Novel Benzothiazole Derivatives as Fluorescent Probes for Detection

May 3, 2017 - Hiroyuki Watanabe, Masahiro Ono , Taisuke Ariyoshi, Rikako Katayanagi, and Hideo Saji. Department of Patho-Functional Bioanalysis, Gradu...
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Novel Benzothiazole Derivatives as Fluorescent Probes for Detection of β‑Amyloid and α‑Synuclein Aggregates Hiroyuki Watanabe, Masahiro Ono,* Taisuke Ariyoshi, Rikako Katayanagi, and Hideo Saji Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan S Supporting Information *

ABSTRACT: Deposits of β-amyloid (Aβ) and α-synuclein (α-syn) are the hallmark of Alzheimer’s disease (AD) and Parkinson’s disease (PD), respectively. The detection of these protein aggregates with fluorescent probes is particularly of interest for preclinical studies using fluorescence microscopy on human brain tissue. In this study, we newly designed and synthesized three push−pull benzothiazole (PP-BTA) derivatives as fluorescent probes for detection of Aβ and α-syn aggregates. Fluorescence intensity of all PP-BTA derivatives significantly increased upon binding to Aβ(1−42) and α-syn aggregates in solution. In in vitro saturation binding assays, PP-BTA derivatives demonstrated affinity for both Aβ(1−42) (Kd = 40−148 nM) and α-syn (Kd = 48−353 nM) aggregates. In particular, PP-BTA-4 clearly stained senile plaques composed of Aβ aggregates in the AD brain section. Moreover, it also labeled Lewy bodies composed of α-syn aggregates in the PD brain section. These results suggest that PP-BTA-4 may serve as a promising fluorescent probe for the detection of Aβ and α-syn aggregates. KEYWORDS: Alzheimer’s disease, Parkinson’s disease, β-amyloid, α-synuclein, fluorescent probe he abnormal association of misfolded proteins, such as βamyloid (Aβ), α-synuclein (α-syn), prion, and polyglutamine, usually leads to the formation of aggregates.1 These aggregates are linked to many neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), prion disease, and Huntington’s disease. Furthermore, the increase in the number of patients with AD and PD has become a social problem. The deposition of senile plaques is one of the neuropathological hallmarks in AD brains. Aβ(1−40) and Aβ(1−42) peptides, which are major components of senile plaques, are derived from the amyloid precursor protein after cleavage by βand γ-secretase.2 Thereafter, fibrillation and aggregation of these peptides occurs. As this event is considered to be the initial and specific neurodegenerative change in AD brains, the detection of Aβ aggregates may contribute to the diagnosis of AD.3 In the past decades, many positron emission tomography (PET) and single photon emission computed tomography (SPECT) probes for imaging Aβ aggregates have been developed. 4,5 Compared with nuclear medical imaging techniques, the optical imaging technique is easy to use. Therefore, more recently, some fluorescence probes targeting Aβ aggregates have also been reported.6 α-Syn is the major fibrillary component of Lewy bodies, which is the hallmark of PD and dementia with Lewy bodies (DLB). Therefore, the neuropathological diagnosis of these diseases is based on the detection and quantification of Lewy

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bodies in the brain section.7,8 In addition, the deposition of Lewy bodies occurs before the onset of clinical symptoms.9 Therefore, α-syn aggregates are regarded as one of the attractive targets for diagnosis of PD and DLB. However, useful PET, SPECT, and fluorescent probes for detection of αsyn aggregates have not been reported.10,11 The detection of Aβ and α-syn aggregates with fluorescent probes is particularly of interest for preclinical studies using fluorescence microscopy on human brain tissue. In general, an appropriate fluorescent probe for staining Aβ and α-syn aggregates should have the following properties: (1) emission wavelength above 650 nm to minimize background fluorescence from brain tissues, (2) sufficient binding affinity with Aβ and α-syn aggregates, and (3) upon binding to Aβ or α-syn aggregates, a significant change in fluorescent properties should be observed.12,13 Thioflavin-S, Thioflavin-T, and Congo Red are commonly used to stain amyloid aggregates including Aβ and α-syn. Polythiophene derivatives are also reported as dyes that can stain several amyloid aggregates.14−16 However, these fluorescent dyes exhibit emission wavelengths under 650 nm, suggesting that their fluorescence may be affected by intrinsic fluorescence in brain tissues. More recently, near-infrared fluorescent probes, such as CRANNAD-2, BBTOM-3, and Received: December 22, 2016 Accepted: April 28, 2017

A

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Figure 1. Chemical structures of PP-BTA derivatives.

