH 7.5. To ensure that our experimental settings were appropriate, we performed an excitation−emission fluorescence scan depicted as a 3D plot (Supporting Figure 1A−D). We corroborated that by using excitation at 330 nm, we were exciting only the probe and avoiding protein excitation (TTR contains two tryptophan residues) and the 3D scan clearly distinguished the well separated emission peaks of SB 11 from the different conformations (Supporting Figure 1A−D). We generated a standard curve in a suitable concentration range for native TTR (Figure 9A) binding to SB 11 (5 μM) in PBS buffer, pH 7.5, showing the concentration-dependent J


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ACS Chemical Neuroscience increase in fluorescence with a red-shifted peak at 525 nm (Figure 9A; inset shows the binding curve). Then, we proceeded to generate the corresponding standard binding curve for TTR protofibrils and SB 11 in pH 3.0 buffer (Figure 9B). As expected, this showed a concentration-dependent increase in fluorescence with a blue-shifted peak at ∼400 nm. As a control, the stilbene probe was mixed with unfolded TTR (11% acetic acid) (Figure 9C). As expected this did not show substantial response at either peak wavelength used to elaborate the binding curves. The probe showed poor affinity of the molecule for this unfolded species. In order to assess the amount of retained fibril and refolded tetramer (i.e., presence of unfolded monomer), we diluted a stock of protofibrils (pH 3.0) into PBS at the same concentrations used for elaborating the binding curves. The presence of both protofibrils and native TTR components in these spectra was clearly visible providing mixed fluorescence spectra (Figure 9D). Thereafter, we used the intensity (corrected for free probe) at both the tetramer peak (525 nm) and the protofibril peak (425 nm). We assessed from these data, based on the relative intensity at 425 and 525 nm, the % maintained fibril and % refolded tetramer. For these purpose, the intensities in our binding curves (Figure 9A,B, insets) were considered as 100% for each respective concentration. We were hereby able to determine the relative amount of native TTR and protofibrils in each sample as dependent on protein concentration (monomer basis) (Figure 9E). The amount of refolded native TTR was higher at 10 μM TTR than at 1 μM. This decreased the amount of TTR protofibrils maintained in the sample. The reason for the higher proportion of refolded tetramer present as the concentration increased is likely due to (i) a higher proportion of unfolded monomer present at higher concentration and (ii) the stronger driving force toward the tetramerization expected at higher concentrations through mass action. Hence these data fully support our previous notion of a high proportion of locally exchanging unfolded monomers on the periphery of protofibrils.33 Our data show that it is possible to quantitatively determine the relative amounts of native TTR and protofibrils in the same sample. These results demonstrate that SB 11 allows for spectral discrimination of native TTR and protofibrils in a mixture because there are different amounts of these conformational species in these preparations that bind to the probe and give different spectral signatures.

have the potential to be initial scaffolds for further development toward positron emission tomography tracers to visualize amyloid deposition in vivo.

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METHODS

Synthesis of trans-Stilbenes: General Information. Chemical reagents and solvents were purchased from Sigma-Aldrich, Fluka, Merck, Alfa Aesar, AK Scientific, Kebo, and Ega Chimie. All the solvents and reagents were used as received. Microwave heated reactions were run in an Initiator instrument from Biotage. Analytical thin-layer chromatography was performed on Merk silica gel 60F254 glass-backed plates. Flash chromatography was performed with silica gel 60 (particles size 0.040−0.063 mm). Preparative liquid chromatography was run on a Gilson Unipoint system with a Gemini C18 column (100 mm × 21.20 mm, 5 μm) under neutral conditions using gradient CH3CN/water as eluent (water phase (A), 95:5 water/acetonitrile, 10 mM NH4OAc; organic phase (B), 90:10 acetonitrile/water, 10 mM NH4OAc). NMR spectra were recorded on a Varian Avance 300 or 500 MHz with solvent indicated. Chemical shift was reported in ppm on the δ scale and referenced to the solvent peak. The stilbenes 1−4 were afforded as an E/Z mixture, predominantly (90%) composed of the E-isomer and were used as is for the biochemical experiments. Synthesis of trans-Stilbenes: General Procedures. General Procedure A.

The phosphonates 1a−4a (required for the synthesis of the corresponding stilbenes 1−4) were synthesized by means of a microwave-mediated Michaelis−Arbuzov reaction between a suitably substituted benzyl bromide and trimethyl phosphite.43 A sealed vial containing the appropriate benzyl bromide (2.00 mmol) and trimethyl phosphite (6.00 mmol) was microwave irradiated at 120 °C for 15 min. The reaction mixture was allowed to cool to rt. Excess trimethyl phosphite was then coevaporated with toluene in vacuo. The crude phosphonate was purified by flash column chromatography on silica gel (eluent EtOAc) to afford the desired product. General Procedure B.