Scheme 1. Synthetic Route of PP-BTA Derivativesa

a

Reagents: (a) (1,3-dioxan-2-yl-methyl)triphenyl-phosphoniumbromide, 18-crown-6, NaH, THF; (b) malononitrile, pyridine, 2-propanol.

(Figure 1) and evaluated their utility for detection of Aβ and αsyn aggregates.

DTA-2, have been reported.13,17,18 These probes demonstrated high binding affinity for Aβ aggregates, but almost all of the probes have not been evaluated for affinity for other protein aggregates including α-syn aggregates. Previously, we reported the fluorescent probe (PP-BTA-1), which was designed based on a push−pull benzothiazole derivative.19 The emission wavelength of PP-BTA-1 was under the near-infrared region (634 nm), but it demonstrated high binding affinity for Aβ(1−42) aggregates, and clearly stained senile plaques in human brain section. This means that it may be an attractive scaffold for development of novel near-infrared fluorescent probes to detect amyloid aggregates. In general, the emission wavelength of the push−pull type dye could be lengthened by increasing the number of conjugated double bonds.18,20 In addition, some reports suggested that the molecular length affects the binding affinity for amyloid aggregates.11,20,21 Taken together, we newly designed and synthesized three benzothiazole derivatives (PP-BTA-3, PPBTA-4, and PP-BTA-5) with different conjugated double bonds

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RESULTS AND DISCUSSION Synthesis of PP-BTA Derivatives. The target compounds (PP-BTA-3, PP-BTA-4, and PP-BTA-5) were synthesized as shown in Scheme 1. Compound 1 was synthesized according to methods reported previously.19,22 Compounds 2, 4, and 6 were prepared by the Wittig reaction from 1, 2, and 4, respectively (42−74% yields). Then, 3 (PP-BTA-3), 5 (PP-BTA-4), and 7 (PP-BTA-5) were prepared by condensation of the corresponding aldehydes with malononitrile in 2-propanol (29−51% yields). Fluorescence Characterization. First, we evaluated the fluorescence properties of PP-BTA derivatives (Table 1, Table S1, Figures S1 and S2). The excitation/emission wavelengths in CHCl3 of PP-BTA-3, PP-BTA-4, and PP-BTA-5 were 556/671, 559/727, and 549/770 nm, respectively. The quantum yields were 5.3% (PP-BTA-3), 2.7% (PP-BTA-4), and 0.6% (PP-BTA5). The emission wavelengths lengthened by the extension of B

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ACS Chemical Neuroscience Table 1. Fluorescence Characterization of PP-BTA Derivativesa PP-BTA-3 PP-BTA-4 PP-BTA-5

Abs (nm)

Ex (nm)

Em (nm)

quantum yield (%)

557 559 564

556 559 549

671 727 770

5.3 2.7 0.6

Absorbance, fluorescence excitation and emission, and quantum yield of PP-BTA derivatives were determined with 10 μM of the compounds in CHCl3. a

double bonds, and were longer than the amyloid staining agents, such as Thioflavin-S, Congo Red, or polythiophene derivatives. As the background autofluorescence was low in the near-infrared region (650−900 nm), the emission wavelength of all PP-BTA derivatives should be suitable for detection of Aβ and α-syn aggregates in brain sections. Fluorescence Measurements Using Aβ(1−42) and αSyn Aggregates. The significant increase in intensity of fluorescence emission wavelength upon binding to amyloid aggregates is one of the essential properties of useful fluorescent probes for detection of Aβ and α-syn aggregates.12,13 Therefore, we compared the fluorescence intensity of PP-BTA derivatives in an aqueous solution to that in the presence of Aβ(1−42) or α-syn aggregates. The fluorescence intensity of PP-BTA derivatives significantly increased with the concentration of Aβ(1−42) aggregates, while it did not change markedly in the solution without Aβ(1−42) aggregates (Figures 2 and S3). This increase in fluorescence of PP-BTA derivatives was also observed in the presence of α-syn aggregates, whereas their fluorescence intensity was very weak in an aqueous solution (Figures 3 and S4). Conversely, we found very low fluorescence intensity during the incubation with Crystallin, indicating that there is little interaction between all PP-BTA derivatives and crystalline (Figures S5 and S6). These fluorescence properties suggested that all PP-BTA derivatives may specifically bind to Aβ(1−42) and α-syn aggregates. These fluorescence measurements also revealed that the emission wavelength of PP-BTA derivatives in the presence of α-syn aggregates (682−782 nm) was lengthened in comparison with that in the presence of Aβ(1−42) aggregates (656−714 nm). Although similar results were reported in the in vitro study with synthetic Aβ(1−42) and α-syn aggregates,23 the precise mechanism of emission wavelength shifting is unclear. This phenomenon may be involved in the difference in binding mode of PP-BTA derivatives between Aβ(1−42) and α-syn aggregates. Binding Affinity for Aβ(1−42) and α-Syn Aggregates in Vitro. To quantitatively evaluate the binding affinity for Aβ and α-syn aggregates, we performed saturation binding assays and measured the apparent binding dissociation constant (Kd). Table 2 summarizes the Kd values of PP-BTA derivatives for Aβ(1−42) and α-syn aggregates. The Kd values of PP-BTA-3, PP-BTA-4, and PP-BTA-5 for Aβ(1−42) aggregates were 148, 40, and 57 nM, respectively. These values were lower or comparable to that of fluorescent probes for imaging Aβ aggregates reported previously,12,24 indicating that PP-BTA derivatives have enough affinity for detection of Aβ plaques in AD brain sections. All PP-BTA derivatives also demonstrated the high binding affinity for α-syn aggregates ranging from 48 to 353 nM. As shown in some previous reports, the Kd value correlated poorly with the fluorescence intensity in the presence of amyloid aggregates.12,25 This result suggested that the molecular length of PP-BTA derivatives affects the binding