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CONCLUDING REMARKS In conclusion, we have successfully synthesized and identified fluorescent molecules that can distinguish native tetrameric and protofibrillar TTR. Our best probes for this purpose, SB 11 and 14, provided fluorescent excited states with a ∼100 nm spectral separation between these conformational states of TTR, which is highly desirable to simplify discrimination of both conformational species simultaneously. Our data shows that the protofibril binding site for both probes is nonpolar with an apparent dielectric constant of 9, while the binding pocked in native TTR is solvent exposed. While our reasoning on probe binding sites within the protofibril and native TTR is hypothetical, our data implicates the power of fluorescence spectroscopy for structural interpretation of misfolded protein structures. Our data also renders strong support for profound conformational differences of these two states of TTR. We foresee that this class of molecules will be useful as research tools for understanding complex folding and misfolding equilibria of TTR. This class of probes are particularly interesting for cell culture experiments expressing TTR of various quality,35,40 tissue samples of transgenic mice,41 or Drosophila expressing amyloidogenic TTR.42 For a more clinical application, these novel molecules

Stilbenes 1−4 were synthesized by means of a Horner−Wadsworth− Emmons reaction between the aldehyde (1H-benzo[d]imidazole-5carbaldehyde) and the suitably substituted benzyl phosphonate (1a−4a) according to a reported literature procedure.44 The stilbenes were afforded as an E/Z mixture, predominantly composed of the E-isomer. The E/Z ratio was determined by calculating the integral ratio for a trans- and a cis-olefinic signal (J = 16.4 and 12.2 Hz, respectively) in 1 H NMR. Potassium tert-butoxide (0.80 mmol) was added to a stirred solution of phosphonate (0.40 mmol) and aldehyde (0.38 mmol) in DMF (2.0 mL) at 0 °C. After 1 h, the reaction mixture was allowed to attain rt and stirred for an additional 3 h. The product was subsequently isolated using preparative HPLC (flow rate 10 mL/min, gradient component A/B from 65:35 to 25:75 over 15 min, then linger on for 10 min). K


Research Article


(E)-6-(3,4,5-Trifluorostyryl)-1H-benzo[d]imidazole (1).

Following lyophilization, the stilbenes 1−4 were afforded as white amorphous solids. Dimethyl 3,4,5-Trifluorobenzyl Phosphonate (1a).

According to general procedure B, reaction between phosphonate 1a and 1H-benzo[d]imidazole-5-carbaldehyde gave 15.0 mg of stilbene 1 (14% yield). E/Z = 7:1 1H NMR (300 MHz, DMSO-d6) δ 8.18 (s, 1H), 7.71 (s, 1H), 7.57−7.45 (m, 3H), 7.41 (dd, J = 8.5, 1.6 Hz, 1H), 7.41 (d, J = 16.4 Hz, 1H), 7.10 (d, J = 16.4 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) δ 150.5 (ddd, JCF = 246.0, 10.0, 4.3 Hz), 143.0, 137.5 (dt, JCF = 248.7, 16.0 Hz), 134.9 (td, JCF = 8.4, 4.5 Hz), 132.3 (d, JCF = 2.7 Hz), 130.4, 123.6 (d, JCF = 2.7 Hz), 120.7, 110.5−110.0 (m, 2C). MS (ESI) Calcd for C15H9F3N2 [M + H]+: 275.08. Found: 275.20. (E)-6-(3,5-Difluorostyryl)-1H-benzo[d]imidazole (2).

According to general procedure A, 3,4,5-trifluorobenzyl bromide was reacted with trimethyl phosphite to afford the title product as a white solid in 49% yield (250 mg, 0.983 mmol). TLC (EtOAc), Rf = 0.26. 1 H NMR (300 MHz, CDCl3) δ 6.98−6.86 (m, 2H), 3.72 (d, 3JHP = 10.9 Hz, 6H), 3.06 (dd, 2JHP = 21.7, JHF = 0.7 Hz, 2H). 13C NMR (75 MHz, chloroform-d) δ 153.7−148.7 (m, 2C), 141.3−137.1 (m, 1C), 128.4−127.2 (m, 1C), 114.4−113.5 (m, 2C), 53.2, 53.1, 32.4 (d, 1JCP = 140.0 Hz). MS (ESI) Calcd for C9H10F3O3P [M + H]+: 255.04. Found: 255.20. Dimethyl 3,5-Difluorobenzyl Phosphonate (2a).

According to general procedure B, reaction between phosphonate 2a and 1H-benzo[d]imidazole-5-carbaldehyde gave 39.0 mg of stilbene 2 (40% yield). E/Z ratio 20:1. 1H NMR (300 MHz, DMSO-d6) δ 12.57 (br, 1H, NH), 8.23 (s, 1H), 7.79 (s, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 16.4 Hz, 1H), 7.49 (dd, J = 8.4, 1.6 Hz, 1H), 7.33 (dt, J = 7.6, 2.2 Hz, 2H), 7.20 (d, J = 16.4 Hz, 1H), 7.05 (tt, J = 9.3, 2.3 Hz, 1H). 13 C NMR (75 MHz, DMSO-d6) δ 163.2 (dd, J = 244.9, 13.7 Hz), 143.4, 142.1 (t, J = 9.8 Hz), 133.0, 130.9, 124.8, 121.3, 110.1−108.4 (m, 2C), 102.5 (t, J = 26.2 Hz). MS (ESI) Calcd for C15H10F2N2 [M + H]+: 257.09. Found: 257.53. (E)-6-(3,5-Bis(trifluoromethyl)styryl)-1H-benzo[d]imidazole (3).

According to general procedure A, 3,5-difluorobenzyl bromide was reacted with trimethyl phosphite to afford the title product as a white solid in 68% yield (352 mg, 1.35 mmol). TLC (EtOAc), Rf = 0.34. 1 H NMR (300 MHz, CDCl3) δ 6.90−6.78 (m, 2H), 6.78−6.65 (m, 1H), 3.72 (d, 3JHP = 10.9 Hz, 6H), 3.13 (d, 2JHP = 21.8 Hz, 2H). 13C NMR (75 MHz, chloroform-d) δ 163.1 (ddd, J = 248.6, 13.0, 3.3 Hz), 135.6− 134.9 (m, 1C), 114.4−112.0 (m, 2C), 102.8 (td, 2JCF = 25.2, 5JCP = 3.5 Hz), 53.2, 53.1, 32.9 (d, 1JCP = 139.3 Hz). MS (ESI) Calcd for C9H11F2O3P [M + H]+: 237.05. Found: 237.20. Dimethyl 3,5-bis(trifluoromethyl)benzyl phosphonate (3a).

According to general procedure B, reaction between phosphonate 3a and 1H-benzo[d]imidazole-5-carbaldehyde gave 24.0 mg of stilbene 3 (18% yield). E/Z ratio 9:1 1H NMR (300 MHz, DMSO-d6) δ 12.44 (br, 1H, NH), 8.26−8.22 (m, 2H), 8.19 (s, 1H), 7.87−7.77 (m, 2H), 7.72 (d, J = 16.5 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.50 (dd, J = 8.5, 1.5 Hz, 1H), 7.39 (d, J = 16.5 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) δ 143.5, 140.9, 134.2, 131.1 (q, JCF = 32.6 Hz), 130.8, 129.7−129.5 (m, 2C), 127.1−126.6 (m, 2C), 125.4, 123.9, 123.9 (q, JCF = 272.8 Hz), 121.8, 121.4, 120.6−119.8 (m, 1C). MS (ESI) Calcd for C17H10F6N2 [M + H]+: 357.08. Found: 357.10. (E)-6-(3,5-Dimethoxystyryl)-1H-benzo[d]imidazole (4).

According to general procedure A, 3,5-bis(trifluoromethyl)benzyl bromide was reacted with trimethyl phosphite to afford the title product as a clear oil in 73% yield (490 mg, 1.46 mmol). TLC (EtOAc), Rf = 0.57. 1 H NMR (300 MHz, CDCl3) δ 7.80−7.71 (m, 3H), 3.73 (d, 3JHP = 11.0 Hz, 6H), 3.26 (d, 2JHP = 22.0 Hz, 2H). 13C NMR (75 MHz, chloroform-d) δ 134.5 (d, 2JCP = 8.9 Hz), 132.1 (qd, 2JCF = 33.4, 4 JCP = 3.0 Hz), 130.4−129.4 (m, 2C), 123.3 (q, 1JCF = 272.6 Hz), 121.4− 121.0 (m, 1C), 53.3, 53.2, 32.9 (d, 1JCP = 139.6 Hz). MS (ESI) Calcd for C11H11F6O3P [M + H]+: 337.04. Found: 337.10. Dimethyl 3,5-Dimethoxybenzyl Phosphonate (4a).

According to general procedure B, reaction between phosphonate 4a and 1H-benzo[d]imidazole-5-carbaldehyde gave 12.0 mg of stilbene 4 (11% yield). E/Z = 12:1. 1H NMR (300 MHz, DMSO-d6) δ 8.13 (s, 1H), 7.70 (s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.42 (dd, J = 8.6, 1.3 Hz, 1H), 7.32 (d, J = 16.4 Hz, 1H), 7.07 (d, J = 16.4 Hz, 1H), 6.72 (d, J = 2.2 Hz, 2H), 6.33 (t, J = 2.2 Hz, 1H), 3.72 (s, 6H). 13C NMR (75 MHz, chloroform-d) δ 160.5, 141.1, 139.1, 132.0, 128.9, 126.9, 120.9, 103.7, 98.9, 54.1. MS (ESI) Calcd for C17H16N2O2 [M + H]+: 281.13. Found: 281.20.