Figure 2. Fluorescence intensity of PP-BTA-3 (A), PP-BTA-4 (B), and PP-BTA-5 (C) upon interaction with Aβ(1−42) aggregates.

affinity for both Aβ and α-syn aggregates, similarly to some previous reports.11 The Kd values of Thioflavin T for Aβ and αsyn aggregates are 430 and 1012 nM, respectively. As the binding affinity of all PP-BTA derivatives was higher than Thioflavin T, these compounds may be excellent dyes for staining senile plaques and lewy bodies. As PP-BTA-4 exhibited the highest affinity for both Aβ and α-syn aggregates among the PP-BTA derivatives, we conducted further characterization of PP-BTA-4. Neuropathologic Staining of AD and PD Brain Sections. To confirm the usefulness of PP-BTA-4 for detection of senile plaques composed of Aβ aggregates and Lewy bodies composed of α-syn aggregates in the human brain sections, we carried out in vitro neuropathological fluorescence staining using AD and PD brain sections. In the AD brain C

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(Figure 5). These results suggested that PP-BTA-4 could detect both senile plaques and Lewy bodies in human brain sections. As PP-BTA-4 has no charge, it has the potential to penetrate the blood-brain barrier and detect Aβ and α-syn aggregates in the brain.

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CONCLUSIONS

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METHODS

We designed and synthesized three novel PP-BTA derivatives for detection of Aβ and α-syn aggregates. The maximum emission wavelength of PP-BTA-4 and PP-BTA-5 fell within the near-infrared region. Upon binding to Aβ(1−42) and α-syn aggregates, fluorescence intensity of all PP-BTA derivatives significantly increased. In vitro saturation binding assays, PPBTA derivatives demonstrated high binding affinity with Aβ(1− 42) and α-syn aggregates. Furthermore, in in vitro fluorescence staining with AD and PD brain sections, PP-BTA-4, which had the highest binding affinity for these aggregates among the PPBTA derivatives, clearly stained senile plaques and Lewy bodies, respectively. These results indicate that PP-BTA-4 may be a promising fluorescent probe for detection of Aβ and α-syn aggregates in human brain sections.