According to general procedure A, 3,5-dimethoxybenzyl bromide was reacted with trimethyl phosphite to afford the title product as a yellow oil in 68% yield (352 mg, 1.35 mmol). TLC (EtOAc), Rf = 0.24. 1H NMR (300 MHz, CDCl3) δ 6.47−6.44 (m, 2H), 6.38−6.34 (m, 1H), 3.69 (d, 3JHP = 10.8 Hz, 6H), 3.67 (s, 3H), 3.10 (d, 2JHP = 21.7 Hz, 2H). 13 C NMR (75 MHz, chloroform-d) δ 160.8 (d, 4JCP = 3.2 Hz), 133.3 (d, 2JCP = 9.0 Hz), 107.9 (d, 3JCP = 6.6 Hz), 99.1 (d, 5JCP = 3.4 Hz), 55.3, 53.0, 52.9, 33.1 (d, 1JCP = 138.2 Hz). MS (ESI) Calcd for C11H17O5P [M + H]+: 261.09. Found: 261.23. L


Research Article

ACS Chemical Neuroscience General Procedure C.

Following general procedure C, the reaction was run using PEG 400 as solvent, and the product was purified with EA/n-heptane/HCOOH (30:70:0.5 to 45:55:0.5) on silica gel to give 16.7 mg of 6 (16% yield). 1 H NMR (CDCl3, 500 MHz) δ 7.48 (d, J = 7.5 Hz, 2H), 7.34 (t, J = 7.5 Hz, 2H), 7.24 (t, J = 7.5 Hz, 1H), 7.08 (d, J = 1.5 Hz, 1H), 7.00 (d, J = 15.5 Hz, 1H), 6.97 (dd, J = 8.0, 2.0 Hz, 1H), 6.94 (d, J = 15.5 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H). MS (ESI) Calcd for C14H12O2: 211.08 [M − H]−. Found: 211.30. (E)-2-Methoxy-4-styrylphenol (7).

Following a method reported from the literature,45 to a 0.5−2.0 mL microwave vial aryl aldehyde (0.5 mmol), aryl acetic acid (0.55 mmol), piperidine (0.75 mmol), N-methyl imidazole (0.75 mmol) and 1 mL PEG 400 or dioxane or THF were added. The mixture was heated under microwave irradiation (MW) at 160 °C for 15−30 min. The reaction mixture was diluted with 2 mL of water, adjusted to a pH value of about 5−6 with 1 N HCl, and extracted with ethyl acetate until no fluorescent substance could be detected in the organic layer according to TLC, monitored with 365 nm UV lamp. The organic layer was concentrated and purified with flash chromatography. If not pure after this procedure, the product was purified further with preparative HPLC to give compounds 5−15 (2−21% yield). General Procedure D.

Following general procedure C, 1.0 mmol scale, THF was used as solvent and reaction was run under MW at 140 °C for 1 h. The product was purified with EA/n-heptane (10:90 to 20:80) on silica gel to give 20.5 mg of 7 (9% yield). 1H NMR (CDCl3, 500 MHz) δ 7.49 (dd, J = 8.0, 1.0 Hz, 2H), 7.35 (t, J = 8.0 Hz, 2H), 7.24 (t, J = 8.0 Hz, 1H), 7.09−7.05 (m, 3H), 6.95 (d, J = 16.0 Hz, 1H), 6.91 (d, J = 8.5 Hz, 1H), 5.65 (s, 1H), 3.96 (s, 3H). MS (ESI) Calcd for C15H14O2: 225.09 [M − H]−. Found: 225.24. (E)-4-(4-Hydroxystyryl)-2-methoxyphenol (8).

Following general procedure C, PEG 400 was used as solvent, and product was purified using DCM with flash chromatography to give 5.6 mg of compound 8 (5% yield). 1H NMR (CD3OD, 500 MHz) δ 7.34 (d, J = 9.0 Hz, 2H), 7.08 (d, J = 1.5 Hz, 1H), 6.95−6.85 (m, 3H), 6.78−6.70 (m, 3H), 3.89 (s, 3H). MS (ESI) Calcd for C15H14O3: 241.09 [M − H]−. Found: 241.25. (E)-4-(2-(1H-Indol-3-yl)vinyl)-2-methoxyphenol (9).

For the synthesis of compounds 16−22, a similar method was used but the amounts of the reagents were different. To a 0.5−2.0 mL microwave vial were added aryl diacetic acid (0.5 mmol), aryl aldehyde (1.05 mmol), piperidine (3.0 mmol), N-methyl imidazole (3.0 mmol), and 1 mL of PEG 400 or dioxane. The mixture was heated under microwave irradiation at 160 °C for 30 min. The reaction mixture was diluted with 2 mL of water, adjusted to pH value of about 5−6 with 1 N HCl, and extracted with ethyl acetate until no fluorescent substance could be detected in the organic layer according to TLC, monitored with 365 nm UV lamp. The organic layer was concentrated and purified with flash chromatography. If not pure after this procedure, the product was purified further with preparative HPLC to give compounds 16−22 (1−42% yield). (E)-4-(4-Hydroxystyryl)benzene-1,2-diol (5).