General. All reagents were obtained commercially and used without further purification unless otherwise indicated. W-Prep 2XY (Yamazen Corporation, Osaka, Japan) was used for silica gel column chromatography on a Hi Flash silica gel column (40 μm, 60 Å, Yamazen Corporation). 1H NMR and 13C NMR spectra were obtained on a JEOL JNM-LM400 spectrometer with TMS as an internal standard. Coupling constants are reported in hertz. Multiplicity was defined by s (singlet), d (doublet), and m (multiplet). Mass spectra were obtained on a JEOL JMS-GC-mate mass spectrometer. (E)-3-(6-(Dimethylamino)benzo[d]thiazol-2-yl)acrylaldehyde (2). To a stirred solution of 1 (78.9 mg, 0.38 mmol), 18-crown-6 (5.0 mg) and (1,3-dioxolan-2-ylmethyl)-triphenylphosphonium bromide in THF (23 mL) under Ar, NaH (38.1 mg, 1.6 mmol) were added in one portion. After 1 h stirring at room temperature, the reaction mixture was quenched with water and extracted with EtOAc. After evaporation of EtOAc, the residue was dissolved in a mixture of THF and 10% oxalic acid and then stirred for 2 h at room temperature. After the mixture was turned basic with an aqueous saturated solution of NaHCO3, the organic solvent was evaporated on a rotary vacuum evaporator and extracted with EtOAc. The combined EtOAc extracts were washed with a saturated aqueous solution of NaCl and dried over Na2SO4. The solvent was removed on a rotary vacuum evaporator, and the residue was purified by silica gel chromatography (CHCl3/MeOH = 49:1) to give 2 (36.7 mg, 41.6%). 1H NMR (400 MHz, CDCl3) δ 9.76 (d, J = 8 Hz, 1H), 7.91 (d, J = 9.2 Hz, 1H), 7.68 (d, J = 16 Hz, 1H), 7.04−6.97(m, 2H), 6.82−6.76 (m, 1H), 3.09 (s, 6H). (E)-2-(3-(6-(Dimethylamino)benzo[d]thiazol-2-yl)allylidene)malononitrile (3). A solution of 2 (25.0 mg, 0.11 mmol), malononitrile (10.9 mg, 0.165 mmol), and pyridine (0.10 mL) in 2propanol (10 mL) was stirred and refluxed for 30 min. The mixture was poured into H2O and extracted with EtOAc. The combined extracts were dried over Na2SO4 and the solvent was removed under vacuum. The residue was purified by silica gel chromatography (hexane/EtOAc = 1:1) to give 3 (15.6 mg, 50.6%). 1H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 9.2 Hz, 1H), 7.59 (d, J = 12 Hz, 1H), 7.47 (d, J = 14.8 Hz, 1H), 7.37−7.34 (m, 1H), 7.06−7.01 (m, 2H), 3.12 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 158.1, 157.0, 150.2, 146.3, 141.5, 139.4, 125.7, 125.0, 114.5, 113.5, 111.6, 101.5, 84.0, 40.7, 29.7. HRMS (EI): m/z calcd for C15H12N4S 280.0782; found 280.0775. (2E,4E)-5-(6-(Dimethylamino)benzo[d]thiazol-2-yl)penta-2,4-dienal (4). The same reaction as described above to prepare 2 was used and 100.4 mg of 4 was obtained in a 42.3% yield from 2. 1H NMR (400 MHz, CDCl3) δ 9.66 (d, J = 8 Hz, 1H), 7.85 (d, J = 9.2 Hz, 1H),

Figure 3. Fluorescence intensity of PP-BTA-3 (A), PP-BTA-4 (B), and PP-BTA-5 (C) upon interaction with α-syn aggregates.

Table 2. Apparent Binding Constant (Kd) of PP-BTA-3, PPBTA-4, PP-BTA-5, and Thioflavin-T Kd (nM)a compounds PP-BTA-3 PP-BTA-4 PP-BTA-5 Thioflavin-T

α-Syn

Aβ 148.0 40.1 57.3 430.4

± ± ± ±

18.1 3.9 8.7 33.5

352.7 48.0 91.8 1012

± ± ± ±

4.8 0.6 10.5 43.1

Values are the mean ± standard error of the mean for 3−9 independent experiments. a

section, many fluorescent spots were observed (Figure 4A). These fluorescent spots corresponded well with the results of immunohistochemical staining with an antibody against Aβ(1− 42) (Figure 4B). In the PD brain section, PP-BTA-4 clearly stained some regions that were the positive regions of immunohistochemical staining with an antibody against α-syn D

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Figure 4. Neuropathological staining of PP-BTA-4 in the AD brain section (A). Labeled plaques were confirmed by staining of the adjacent sections with anti-Aβ antibody (B).

Figure 5. Neuropathological staining of PP-BTA-4 in the PD brain section (A). Labeled plaques were confirmed by staining of the adjacent sections with anti-α-syn antibody (B). by incubating recombinant α-syn monomer (1.67 mg/mL in 20 mM Tris−HCl, 100 mM NaCl, pH 7.48) at 37 °C for 144 h with shaking at 1000 rpm. A mixture (10% EtOH) containing PP-BTA derivatives (10 μM) and Aβ(1−42) or α-syn aggregates (0.55, 1.1, and 2.2 μM) was incubated at room temperature for 30 min. After incubation, fluorescence emission spectra were collected (Infinite M200PRO, TECAN, Männedorf, Switzerland). Measurement of the Constant for Binding of Aβ and α-Syn Aggregates. A mixture (100 μL of 10% EtOH) containing PP-BTA3, PP-BTA-4, PP-BTA-5, and Thioflavin-T (final concentration 0− 3.75 μM) and Aβ(1−42) or α-syn aggregates (final concentration 2.2 μM) was incubated at room temperature for 30 min. Fluorescence intensity was recorded (Infinite M200PRO), and the Kd binding curve was generated with Prism 4.0 software (Graphpad Software, San Diego, CA). Fluorescence Staining of an AD Brain Section. Experiments involving human subjects were performed in accordance with relevant guidelines and regulations, and were approved by the ethics committee of National Cerebral and Cardiovascular Center (M25-107-2). Informed consent was secured from all subjects in this study. Postmortem brain tissue from an autopsy-confirmed case of AD (male, 76 years old) was obtained from the National Cerebral and Cardiovascular Center. The 6 μm thick serial section of a paraffinembedded block was used for staining. The section was subjected to two 15 min incubations in xylene, two 1 min incubations in 100% EtOH, one 1 min incubation in 90% EtOH, and one 1 min incubation in 70% EtOH to completely deparaffinize, followed by two 2.5 min washes in water. The section was incubated with PP-BTA-4 (50 μM, 50% EtOH) for 10 min. After two 1 min incubations in 50% EtOH, the section was observed with a fluorescence microscope (Eclipse 80i, Nikon, Tokyo, Japan) equipped with a Cy5 filter set. Immunohistochemical Staining of Aβ in a Human AD Brain Section. We conducted immunohistopathological staining according to a method reported previously.29 After two 5 min incubations in PBS-Tween 20, the section used in the fluorescence staining study was incubated at room temperature with an Aβ(1−42) (BC05, Wako) primary antibody for 1 h. After three 5 min incubations in PBS-Tween 20, it was incubated with biotinylated goat antimouse IgG (Histofine Simple Stain Mouse MAX-PO (MULTI), Nichirei Biosciences inc.) at room temperature for 30 min. After three 3 min incubations in PBS-

7.25−7.05 (m, 4H), 6.96 (dd, J = 9.2, 2.4 Hz, 1H), 6.38−6.33 (m, 1H), 3.07 (s, 6H). 2-((2E,4E)-5-(6-(Dimethylamino)benzo[d]thiazol-2-yl)penta-2,4dien-1-ylidene)malononitrile (5). The same reaction as described above to prepare 3 was used, and 7.9 mg of 5 was obtained in a 28.5% yield from 4. 1H NMR (400 MHz, CDCl3) δ 7.85 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 11.6 Hz, 1H), 7.22 (d, J = 14.8 Hz, 1H),7.13−7.02 (m, 3H), 6.97 (dd, J = 9.2, 2.4 Hz, 1H), 6.91−6.85 (m, 1H), 3.09 (s, 6H). 13 C NMR (100 MHz, CDCl3) δ 158.9, 158.5, 149.7, 148.1, 146.1, 138.2, 136.3, 131.5, 127.8, 124.2, 114.0, 113.6, 111.7, 101.9, 83.0, 40.8, 29.7. HRMS (EI): m/z calcd for C17H14N4S 306.0939; found 306.0930. (2E,4E,6E)-7-(6-(Dimethylamino)benzo[d]thiazol-2-yl)hepta2,4,6-trienal (6). The same reaction as described above to prepare 2 was used, and 75.8 mg of 6 was obtained in a 74.0% yield from 4. 1H NMR (400 MHz, CDCl3) δ 10.21−9.58 (m, 1H), 7.81 (d, J = 9.2 Hz, 1H), 7.43−6.6 (m, 7H), 6.24−5.91 (m, 1H), 3.04 (s, 6H). 2-((2E,4E,6E)-7-(6-(Dimethylamino)benzo[d]thiazol-2-yl)hepta2,4,6-trien-1-ylidene)malononitrile (7). The same reaction as described above to prepare 3 was used, and 38.1 mg of 7 was obtained in a 45.1% yield from 5. 1H NMR (400 MHz, CDCl3) δ 7.82 (d, J = 9.6 Hz, 1H), 7.45 (d, J = 11.6 Hz, 1H), 7.1−6.93 (m, 5H), 6.89−6.75 (m, 2H), 6.67−6.6 (m, 1H), 3.07 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 160.0, 158.8, 149.4, 149.0, 146.0, 143.0, 137.7, 133.2, 133.1, 132.1, 127.0, 123.7, 113.8, 113.7, 111.9, 106.8, 102.1, 82.1, 40.8, 29.7. HRMS (EI): m/z calcd for C19H16N4S 332.1096; found 332.1090 Fluorescence Characterization. PP-BTA-3, PP-BTA-4, and PPBTA-5 were dissolved in chloroform at 10 μM. Absorption wavelengths, fluorescence excitation and emission wavelengths, and quantum yields were determined with 10 μM of the compounds in CHCl3 (UV-1800, SHIMADZU Corp, Kyoto, Japan or RF-6000, SHIMADZU Corp). Rhodamine B was taken as a reference for determining quantum yields. Fluorescence Measurement Using Aβ(1−42) and α-Syn Aggregates. Solid forms of Aβ and α-syn were purchased from the Peptides Institute, Inc. (Osaka, Japan) and rPeptide (Bogart, GA), respectively. Aggregation of Aβ(1−42) was carried out by gently dissolving the Aβ(1−42) peptide (0.25 mg/mL) in phosphate buffered saline (PBS) (pH 7.4).26−28 The solution was incubated at 37 °C for 42 h with gentle and constant shaking. α-Syn aggregates were prepared E