Following general procedure C, PEG 400 was used as solvent, and the reaction mixture was heated under MW at 160 °C for 15 min. The product was purified with CH2Cl2 on silica gel to give 5.2 mg of compound 9 (4% yield). 1H NMR (CD3OD, 500 MHz) δ 7.92 (d, J = 7.5 Hz, 1H), 7.40−7.34 (m, 2H), 7.19 (d, J = 15.5 Hz, 1H), 7.17−7.05 (m, 3H), 7.01 (d, J = 15.5 Hz, 1H), 6.96 (dd, J = 8.5, 2.0 Hz, 1H), 6.77 (d, J = 7.5 Hz, 1H), 3.91 (s, 3H). 13C NMR (CD3OD, 75 MHz) δ 149.2, 146.6, 138.9, 132.7, 126.9, 125.6, 125.3, 122.8, 121.1, 120.8, 120.6, 120.0, 116.4, 116.0, 112.5, 109.9, 56.4. MS (ESI) Calcd for C17H15NO2: 264.10 [M − H]−. Found: 264.29. (E)-N,N-Dimethyl-4-(2-(naphthalen-1-yl)vinyl)aniline (10).

Following general procedure C, the reaction was run under microwave for 20 min using PEG 400 as solvent and purified with flash chromatography and preparative HPLC to give 8 mg of compound 5 (7% yield). 1H NMR (acetone-d6, 500 MHz) δ 7.38 (d, J = 8.0 Hz, 2H), 7.06 (s, 1H), 6.92−6.86 (m, 3H), 6.84−6.76 (m, 3H). MS (ESI) Calcd for C14H12O3: 227.07 [M − H]−. Found: 227.11. (E)-4-Styrylbenzene-1,2-diol (6).

Following general procedure C, PEG 400 was used as solvent, and the reaction mixture was heated under MW at 160 °C for 20 min. The product was purified with EA/n-heptane (30:70) on silica gel to give 24.4 mg of compound 10 (18% yield). 1H NMR (300 MHz, CDCl3) δ 8.25 (d, J = 8.5 Hz, 1H), 7.86 (dd, J = 8.0, 1.5 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 7.0 Hz, 1H), 7.69 (d, J = 16.0 Hz, 1H), 7.55−7.44 (m, 5H), 7.10 (d, J = 16.0 Hz, 1H), 6.77 (d, J = 8.5 Hz, 2H), 3.01 (s, 6H). MS (ESI) Calcd for C20H19N: 274.16 [M + H]+. Found: 274.30. M


Research Article


(E)-2-methoxy-5-(2-(naphthalen-2-yl)vinyl)phenol (15).

(E)-4-(2-(Naphthalen-1-yl)vinyl)benzene-1,2-diol (11).

Following general procedure C, PEG 400 was used as solvent, and the reaction mixture was heated under MW at 160 °C for 30 min. The product was purified with EA/n-heptane (20:80 to 40:60) on silica gel to give 6.2 mg of compound 15 (4% yield). 1H NMR (300 MHz, acetone-d6) δ 7.94 (br s, 1H), 7.90−7.80 (m, 4H), 7.53−7.40 (m, 2H), 7.29 (d, J = 16.2, 1H), 7.22 (d, J = 16.2 Hz, 1H), 7.19 (d, J = 2.1 Hz, 1H), 7.08 (d, J = 8.4, 2.1 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 3.88 (s, 3H). MS (ESI) Calcd for C19H16O2: 275.11 [M − H]−. Found: 275.31. 4,4′-((1E,1′E)-1,4-Phenylenebis(ethene-2,1-diyl))bis(2-methoxyphenol) (16) and (E)-2-(4-(4-Hydroxy-3-methoxystyryl)phenyl)acetic Acid (17).

Following general procedure C, PEG 400 was used as solvent, and the reaction mixture was heated under MW at 160 °C for 15 min. The product was purified with EA/n-heptane (30:70 to 50:50) on silica gel to give 18.0 mg of compound 11 (14% yield). 1H NMR (300 MHz, CDCl3) δ 8.21−8.16 (m, 1H), 7.87−7−82 (m, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.71−7.65 (m, 2H), 7.53−7.40 (m, 3H), 7.16 (d, J = 1.5 Hz, 1H), 7.03−6.98 (m, 2H), 6.88 (d, J = 8.1 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 144.2, 144.0, 135.2, 133.9, 131.5, 131.4, 131.3, 128.7, 127.8, 126.1, 125.9, 125.8, 124.1, 123.9, 123.5, 120.3, 115.9, 113.6. MS (ESI) Calcd for C18H14O2: 261.09 [M − H]−. Found: 261.27. (E)-2-Methoxy-5-(2-(naphthalen-1-yl)vinyl)phenol (12).

Following general procedure C, PEG 400 was used as solvent, and the reaction mixture was heated under MW at 160 °C for 30 min. The product was purified with EA./n-heptane (20:80 to 40:60) on silica gel to give 3.1 mg of compound 12 (2% yield). 1H NMR (300 MHz, CDCl3) δ 8.22 (dd, J = 6.9, 1.8 Hz, 1H), 7.87 (dd, J = 7.2, 2.4 Hz, 1H), 7.80−7.70 (m, 3H), 7.58−7.44 (m, 3H), 7.27 (d, J = 2.4 Hz, 1H), 7.15− 7.02 (m, 2H), 6.88 (d, J = 8.1 Hz, 1H), 5.63 (s, 1H), 3.94 (s, 3H). 13 C NMR (75 MHz, CDCl3) δ 146.7, 146.0, 135.3, 133.9, 131.7, 131.6, 131.4, 128.7, 127.9, 126.2, 125.9, 125.8, 124.3, 124.0, 123.5, 119.6, 112.1, 110.9, 56.2. MS (ESI) Calcd for C19H16O2: 277.12 [M + H]+. Found: 277.17. (E)-2-Methoxy-4-(2-(naphthalen-1-yl)vinyl)phenol (13).

Following general procedure D, PEG 400 was used as solvent, and the reaction mixture was heated under MW at 160 °C for 30 min The products were purified with EA/n-heptane/HCOOH (30:80:0.5 to 50:50:0.5) on silica gel, followed by further purification with preparative HPLC, to give 1.8 mg of compound 16 (1% yield) and 3.5 mg of compound 17 (2% yield). Compound 16: 1H NMR (300 MHz, acetone-d6) δ 7.53 (s, 4H), 7.26 (d, J = 1.8 Hz, 2H), 7.18 (d, J = 16.2 Hz, 2H), 7.08 (d, J = 16.2 Hz, 2H), 7.06 (dd, J = 8.1, 1.8 Hz, 2H), 6.83 (d, J = 8.1 Hz, 2H), 3.91 (s, 6H). MS (ESI) Calcd for C24H22O4: 373.14 [M − H]−. Found: 373.48. Compound 17: 1H NMR (300 MHz, acetone-d6) δ 7.50 (d, J = 8.1 Hz, 2H), 7.30 (d, J = 8.1 Hz, 2H), 7.25 (d, J = 1.8 Hz, 1H), 7.15 (d, J = 16.2 Hz, 1H), 7.07 (d, J = 16.2 Hz, 1H), 7.04 (dd, J = 8.1, 1.8 Hz, 1H), 6.82 (d, J = 8.1 Hz, 1H), 3.90 (s, 3H), 3.62 (s, 2H). 13C NMR (75 MHz, acetone-d6) δ 172.6, 148.6, 147.6, 137.5, 134.7, 130.6, 129.5, 127.0, 126.4, 121.2, 115.9, 110.2, 109.7, 56.3, 41.0. MS (ESI) Calcd for C17H16O4: 283.10 [M − H]−. Found: 283.26. 4,4′-((1E,1′E)-1,4-phenylenebis(ethene-2,1-diyl))bis(benzene-1,2diol) (18) and (E)-2-(4-(3,4-dihydroxystyryl)phenyl)acetic acid (19).

Following general procedure C, PEG 400 was used as solvent, and the reaction mixture was heated under MW at 160 °C for 15 min. The product was purified with EA/n-heptane (20:80 to 25:75) on silica gel to give 28.4 mg of compound 13 (21% yield). 1H NMR (500 MHz, CDCl3) δ 8.23 (d, J = 7.5 Hz, 1H), 7.87 (dd, J = 8.0, 1.0 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.75−7.66 (m, 2H), 7.56−7.44 (m, 3H), 7.17−7.05 (m, 3H), 6.96 (d, J = 9.0 Hz, 1H), 5.69 (s, 1H), 3.99 (s, 3H). MS (ESI) Calcd for C19H16O2: 275.11 [M − H]−. Found: 275.28. (E)-4-(2-(Naphthalen-2-yl)vinyl)benzene-1,2-diol (14).

Following general procedure C, PEG 400 was used as solvent, and the reaction mixture was heated under MW at 160 °C for 30 min. The product was purified with EA/n-heptane (30:70 to 50:50) on silica gel to give 25.2 mg of compound 14 (20% yield). 1H NMR (300 MHz, CD3OD) δ 7.81−7.68 (m, 5H), 7.46−7.34 (m, 2H), 7.14 (d, J = 16.5, Hz, 1H), 7.08 (s, 1H), 7.06 (d, J = 16.5 Hz, 1H), 6.92 (dd, J = 2.4 and 8.1 Hz, 1H), 6.78 (d, J = 8.4 Hz, 1H). 13C NMR (75 MHz, CD3OD) δ 146.7, 146.5, 136.8, 135.3, 134.2, 131.2, 130.4, 129.1, 128.8, 128.6, 127.2, 126.84, 126.81, 126.5, 124.4, 120.4, 116.5, 114.0. MS (ESI) Calcd for C18H14O2: 261.09 [M − H]−. Found: 261.27.

Following general procedure D, PEG 400 was used as solvent, and the reaction mixture was heated under MW at 160 °C for 30 min. The products were purified with EA/n-heptane/HCOOH (20:80:0.5 to 80:20:0.5) on silica gel, followed by further purification with preparative HPLC to give 3.8 mg of compound 18 (2% yield) and 8.5 mg of compound 19 (6% yield). Compound 18: 1H NMR (300 MHz, CD3OD) δ 7.45 (s, 4H), 7.05− 6.99 (m, 4H), 6.91 (d, J = 16.2 Hz, 2H), 6.88 (dd, J = 8.1, 2.4 Hz, 2H), N


Research Article

ACS Chemical Neuroscience 6.75 (d, J = 8.1 Hz, 2H). 13C NMR (75 MHz, CD3OD) δ 146.6, 146.5, 138.1, 131.2, 129.5, 127.4, 126.5, 120.2, 116.5, 113.9. MS (ESI) Calcd for C22H18O4: 345.11 [M − H]−. Found: 345.37. Compound 19: 1H NMR (300 MHz, CD3OD) δ 7.39 (d, J = 8.1 Hz, 2H), 7.26 (d, J = 8.1 Hz, 2H), 6.99 (d, J = 2.1 Hz, 1H), 6.96 (d, J = 16.8 Hz, 1H), 6.87 (d, J = 16.5 Hz, 1H), 6.84 (dd, J = 8.1, 2.1 Hz, 1H), 6.73 (d, J = 8.1 Hz, 1H), 3.47 (s, 2H). 13C NMR (75 MHz, CD3OD) δ 179.6, 146.5, 146.4, 137.8, 137.1, 131.3, 130.5, 129.1, 127.0, 126.7, 120.1, 116.4, 113.8, 45.6. MS (ESI) Calcd for C16H14O4: 269.08 [M − H]−. Found: 269.22. 4,4′-((1E,1′E)-1,3-Phenylenebis(ethene-2,1-diyl))bis(benzene-1, 2-diol) (20).

Protein Production and Purification. WT TTR expressed in BL21(DE3) cells transformed with the pmm Hα:WT TTR plasmid was isolated and purified as previously described.32 For concentration determination, ε = 77600 M−1 cm−1, based on a 55 kDa molecular mass for native tetrameric TTR at 280 nm, was applied.46 Preparation of TTR Protofibrils. The protein was dialyzed against milli-Q water at 4 °C using a dialysis tube (Spectra/Por, Spectrum Laboratories) with a volume/length of 1 mL/cm and a molecular weight cutoff (MWCO) of 12−14 kDa and concentrated to 5.2 mg/mL using spin-filters (Centriprep, Millipore) with a MWCO of 10 kDa at 4 °C. The sample was centrifuged for 60 s at 18000 rpm (Denville 260D, Denville Scientific Inc.) prior to the initiation of TTR fibrillation process. The fibrillation of 5 mg/mL TTR was induced by adding a stock solution of 1 M NaAc and 2 M NaCl to a final concentration of 50 mM NaAc and 100 mM NaCl (final pH 3.0). The sample was mixed using a vortex for 5 s and then stored at 4 °C. The mature protofibrils used for our experiments were obtained by incubation at these conditions at least 1 month.33 Fluorescence Spectroscopy Measurements on Probes. Fluorescence analysis for each probe upon binding native TTR or protofibrils was performed by mixing 1.8 μM of each molecule from a dimethyl sulfoxide (DMSO) stock with 0.1 mg/mL protein in PBS buffer, pH 3, for native TTR or PF. The samples were aliquoted in 96 well plates, and emission scans were captured in a Tecan Saphire2 microplate reader (Tecan, Männerdorf, Switzerland) at 22 °C, after excitation at 330 nm. Slits were set to 5/5 nm (Ex/Em) and gain to 100. For the 2D excitation/emission scans, we used Ex = 250−350 and Em 370−480 (blue setting) and Ex 330−430 and Em 450−610 (red setting), which we combined using Origin software (Origin Lab Corporation). For the rest of the data analysis, including curve fittings of titrations, we used GraphPad Prism v6.0 (San Diego, CA). To capture the images of stilbene 11 on a UV table, we used a benchtop UV transilluminator, 302 nm (UVP, Upland, CA, USA), and quartz fluorescence cuvettes (Hellma). The different solvents used to measure polarity and viscosity effects were purchased from Sigma-Aldrich Chemical Co. (Sigma-Aldrich, Stockholm, Sweden). Fibril Inhibition Assay. The fibrillation was initiated by mixing 20 μL of 1 mg/mL native TTR (with and without 2 equiv of the compounds) with 80 μL of 10 mM NaAc buffer with 100 mM KCl, 1 mM EDTA, and 1 mM DTT. The pH value of the NaAc buffer was adjusted to yield a final pH 4.4. Aggregate formation (Turbidity) was assessed by optical density at 400 nm measured on a Nanodrop ND-1000 spectrophotometer (path length 1 mm) (Thermo Fisher, USA) after 72 h incubation of the TTR samples at pH 4.4 in 37 °C during stagnant conditions. For calculation of fibril formation inhibition, the turbidity of TTR in the absence of probe was used as a reference of 100% fibril formation. The classification of inhibitor activity was set to good inhibition for 80%.

Following general procedure D, PEG 400 was used as solvent, and the reaction mixture was heated under MW at 160 °C for 30 min. The product was purified with EA/n-heptane/HCOOH (40:60:0.5 to 80:20:0.5) on silica gel, followed by further purification with preparative HPLC to give 3.8 mg of compound 20 (2% yield). 1H NMR (300 MHz, CD3OD) δ 7.59 (br s, 1H), 7.38−7.24 (m, 3H), 7.06 (d, J = 16.5 Hz, 2H), 7.04 (d, J = 1.8 Hz, 2H), 6.94 (d, J = 16.5 Hz, 2H), 6.90 (dd, J = 8.4, 2.1 H, z2H), 6.76 (d, J = 7.5 Hz, 2H). 13C NMR (75 MHz, CD3OD) δ 146.9, 146.8, 139.9, 131.4, 130.4, 130.2, 127.0, 126.0, 125.4, 120.6, 116.7, 114.2. MS (ESI) Calcd for C22H18O4:345.11 [M − H]−. Found: 345.34. (E)-5-(3-(Carboxymethyl)styryl)-2-hydroxybenzoic acid (21) and 5,5′-((1E,1′E)-1,3-Phenylenebis(ethene-2,1-diyl))bis(2-hydroxybenzoic acid) (22).

Following general procedure D, dioxane was used as solvent, and the reaction mixture, at 0.25 mmol scale and with 4.0 mmol of piperidine and 4.0 mmol of N-methylimidazole, was heated under MW at 160 °C for 30 min. The products were purified with EA/n-heptane/HCOOH (40:60:0.5 to 100:0:0.5), then with EA/MO/HCOOH (2:98:0.5 to 8:92:0.5) on silica gel, followed by further purification with crystallization and preparative HPLC to give 20.7 mg of compound 21 (28% yield) and 25.4 mg of compound 22 (25% yield). Compound 21: 1H NMR (300 MHz, CD3OD) δ 8.03 (d, J = 2.1 Hz, 1H), 7.51 (dd, J = 2.1 and 8.1 Hz, 1H), 7.47 (s, 1H), 7.35 (d, J = 7.5 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 7.16 (d, J = 8.1 Hz, 1H), 7.11 (d, J = 16.5 Hz, 1H), 6.99 (d, J = 16.5 Hz, 1H), 6.80 (d, J = 8.4 Hz, 1H) 3.53 (s, 2H). 13C NMR (75 MHz, CD3OD) δ 178.5, 176.1, 162.5, 139.4, 138.8, 131.8, 129.9, 129.5, 129.4, 129.1, 129.0, 128.1, 127.0, 125.2, 120.2, 117.6, 45.2. MS (ESI) Calcd for C17H14O5: 297.08 [M − H]−. Found: 297.14. Compound 22: 1H NMR (300 MHz, CD3OD) δ 8.0 (d, J = 2.4 Hz, 2H), 7.87−7.80 (m, 3H), 7.46 (d, J = 8.4 Hz, 2H), 7.38−7.28 (m, 3H), 7.14 (d, J = 17.1 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 171.7, 160.7, 137.5, 133.1, 129.0, 128.5, 127.6, 126.7, 125.6, 123.8, 117.7, 113.2. MS (ESI) Calcd for C24H18O6:401.10 [M − H]−. Found: 401.39.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.6b00062. Solvent properties (Table S1) and fluorescence excitation−emission plots of SB 11 bound to native TTR and TTR protofibrils (Figure S1) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone +4613285690. Funding

The work was supported by Göran Gustafsson’s Foundation (PH), The Swedish Research Council (PH), The Linköping center for systemic neuroscience, LiU-Neuro, (XW), and Sven and Lilly Lawski’s foundation (ME). O


Research Article

ACS Chemical Neuroscience Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Mikael Lindgren and Bengt-Harald Jonsson for discussions of the work and Sofie Nyström for assistance with Figures. We thank Jeffery W. Kelly and David Wemmer for the pmm Hα:WT TTR plasmid.

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