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ACS Chemical Neuroscience Tween 20 and one 5 min incubation in TBS, the section was incubated with DAB as a chromogen for 1 min. After washing with water, the section was observed under a microscope (FSX100, Olympus Corp). Fluorescence staining of a PD Brain Section. Experiments involving a human subject were performed in accordance with relevant guidelines and regulations, and were approved by the ethics committee of Osaka Saiseikai Nakatsu Hospital. Informed consent was obtained from all subjects in this study. Postmortem brain tissue from an autopsy-confirmed case of PD (female, 79 years old) was obtained from Osaka Saiseikai Nakatsu Hospital. The 8 μm thick serial section of a paraffin-embedded block was used for staining. After deparaffinization of the section according to the same method as described in the Fluorescence Staining of an AD Brain Section subsection, the section was incubated with PP-BTA-4 (50 μM, 50% EtOH) for 10 min. After two 1 min incubations in 50% EtOH, the section was observed with a fluorescence microscope (FSX100) equipped with a U-MWIG3 filter set. Immunohistochemical Staining of α-Syn in a Human PD Brain Section. We conducted immunohistopathological staining according to a method reported previously.11 After fluorescence staining, the section was activated by 90% formic acid and incubated with anti-phosphorylated α-syn primary antibody (pSyn#64) at 4 °C for 2 h. After three 5 min incubations in PBS-Tween 20, it was incubated with a fluorescent secondary antibody labeled with Alexa Fluor 647 at 4 °C for 1 h. After three 5 min incubations in PBS-Tween 20, the section was observed with a fluorescence microscope (FSX100) equipped with a U-DM-CY5-3 filter set.

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Policy (CSTP), JSPS KAKENHI Grant Number 15K19785, Takeda Science Foundation, and the Mochida Memorial Foundation for Medical Pharmaceutical Research. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Masafumi Ihara (National Cerebral and Cardiovascular Center) and Akihiko Ozaki (Osaka Saiseikai Nakatsu Hospital) for providing brain samples of AD and PD cases, and Dr. Hiroyuki Kimura (Kyoto Pharmaceutical University) for measuring the high resolution mass of PPBTA derivatives.

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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.6b00450. Fluorescence characterization of PP-BTA derivatives; absorption, excitation, and emission spectra of PP-BTA derivatives in CHCl3; approximate curves between Aβ(1−42) concentration and fluorescence intensity of PP-BTA derivatives; approximate curves between α-syn concentration and fluorescence intensity of PP-BTA derivatives; fluorescence intensity of PP-BTA derivatives in the presence of Aβ(1−42) aggregates or crystalline; fluorescence intensity of PP-BTA derivatives in the presence of α-syn aggregates or crystalline; saturation curves of PP-BTA derivatives for Aβ aggregates and αsyn aggregates; 1H and 13C NMR spectra of PP-BTA derivatives (PDF)

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REFERENCES

(1) Invernizzi, G., Papaleo, E., Sabate, R., and Ventura, S. (2012) Protein aggregation: mechanisms and functional consequences. Int. J. Biochedm. Cell Biol. 44, 1541−1554. (2) Holtzman, D. M., Morris, J. C., and Goate, A. M. (2011) Alzheimer’s disease: the challenge of the second century. Sci. Transl. Med. 3, 77sr1. (3) Jack, C. R., Jr., Knopman, D. S., Jagust, W. J., Petersen, R. C., Weiner, M. W., Aisen, P. S., Shaw, L. M., Vemuri, P., Wiste, H. J., Weigand, S. D., Lesnick, T. G., Pankratz, V. S., Donohue, M. C., and Trojanowski, J. Q. (2013) Tracking pathophysiological processes in Alzheimer’s disease: an updated hypothetical model of dynamic biomarkers. Lancet Neurol. 12, 207−216. (4) Cui, M. (2014) Past and recent progress of molecular imaging probes for β-amyloid plaques in the brain. Curr. Med. Chem. 21, 82− 112. (5) Ono, M., and Saji, H. (2015) Recent advances in molecular imaging probes for β-amyloid plaques. MedChemComm 6, 391−402. (6) Staderini, M., Martin, M. A., Bolognesi, M. L., and Menendez, J. C. (2015) Imaging of β-amyloid plaques by near infrared fluorescent tracers: a new frontier for chemical neuroscience. Chem. Soc. Rev. 44, 1807−1819. (7) Schulz-Schaeffer, W. J. (2010) The synaptic pathology of αsynuclein aggregation in dementia with Lewy bodies, Parkinson’s disease and Parkinson’s disease dementia. Acta Neuropathol. 120, 131− 143. (8) Beach, T. G., Adler, C. H., Lue, L., Sue, L. I., Bachalakuri, J., Henry-Watson, J., Sasse, J., Boyer, S., Shirohi, S., Brooks, R., Eschbacher, J., White, C. L., 3rd, Akiyama, H., Caviness, J., Shill, H. A., Connor, D. J., Sabbagh, M. N., and Walker, D. G. (2009) Unified staging system for Lewy body disorders: correlation with nigrostriatal degeneration, cognitive impairment and motor dysfunction. Acta Neuropathol. 117, 613−634. (9) Vekrellis, K., Xilouri, M., Emmanouilidou, E., Rideout, H. J., and Stefanis, L. (2011) Pathological roles of α-synuclein in neurological disorders. Lancet Neurol. 10, 1015−1025. (10) Bagchi, D. P., Yu, L., Perlmutter, J. S., Xu, J., Mach, R. H., Tu, Z., and Kotzbauer, P. T. (2013) Binding of the radioligand SIL23 to αsynuclein fibrils in Parkinson disease brain tissue establishes feasibility and screening approaches for developing a Parkinson disease imaging agent. PLoS One 8, e55031. (11) Ono, M., Doi, Y., Watanabe, H., Ihara, M., Ozaki, A., and Saji, H. (2016) Structure−activity relationships of radioiodinated diphenyl derivatives with different conjugated double bonds as ligands for αsynuclein aggregates. RSC Adv. 6, 44305−44312. (12) Chang, W. M., Dakanali, M., Capule, C. C., Sigurdson, C. J., Yang, J., and Theodorakis, E. A. (2011) ANCA: A Family of Fluorescent Probes that Bind and Stain Amyloid Plaques in Human Tissue. ACS Chem. Neurosci. 2, 249−255. (13) Ran, C., Xu, X., Raymond, S. B., Ferrara, B. J., Neal, K., Bacskai, B. J., Medarova, Z., and Moore, A. (2009) Design, synthesis, and testing of difluoroboron-derivatized curcumins as near-infrared probes

AUTHOR INFORMATION

Corresponding Author

*Phone +81-75-753-4608. Fax: +81-75-753-4568. E-mail: [email protected]. ORCID

Masahiro Ono: 0000-0002-2497-039X Hideo Saji: 0000-0002-3077-9321 Author Contributions

H.W., M.O., and H.S. designed the study. H.W., T.A., and R.K. carried out the experiments. H.W., M.O., T.A., and R.K. analyzed the data. H.W. and M.O. wrote the paper. All authors discussed the results and reviewed the manuscript. Funding

The study was supported by a grant from the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for Next Generation World-Leading Researchers (LS60),” initiated by the Council for Science and Technology F

DOI: 10.1021/acschemneuro.6b00450 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Letter

ACS Chemical Neuroscience for in vivo detection of amyloid-β deposits. J. Am. Chem. Soc. 131, 15257−15261. (14) Aslund, A., Sigurdson, C. J., Klingstedt, T., Grathwohl, S., Bolmont, T., Dickstein, D. L., Glimsdal, E., Prokop, S., Lindgren, M., Konradsson, P., Holtzman, D. M., Hof, P. R., Heppner, F. L., Gandy, S., Jucker, M., Aguzzi, A., Hammarstrom, P., and Nilsson, K. P. (2009) Novel pentameric thiophene derivatives for in vitro and in vivo optical imaging of a plethora of protein aggregates in cerebral amyloidoses. ACS Chem. Biol. 4, 673−684. (15) Klingstedt, T., Blechschmidt, C., Nogalska, A., Prokop, S., Haggqvist, B., Danielsson, O., Engel, W. K., Askanas, V., Heppner, F. L., and Nilsson, K. P. (2013) Luminescent conjugated oligothiophenes for sensitive fluorescent assignment of protein inclusion bodies. ChemBioChem 14, 607−616. (16) Magnusson, K., Simon, R., Sjolander, D., Sigurdson, C. J., Hammarstrom, P., and Nilsson, K. P. (2014) Multimodal fluorescence microscopy of prion strain specific PrP deposits stained by thiophenebased amyloid ligands. Prion 8, 319−329. (17) Zhou, K., Fu, H., Feng, L., Cui, M., Dai, J., and Liu, B. (2015) The synthesis and evaluation of near-infrared probes with barbituric acid acceptors for in vivo detection of amyloid plaques. Chem. Commun. (Cambridge, U. K.) 51, 11665−11668. (18) Watanabe, H., Ono, M., and Saji, H. (2015) In vivo fluorescence imaging of β-amyloid plaques with push-pull dimethylaminothiophene derivatives. Chem. Commun. (Cambridge, U. K.) 51, 17124−17127. (19) Ono, M., Hayashi, S., Kimura, H., Kawashima, H., Nakayama, M., and Saji, H. (2009) Push-pull benzothiazole derivatives as probes for detecting β-amyloid plaques in Alzheimer’s brains. Bioorg. Med. Chem. 17, 7002−7007. (20) Cui, M., Ono, M., Watanabe, H., Kimura, H., Liu, B., and Saji, H. (2014) Smart Near-Infrared Fluorescence Probes with DonorAcceptor Structure for in Vivo Detection of β-Amyloid Deposits. J. Am. Chem. Soc. 136, 3388−3394. (21) Maruyama, M., Shimada, H., Suhara, T., Shinotoh, H., Ji, B., Maeda, J., Zhang, M. R., Trojanowski, J. Q., Lee, V. M., Ono, M., Masamoto, K., Takano, H., Sahara, N., Iwata, N., Okamura, N., Furumoto, S., Kudo, Y., Chang, Q., Saido, T. C., Takashima, A., Lewis, J., Jang, M. K., Aoki, I., Ito, H., and Higuchi, M. (2013) Imaging of tau pathology in a tauopathy mouse model and in Alzheimer patients compared to normal controls. Neuron 79, 1094−1108. (22) Hrobarik, P., Sigmundova, I., and Zahradnik, P. (2005) Preparation of novel push-pull benzothiazole derivatives with reverse polarity: Compounds with potential non-linear optic application. Synthesis 2005, 600−604. (23) Ribeiro Morais, G., Vicente Miranda, H., Santos, I. C., Santos, I., Outeiro, T. F., and Paulo, A. (2011) Synthesis and in vitro evaluation of fluorinated styryl benzazoles as amyloid-probes. Bioorg. Med. Chem. 19, 7698−7710. (24) Tong, H., Lou, K., and Wang, W. (2015) Near-infrared fluorescent probes for imaging of amyloid plaques in Alzheimers disease. Acta Pharm. Sin. B 5, 25−33. (25) Fu, H., Cui, M., Zhao, L., Tu, P., Zhou, K., Dai, J., and Liu, B. (2015) Highly Sensitive Near-Infrared Fluorophores for in Vivo Detection of Amyloid-β Plaques in Alzheimer’s Disease. J. Med. Chem. 58, 6972−6983. (26) Kung, M. P., Hou, C., Zhuang, Z. P., Zhang, B., Skovronsky, D., Trojanowski, J. Q., Lee, V. M., and Kung, H. F. (2002) IMPY: an improved thioflavin-T derivative for in vivo labeling of β-amyloid plaques. Brain Res. 956, 202−210. (27) Chauhan, K., Datta, A., Adhikari, A., Chuttani, K., Kumar Singh, A., and Mishra, A. K. (2014) 68Ga based probe for Alzheimer’s disease: synthesis and preclinical evaluation of homodimeric chalcone in βamyloid imaging. Org. Biomol. Chem. 12, 7328−7337. (28) Iikuni, S., Ono, M., Watanabe, H., Matsumura, K., Yoshimura, M., Harada, N., Kimura, H., Nakayama, M., and Saji, H. (2014) Enhancement of binding affinity for amyloid aggregates by multivalent interactions of 99mTc-hydroxamamide complexes. Mol. Pharmaceutics 11, 1132−1139.

(29) Matsumura, K., Ono, M., Kitada, A., Watanabe, H., Yoshimura, M., Iikuni, S., Kimura, H., Okamoto, Y., Ihara, M., and Saji, H. (2015) Structure-Activity Relationship Study of Heterocyclic Phenylethenyl and Pyridinylethenyl Derivatives as Tau-Imaging Agents That Selectively Detect Neurofibrillary Tangles in Alzheimer’s Disease Brains. J. Med. Chem. 58, 7241−7257.

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DOI: 10.1021/acschemneuro.6b00450 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX