Near-Infrared Fluorescent Probes with Rotatable Polyacetylene Chains for the Detection of Amyloid-β Plaques

Article abstract of DOI:10.1021/acs.jpcb.0c08845

The plaques of accumulated β-amyloid (Aβ) in the parenchymal brain are accepted as an important biomarker for the early diagnosis of Alzheimer's disease (AD). Many near-infrared (NIR) probes, which were based on the D-π-A structure and bridged by conjugat

Full text of DOI:10.1021/acs.jpcb.0c08845

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Article
Near-Infrared Fluorescent Probes with Rotatable Polyacetylene
Chains for the Detection of Amyloid‑β Plaques
Longfei Zhang, Xin Gong, Chuan Tian, Hualong Fu,* Hongwei Tan, Jiapei Dai, and Mengchao Cui*
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ABSTRACT: The plaques of accumulated β-amyloid (Aβ) in the
parenchymal brain are accepted as an important biomarker for the
early diagnosis of Alzheimer’s disease (AD). Many near-infrared
(NIR) probes, which were based on the D−π−A structure and
bridged by conjugated double bonds, had been reported and
displayed a high affinity to Aβ plaques. Considering the
isomerization caused by the polyethylene chain, however, the
conjugated polyacetylene chain is a better choice for developing
new NIR Aβ probes. Hence, in this report, a new series of NIR
probes with naphthyl or phenyl rings and different numbers of
conjugated triple bonds were designed, synthesized, and evaluated
as NIR probes for Aβ plaques. Upon interaction with Aβ
aggregates, these probes displayed a significant increase in
fluorescence intensity (45- to 360-fold) and a high to moderate affinity (6.05−56.62 nM). Among them, probe 22b displayed
excellent fluorescent properties with a 183-fold increase in fluorescence intensity and an emission maximum at 650 nm after
incubated with Aβ aggregates. Furthermore, 22b had a high affinity to Aβ aggregates (Kd = 12.96 nM) and could efficiently detect
the Aβ plaques in brain sections from both transgenic mice and AD patients in vitro. In summary, this work may lead to a new
direction in the development of novel NIR probes for the detection of Aβ plaques.
1. INTRODUCTION
Alzheimer’s disease (AD) was significantly associated with
aging.1 It could cause dementia, logagnosia, disorientation, and
lapsus memoriae, which would bring a heavy burden to the
patient’s family and the whole society.2−4 According to the Aβ
cascade hypothesis, deposition of amyloid plaques that consist
of aggregated Aβ peptides is the hallmark of AD.5−9 Develop-
ment of probes targeting to Aβ plaque is essential for monitoring
AD’s condition, distinguish other types of dementia, and help to
test drug efficacy in pharmaceutical development.
In in vivo biological imaging, optical imaging technology using
the near-infrared (NIR) spectroscopy band (650−900 nm) has
many advantages including real-time imaging and sufficient light
penetration depth in biological tissues.6,10,11 Thus, NIR imaging
Figure 1. (A) Chemical structures of previously reported Aβ NIR
probes with polyene chain. (B) The design concept of our new D−π−A
probes with rotatable polyacetylene chain.
has received increasing attention in recent years, and the
research of the NIR fluorescent probe is an important part of
this.12−16 In recent years, a variety of NIR fluorescent probes for
Aβ plaques have been reported by our group, including DANIR
2c and 3c (Figure 1A).11,17 In 2014, DANIR 2c, in which the
electron donor (D) and acceptor (A) moieties were bridged by
Received: September 29, 2020
Revised: December 16, 2020
Published: January 8, 2021
three conjugated CC bonds, was reported to have a moderate
affinity to Aβ aggregates (Kd = 26.9 nM) and has been
successfully used in in vivo NIR imaging.11 In 2015, further
modification of DANIR 2c led to the development of DANIR 3c
by replacing the benzene ring with a naphthalene ring.17 DANIR
3c was demonstrated to be an excellent in vivo NIR probe for Aβ
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Table 1. Spectroscopic and Binding Data for the Synthesized Probes
a
a
b
c
d
e
f
probe
21a
21b
21c
22a
22b
22c
DANIR 2c11
DANIR 3c17
λem1 (nm)
λem2 (nm)
Φfl (%)
fold
Kd (nM)
clog P
E (kcal/mol)
580
576
649
627
520
596
660
720
535
602
649
603
650
665
659
678
0.2/0.2
0.3/0.6
0.3/0.3
0.4/1.8
0.3/0.2
0.4/0.5
4.1/0.2
9.0/0.009
360
167
80
263
183
45
18.74 4.86
6.05 2.02
51.54 3.2
12.19 1.36
12.96 3.19
56.62 11.11
26.9 3.0
2.60
2.91
3.30
3.72
3.80
4.27
2.95
4.05
6.28
3.77
1.88
5.02
2.51
6.28
18.20
16.94
12
280
1.9 1.1
a
b
Emission maxima of probes that were measured in CH2Cl2 (λem1) and upon interaction with Aβ1−42 aggregates in PBS (λem2). Quantum yields
c
d
that were measured in CH2Cl2 and PBS, respectively. Fluorescence enhancement after binding to Aβ1−42 aggregates in PBS. Kd values to Aβ1−42
e
aggregates measured in triplicate with results given as mean standard deviation (SD). Calculated using the online ALOGPS 2.1 program.
f
Rotation energies were calculated using the B3LYP/6-31G* method in CH2Cl2.
a
Scheme 1. Synthesis of the DANIR Probes
a
Reagent and conditions: (a) (1) n-BuLi (1.6 M in hexane), anhydrous tetrahydrofuran (THF), −78 °C, 30 min, (2) I2, room temperature (rt), 30
min; (b) PdCl2(PPh3)2 for 5 or Pd(PPh3)4 for 6, CuI, triethylamine (Et3N), THF, 66 °C, 24 h; (c) tetrabutylammonium fluoride (TBAF), THF,
66 °C, 3 h; (d) ethynyltrimethylsilane, Cu, and CH2Cl2/1,4-dioxane (v/v = 3/1) for 9 or 3-butyn-2-ol, CuCl, and CHCl3 for 10,
tetramethylethylenediamine (TMEDA), O2, 50 °C, overnight; (e) TBAF and THF for 11 or NaOH and 1,4-dioxane for 12, 66 °C, 3 h; (f)
PdCl2(PPh3)2 for 13 or Pd(PPh3)4 for 14, ethynol, CuI, Et3N, THF, N2, rt, overnight; (g) Dess−Martin periodinane reagent (DMP), CH2Cl2, rt,
10 min; (h) ethynol, Cu, TMEDA, O2, CH2Cl2/1,4-dioxane (v/v = 3/1), 50 °C; (i) malononitrile, basic Al2O3 (48−75 μm), CH2Cl2, rt, 5 min.
plaques with improved properties like high quantum yield (QY,
9.0% in dichloromethane (DCM), 0.009% in phosphate-
buffered saline (PBS)) and a high affinity (Kd = 1.9 nM).11,17
In general, these intramolecular charge transfer (ICT) probes
(Figure 1A) share a common D−π−A structure, consisting of an
electron donor (D) group and an electron acceptor (A) group,
which were connected by a conjugated π bridge (π).18−20 As
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reported, the biological and optical properties of these probes
could be efficiently tuned by adjusting the number of conjugated
CC units.21 However, the CC bonds could be isomerized
under light irradiation, which may cause a change in the
molecular conformation in solvent and thus deteriorate their
performance when binding to Aβ plaques. Moreover, these
probes are very sensitive to the polarity of the surrounding
environment. Their quantum yields were remarkably increased
when the solvent altered from polar PBS to nonpolar DCM (e.g.,
DANIR 2c and 3c, Table 1), which may likely induce severe
nonspecific signals when used in in vivo brain imaging.17,22
Similar to the CC bond, polyacetylene chain also possesses
excellent electron transfer capability and has been widely used in
the chemo-sensors of many fields like polarity, viscosity, NO,
and oxygen.23−25 In 2015, Whitten’s and Chi’s group developed
a focused library of molecular rotors based on the phenylene
ethylene backbone, which demonstrated moderate increases in
fluorescence intensity after incubated with hen egg-white
lysozyme-induced amyloid conformer, and the lowest binding
constant was determined as 1 μM.26,27 The polyacetylene chain
endows the probes with distinctive design features (Figure 1B):
(1) Since the CC bond is formed by sp hybridization between
two carbon atoms, its derivatives often share a linear structure, in
which the terminal moieties could rotate freely around the
polyacetylene π bridge. (2) The polyacetylene chain is an
excellent π bridge in D−π−A molecules.28 The emission
maximum of a fluorescent probe might be shifted to the NIR
window with the extension of the polyacetylene chain. (3)
Besides, the probe with polyacetylene chains could avoid
isomerization, which may result in a more precise response to Aβ
plaques. Herein, we designed and synthesized a focused library
of D−π−A fluorescent probes (21a−c and 22a−c, Scheme 1)
that contain polyacetylene chains as conjugated π bridges.
Furthermore, these compounds were assessed as NIR probes for
the detection of Aβ plaques in vitro.
visible (UV−vis) spectra were obtained on a Shimadzu UV-
2450 UV−vis spectrophotometer (Japan) with a Jiangsu Jingbo
quartz absorption cell (751, 10 mm). Fluorescence spectra were
recorded on a Shimadzu RF-5301PC spectrofluorophotometer
(Japan) with a Jiangsu Jingbo quartz absorption cell (751, 10
mm). Fluorescence QYs were measured on a Hamamatsu
C11347 Absolute PL quantum yield spectrometer (Japan).
High-performance liquid chromatography (HPLC) was per-
formed on a Hitachi Primaide system (Japan). Samples were
analyzed on a Bonna-Agela Technologies Venusil MP C18
reverse-phase column (5 μm, 4.6 mm × 250 mm) and eluted at a
flow rate of 1.0 mL/min. Mobile phase A was water, and mobile
phase B was acetonitrile. In vitro fluorescent staining results were
acquired on a Life Technologies EVOS FL imaging system
(equipped with DAPI, GFP, RFP, and CY5 filter sets) or a Leica
DMi8 THUNDER (equipped with DAPI, GFP, and RFP filter
sets, Germany). Alzheimer’s model mouse (C57BL6, APPsw/
PSEN1, 13 months old, male) were purchased from the Institute
of Laboratory Animal Sciences, Chinese Academy of Medical
Sciences (Beijing, P. R. China), and the mouse brain was used to
prepare paraffin sections after sacrifice. The brain tissues of
autopsy-confirmed AD patients (95 years old, female; 64 years
old, female) were obtained from the Chinese Brain Bank Center
(CBBC), South-Central University for Nationalities (Wuhan, P.
R. China).
2.2. Chemistry. 2.2.1. 4-Iodo-N,N-dimethylaniline (3). To
a stirred solution of 1 (10.0 g, 50 mmol) in anhydrous THF, n-
butyllithium (40 mL, 1.6 M hexane, 64 mmol) was added
dropwise at −78 °C under nitrogen atmosphere. The mixture
was stirred for 30 min. Afterward, iodine (13.9 g, 55 mmol) was
dissolved in anhydrous THF and added into the above mixture,
stirred at room temperature for an additional 30 min, and then
the reaction was neutralized by saturated Na2S2O3 aqueous
solution. THF was removed under vacuum, and the residue was
extracted with petroleum ether (3 × 100 mL). The combined
organic layer was dried over anhydrous Na2SO4, and solvents
were evaporated under vacuum to yield 3 as a white crystalline
solid without further purification (12.2 g, 98.5%). 1H NMR (400
MHz, CDCl3) δ 7.47 (d, J = 8.4 Hz, 2H), 6.51 (d, J = 8.4 Hz,
2H), 2.92 (s, 6H). Spectra data are consistent with the
literature.30
2. METHODS
2.1. General Information. All reagents used for synthesis
were of commercial grade and used without further purification.
Thin-layer chromatography (TLC) was used for monitoring
reactions on Merck silica gel 60 F254 plates. Upon loading to the
flash column (Silica-CS, 40−60 μm, 20−80 g, Bonna-Agela
Technologies Co., Ltd., China), the compounds in organic
synthesis were purified using a FLEXA modular preparative
chromatography system (Bonna-Agela Technologies Co., Ltd.,
China). Amyloid-β1−42 peptides were purchased from Peptide
Institute, Inc. (Japan) and aggregated and tested by the reported
method.29 1H and 13C NMR spectra were collected on a Bruker
Avance III (400 for 1H or 100 MHz for 13C, respectively, 5 mm
PABBO BB- probe), a JEOL JNM-ECZ400R/S1 (400 for 1H or
100 MHz for 13C, respectively, 4 mm Cryocoil MAS probe), or a
JNMECZ600R/S3 (600 for 1H or 150 MHz for 13C,
respectively, 4 mm Cryocoil MAS probe) NMR spectrometer
in CDCl3, trifluoroacetic acid-d, or dimethyl sulfoxide (DMSO)-
d6 at room temperature unless further noted. Chemical shifts
were reported in parts per million (ppm) compared with
tetramethylsilane (δ 0.00, s). Coupling constants (J) were
reported in hertz (Hz). Multiplicities were reported as s
(singlet,), d (doublet), t (triplet), and m (multiplet). Mass
spectrometry (MS) spectra were acquired on a Thermo
Scientific LCQ FLEET (electrospray ionization (ESI)) mass
spectrometer. High-resolution mass spectra were acquired on a
Thermo Scientific Q Exactive (ESI) mass spectrometer. UV−
2.2.2. N,N-Dimethyl-4-((trimethylsilyl)ethynyl)aniline (5).
The preparation of compound 5 was in accordance with a
reported study with slight modification.31 To a stirred solution
of 3 (2.0 g, 8.0 mmol) in THF, PdCl2(PPh3)2 (168.5 mg, 0.24
mmol), CuI (91.4 mg, 0.48 mmol), and Et3N (1.7 mL, 12.0
mmol) were added, and finally the solution of ethynyltrime-
thylsilane in THF was added slowly under nitrogen atmosphere.
The reaction mixture was maintained for 24 h at room
temperature. The mixture was filtered, and the solvent was
removed by vacuum. The residue was purified by flash column
chromatography (petroleum ether/CH2Cl2 = 3/1, v/v) to give 5
1
as a pale yellow solid (1.2 g, 66.7%). H NMR (600 MHz,
CDCl3) δ 7.33 (d, J = 8.6 Hz, 2H), 6.59 (d, J = 8.6 Hz, 2H), 2.96
(s, 6H), 0.22 (s, 9H).
2.2.3. 4-Ethynyl-N,N-dimethylaniline (7). The preparation
of compound 7 was in accordance with a reported study with
slight modification.31 To a solution of 5 (1.1 g, 5.3 mmol) in
THF, tetrabutylammonium fluoride (TBAF, 10.6 mL, 1 M in
THF, 10.6 mmol) was added, and the mixture was refluxed for 3
h. After cooling to room temperature, the mixture was washed
with water and extracted with CH2Cl2 (3 × 100 mL). The
combined organic layer was dried over anhydrous Na2SO4,
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concentrated, and purified by flash column chromatography
(petroleum ether/CH2Cl2 = 9/1, v/v) to give 7 as a pale yellow
solid (566.8 mg, 73.6%). 1H NMR (600 MHz, CDCl3) δ 7.38
(d, J = 8.9 Hz, 2H), 6.61 (d, J = 8.9 Hz, 2H), 2.98 (s, 6H).
2.2.4. N,N-Dimethyl-4-((trimethylsilyl)buta-1,3-diyn-1-yl)-
aniline (9). The preparation of compound 9 was in accordance
with a reported study with slight modification.28 A mixture of
copper powder (6.4 mg, 0.1 mmol) and tetramethylethylenedi-
amine (TMEDA, 46.5 mg, 0.4 mmol) in the mixed solution of
CH2Cl2 and 1,4-dioxane (v/v = 3/1) was stirred under air for 2
min. Then, compound 7 (290.4 mg, 2 mmol) and ethynyl-
trimethylsilane (0.15 mL, 2.6 mmol) were added slowly. The
reaction mixture was stirred at 50 °C and monitored by TLC.
After consumption of the starting material, the mixture was
filtered and concentrated under vacuum. The crude product was
further purified by flash column chromatography (petroleum
ether/CH2Cl2 = 5/1, v/v) to give 9 as a tawny solid (52.3 mg,
10.8%). 1H NMR (400 MHz, CDCl3) δ 7.35 (d, J = 8.8 Hz, 2H),
6.59 (d, J = 8.8 Hz, 2H), 2.98 (s, 6H), 0.20 (s, 9H).
chromatography (petroleum ether/ethyl acetate = 3/1, v/v).
Compound 19a was obtained as a pale yellow solid (95.0 mg,
53.0%). 1H NMR (400 MHz, CDCl3) δ 9.26 (s, 1H), 7.43 (d, J =
9.3 Hz, 2H), 6.60 (d, J = 9.3 Hz, 2H), 3.02 (s, 6H).
2.2.10. 7-(4-(Dimethylamino)phenyl)hepta-2,4,6-triyn-1-
ol (17b). The same reaction described above to prepare
compound 9 was used, and the crude product was purified by
flash column chromatography (petroleum ether/ethyl acetate =
6/1, v/v). Compound 17b was obtained as a saffron yellow solid
(44.2 mg, 33.6%). 1H NMR (400 MHz, CDCl3) δ 7.39 (d, J =
8.8 Hz, 2H), 6.62 (d, J = 8.8 Hz, 12H), 4.37 (s, 2H), 3.00 (s,
6H). Spectra data are consistent with the literature.28
2.2.11. 17-(4-(Dimethylamino)phenyl)hepta-2,4,6-triynal
(19b). The same reaction described above to prepare compound
15 was used, and the crude product was purified by flash column
chromatography (petroleum ether/ethyl acetate = 5/1, v/v).
Compound 19b was obtained as a pale yellow solid (42.3 mg,
47.8%). 1H NMR (400 MHz, CDCl3) δ 9.22 (s, 1H), 7.43 (d, J =
9.1 Hz, 2H), 6.60 (d, J = 9.1 Hz, 2H), 3.02 (s, 6H).
2.2.12. 2-(3-(4-(Dimethylamino)phenyl)prop-2-yn-1-yli-
dene) malononitrile (21a). To a solution of 15 (34.1 mg, 0.2
mmol) in CH2Cl2, malononitrile and basic Al2O3 (48−75 μm,
Sinopharm Chemical Reagent Beijing Co., Ltd.) was added.33
The reaction finished at once. The crude product was purified by
flash column chromatography (pure CH2Cl2) and recrystalliza-
tion in petroleum ether. Compound 21a was obtained as red-
purple crystalline solid (42.3 mg, 95.7%). 1H NMR (400 MHz,
CDCl3) δ 7.51 (d, J = 8.9 Hz, 2H), 7.09 (s, 1H), 6.69 (d, J = 8.9
Hz, 2H), 3.08 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 152.52,
141.07, 135.57, 121.60, 113.62, 112.39, 111.73, 105.74, 88.29,
40.03. HRMS found 222.1022; molecular formula C14H11N3
requires [M + H]+ 222.0953. Spectra data are consistent with
the literature.28
2.2.13. 2-(5-(4-(Dimethylamino)phenyl)penta-2,4-diyn-1-
ylidene)malononitrile (21b). The same reaction described
above to prepare compound 21a was used, and the crude
product was purified by flash column chromatography (pure
CH2Cl2). Compound 21b was obtained as a dark purple
crystalline solid (44.1 mg, 90.0%). 1H NMR (400 MHz, CDCl3)
δ 7.48 (d, 2H), 7.01 (s, 1H), 6.63 (d, J = 9.0 Hz, 2H), 3.05 (s,
6H). 13C NMR (101 MHz, CDCl3) δ 151.73, 140.43, 135.24,
112.81, 112.07, 111.64, 105.93, 101.51, 100.02, 93.86, 73.55,
40.29. HRMS found 246.1024; molecular formula C16H11N3
requires [M + H]+ 246.0953. Spectra data are consistent with
the literature.28
2.2.14. 2-(7-(4-(Dimethylamino)phenyl)hepta-2,4,6-triyn-
1-ylidene)malononitrile (21c). The same reaction described
above to prepare compound 21a was used, and the crude
product was purified by flash column chromatography (pure
CH2Cl2). Compound 21c was obtained as a dark purple
crystalline solid (40.0 mg, 78.2%). 1H NMR (600 MHz, CDCl3)
δ 7.44 (d, J = 9.1 Hz, 2H), 6.96 (s, 1H), 6.64 (d, J = 9.0 Hz, 2H),
3.04 (s, 6H). 13C NMR (101 MHz, Trifluoroacetic Acid-d) δ
143.02, 142.33, 135.31, 123.97, 120.04, 98.83, 95.84, 81.58,
78.35, 74.71, 70.08, 63.87, 46.94. HRMS found 270.1023;
molecular formula C18H11N3 requires [M + H]+ 270.0953.
Spectra data are consistent with the literature.28
2.2.5. 4-(Buta-1,3-diyn-1-yl)-N,N-dimethylaniline (11). The
same reaction described above to prepare compound 7 was used,
and the crude product was purified by flash column
chromatography (petroleum ether/ethyl acetate = 3/1, v/v).
Compound 11 was obtained as a pale yellow solid (91.5 mg,
87.1%). 1H NMR (600 MHz, CDCl3) δ 7.37 (d, J = 6.4 Hz, 2H),
6.75 (d, J = 6.4 Hz, 2H), 3.85 (s, 6H), 3.42 (s, 1H). MS found
170.2; molecular formula C12H11N requires [M + H]+ 170.1
2.2.6. 3-(4-(Dimethylamino)phenyl)prop-2-yn-1-ol (13).
To a stirred solution of 3 (1.2 g, 5.0 mmol) and ethynol
(364.5 mg, 6.5 mmol) in THF, PdCl2(PPh3)2 (175.7 mg, 0.25
mmol), CuI (47.6 mg, 0.25 mmol), and Et3N (87.6 mg, 0.5
mmol) were added. The reaction mixture was maintained for 24
h at room temperature under nitrogen atmosphere. The mixture
was filtered, and the solvent was removed by vacuum. The crude
product was purified by flash column chromatography
(petroleum ether/ethyl acetate = 9/1, v/v). Compound 13
1
was obtained as a yellow solid (1305.4 mg, 52.8%). H NMR
(400 MHz, CDCl3) δ 7.30 (d, J = 8.8 Hz, 2H), 6.61 (d, J = 8.8
Hz, 2H), 4.47 (d, J = 5.5 Hz, 2H), 2.96 (s, 6H). Spectra data are
consistent with the literature.32
2.2.7. 3-(4-(Dimethylamino)phenyl)propiolaldehyde (15).
The preparation of compound 15 was in accordance with a
reported study with slight modification.28 To a solution of 13
(697.7 mg, 4.0 mmol) in CH2Cl2, Dess−Martin periodinane
(DMP, 1703.5 mg, 4 mmol) was added in batches. The reaction
was stirred at room temperature and monitored by TLC. After
the reaction was finished, saturated Na2SO3 aqueous solution
and NaHCO3 aqueous solution were added. The resulting
mixture was extracted with CH2Cl2 (3 × 100 mL) and dried over
anhydrous Na2SO4. The solvent was evaporated under vacuum,
and the residue was purified by flash column chromatography
(petroleum ether/CH2Cl2 = 3/1, v/v) to give 15 as a yellow
solid (34.1 mg, 5%).
2.2.8. 5-(4-(Dimethylamino)phenyl)penta-2,4-diyn-1-ol
(17a). The same reaction described above to prepare compound
9 was used, and the crude product was purified by flash column
chromatography (petroleum ether/ethyl acetate = 3/1, v/v).
Compound 17a was obtained as a pale yellow solid (50.3 mg,
12.6%). 1H NMR (400 MHz, CDCl3) δ 7.36 (d, J = 8.8 Hz, 2H),
6.60 (d, J = 8.8 Hz, 2H), 4.40 (s, 2H), 2.99 (s, 6H).
2.2.15. 6-Iodo-N,N-dimethylnaphthalen-2-amine (4). The
same reaction described above to prepare compound 3 was used,
and the crude product was purified by flash column
chromatography (petroleum ether/ethyl acetate = 3/1, v/v) to
give 4 as a pale yellow solid (574.7 mg, 64.5%). MS found 298.3;
molecular formula C12H12IN requires [M + H]+ 298.0.
2.2.9. 5-(4-(Dimethylamino)phenyl)penta-2,4-diynal
(19a). The same reaction described above to prepare compound
15 was used, and the crude product was purified by flash column
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2.2.16. N,N-Dimethyl-6-((trimethylsilyl)ethynyl)-
naphthalen-2-am-5ine (6). The same reaction described
above to prepare compound 5 was used. Compound 6 was
(s, 1H), 7.64 (s, 1H), 7.44 (d, J = 7.4 Hz, 1H), 7.29 (s, 1H), 4.44
(s, 2H), 3.13 (s, 6H).
2.2.23. 5-(6-(Dimethylamino)naphthalen-2-yl)penta-2,4-
diynal (20a). The same reaction described above to prepare
compound 15 was used. Compound 20a was obtained as a
1
obtained as a tawny solid (40.0 mg, 78.2%). H NMR (400
MHz, CDCl3) δ 7.88 (s, 1H), 7.70 (s, 1H), 7.60 (s, 1H), 7.44 (s,
1H), 6.99 (s, 1H), 3.10 (s, 6H), 0.27 (s, 9H). MS found 268.3;
molecular formula C17H21NSi requires [M + H]+ 268.1. Spectra
data are consistent with the literature.34
2.2.17. 6-Ethynyl-N,N-dimethylnaphthalen-2-amine (8).
The same reaction described above to prepare compound 7
was used, and the crude product was purified by flash column
chromatography (petroleum ether/ethyl acetate = 1/1, v/v).
Compound 8 was obtained as a pale yellow solid (292.5 mg,
15.0%). 1H NMR (400 MHz, CDCl3) δ 7.91 (s, 1H), 7.71 (s,
1H), 7.63 (s, 1H), 7.47 (s, 1H), 6.99 (s, 1H), 3.11 (s, 6H).
Spectra data are consistent with the literature.34
2.2.18. 6-(6-(Dimethylamino)naphthalen-2-yl)-2-methyl-
hexa-3,5-diyn-2-ol (10). To a solution of 8 (366 mg, 19
mmol) and 3-butyn-2-ol (236.9 mg, 1.9 mmol) in CHCl3, CuCl
(9.3 mg, 0.09 mmol) and TMEDA (43.7 mg, 0.4 mmol) were
added. The reaction mixture was stirred at 50 °C for 4 h and
monitored by TLC. The solvent was evaporated under vacuum,
and the residue was purified by flash column chromatography
(petroleum ether/ethyl acetate = 5/1, v/v) to give 10 as a pale
yellow solid (364.8 mg, 70.2%).
2.2.19. 6-(Buta-1,3-diyn-1-yl)-N,N-dimethylnaphthalen-2-
amine (12). To a solution of 10 (345.7 mg, 1.3 mmol) in 1,4-
dioxane, NaOH (105.6 mg, 2.64 mmol) was added. The
reaction mixture was refluxed at 60 °C and monitored by TLC.
The above mixture was filtered, and the residue was purified by
flash column chromatography (petroleum ether/CH2Cl2 = 2/1,
v/v) to give 12 was obtained as a pale yellow solid (148.2 mg,
54.1%). 1H NMR (400 MHz, CDCl3) δ 7.90 (s, 1H), 7.65 (d, J =
9.1 Hz, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.40 (dd, J = 8.5, 1.5 Hz,
1H), 7.16 (d, J = 8.0 Hz, 1H), 6.85 (s, 1H), 3.08 (s, 6H), 2.49 (s,
1H). MS found 220.3; molecular formula C16H13N requires [M
+ H]+ 220.1.
2.2.20. 3-(6-(Dimethylamino)naphthalen-2-yl)prop-2-yn-
1-ol (14). The same reaction described above to prepare
compound 13 was used, and the crude product was purified by
flash column chromatography (petroleum ether/ethyl acetate =
3/1, v/v). Compound 14 was obtained as a tawny oil (170.0 mg,
39.7%). 1H NMR (400 MHz, CDCl3) δ 7.79 (s, 1H), 7.63 (d, J =
9.1 Hz, 1H), 7.55 (d, J = 8.5 Hz, 1H), 7.34 (dd, J = 8.5, 1.6 Hz,
1H), 7.13 (dd, J = 9.1, 2.5 Hz, 1H), 6.85 (s, 1H), 4.51 (s, 2H),
3.05 (s, 6H).
2.2.21. 3-(6-(Dimethylamino)naphthalen-2-yl)-
propiolaldehyde (16). The same reaction described above to
prepare compound 15 was used, and the crude product was
purified by flash column chromatography (petroleum ether/
ethyl acetate = 6/1, v/v). Compound 16 was obtained as a pale
yellow solid (50.2 mg, 12.9%). 1H NMR (400 MHz, CDCl3) δ
9.44 (s, 1H), 8.04 (s, 1H), 7.73 (d, J = 9.1 Hz, 1H), 7.63 (d, J =
8.5 Hz, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.23 (d, J = 9.1 Hz, 1H),
6.99 (s, 1H), 3.12 (s, 6H). MS found 224.3; molecular formula
C15H13NO requires [M + H]+ 224.1.
1
reddish-brown solid (55.6 mg, 26.4%). H NMR (400 MHz,
CDCl3) δ 9.30 (s, 1H), 8.00 (s, 1H), 7.72 (d, J = 9.0 Hz, 1H),
7.63 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 8.7 Hz, 1H), 7.06 (s, 1H),
3.13 (s, 6H).
2.2.24. 7-(6-(Dimethylamino)naphthalen-2-yl)hepta-
2,4,6-triyn-1-ol (18b). The same reaction described above to
prepare compound 9 was used, and the crude product was
purified by flash column chromatography (petroleum ether/
ethyl acetate = 3/1, v/v). Compound 18b was obtained as a
brown solid (17.0 mg, 14.1%).
2.2.25. 7-(6-(Dimethylamino)naphthalen-2-yl)hepta-
2,4,6-triynal (20b). The same reaction described above to
prepare compound 15 was used. Compound 20b was obtained
as a reddish-brown solid (55.6 mg, 26.4%). 1H NMR (400 MHz,
CDCl3) δ 9.14 (s, 1H), 7.87 (s, 1H), 7.59−7.51 (m, 1H), 7.46
(d, J = 8.5 Hz, 1H), 7.30 (d, J = 8.5 Hz, 1H), 7.06 (d, J = 9.0 Hz,
1H), 6.75 (s, 1H), 3.00 (s, 6H).
2.2.26. 2-(3-(6-(Dimethylamino)naphthalen-2-yl)prop-2-
yn-1-ylidene)malononitrile (22a). The same reaction de-
scribed above to prepare compound 21a was used, and the
crude product was purified by flash column chromatography
(pure CH2Cl2). Compound 22a was obtained as an orange red
crystalline solid (24.9 mg, 9.2%). 1H NMR (400 MHz, CDCl3) δ
8.04 (s, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 6.7 Hz, 1H),
7.46 (d, J = 8.5 Hz, 1H), 7.22 (s, 1H), 7.17 (s, 1H), 6.99 (s, 1H),
3.15 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 141.15, 136.61,
135.45, 130.09, 128.94, 126.80, 125.81, 119.17, 116.63, 113.13,
111.98, 106.26, 90.93, 86.82, 77.23, 40.71. HRMS found
272.1179; molecular formula C18H13N3 requires [M + H]+
272.1109.
2.2.27. 2-(5-(6-(Dimethylamino)naphthalen-2-yl)penta-
2,4-diyn-1-ylidene)malononitrile (22b). The same reaction
described above to prepare compound 21a was used, and the
crude product was purified by flash column chromatography
(pure CH2Cl2). Compound 22b was obtained as a brown-purple
crystalline solid (24.9 mg, 90.6%). 1H NMR (400 MHz, CDCl3)
δ 8.02 (s, 1H), 7.72 (d, J = 9.1 Hz, 1H), 7.63 (d, J = 8.6 Hz, 1H),
7.45 (d, J = 8.7 Hz, 1H), 7.23 (s, 1H), 7.04 (s, 1H), 6.99 (s, 1H),
3.13 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 140.41, 136.27,
135.06, 129.63, 128.79, 126.66, 125.56, 116.66, 112.60, 111.84,
111.45, 105.64, 100.32, 98.60, 95.10, 75.94, 73.23, 40.54. HRMS
found 296.1179; molecular formula C20H13N3 requires [M +
H]+ 296.1109.
2.2.28. 2-(7-(6-(Dimethylamino)naphthalen-2-yl)hepta-
2,4,6-triyn-1-ylidene)malononitrile (22c). The same reaction
described above to prepare compound 21a was used, and the
crude product was purified by flash column chromatography
(pure CH2Cl2). Compound 22c was obtained as a dark purple
crystalline solid (3.0 mg, 23.5%). 1H NMR (400 MHz, CDCl3) δ
7.99 (s, 1H), 7.69 (d, J = 9.0 Hz, 1H), 7.59 (d, J = 8.5 Hz, 1H),
7.42 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 8.5 Hz, 1H), 6.97 (s, 2H),
3.12 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 139.55, 135.81,
134.96, 129.52, 129.00, 126.77, 116.77, 112.16, 111.01, 98.79,
97.20, 89.04, 88.74, 86.33, 80.85, 77.27, 73.73, 71.60, 64.81,
40.94. HRMS: m/z calcd for [M + H]+ 320.1109, found
320.1179. HRMS found 320.1179; molecular formula C22H13N3
requires [M + H]+ 320.1109.
2.2.22. 5-(6-(Dimethylamino)naphthalen-2-yl)penta-2,4-
diyn-1-ol (18a). The same reaction described above to prepare
compound 9 was used, and the crude product was purified by
flash column chromatography (petroleum ether/ethyl acetate =
3/1, v/v). Compound 18a was obtained as a brown solid (331.0
mg, 88.6%). 1H NMR (400 MHz, CDCl3) δ 7.93 (s, 1H), 7.72
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2.3. Spectroscopic Measurements. Ultraviolet absorp-
tion spectra and fluorescence spectra of probes were recorded
according to the previously reported methods,29 and the
experimental details are given in the Supporting Information
(SI).
2.4. Biological Evaluations. For these new probes, the
fluorescent responses with Aβ1−42 aggregates and bovine serum
albumin (BSA), binding affinity to Aβ1−42 aggregates, in vitro
fluorescent staining on brain sections of transgenic mice and AD
patients, and NMR titration were tested according to the
previously reported methods,29,35,36 and the detailed procedures
are given in the SI.
indicating that the electron density of acceptor moiety increased
with the expansion of the CC units.
As shown in Table 1, the anticipated increase in the
fluorescent maxima of these probes was not observed with the
extension of polyacetylene chain, which is different from the
reported ICT probes with polyene chains.11,17,21 For phenyl
derivatives (21a−c), 21c with the largest conjugated π system
displayed the longest emission maximum at 649 nm, while 21a
and 21b showed nearly the same maxima around 580 nm. In
addition, the naphthalene derivatives (22a−c) exhibited
irregular fluorescent properties that 22a had the longest
emission wavelength of 627 nm in dichloromethane followed
by 22c and 22b (596 and 520 nm, respectively). Moreover, all of
these new probes displayed shorter emission maxima (<650 nm)
than those of DANIR 2c and 3c (660 and 720 nm, respectively,
Table 1). These results may be caused by both charge
delocalization and molecule configuration: (1) The electrons
are held more firmly by carbon atoms in CC bonds (sp
hybridization) than in CC bonds (sp2 hybridization), and
their transformation is obstructed in a polyacetylene chain,
which expands the highest occupied molecular orbital−lowest
unoccupied molecular orbital (HOMO−LUMO) gap and leads
to a blue shift in emission maximum. (2) For our new probes, an
obvious decrease in the rotation energy barrier (1.88−6.28 eV,
Table 1) was observed compared with that of polyene probes
DANIR 2c and 3c (>16 eV, Table 1), which leads to multiple
molecular rotations in free status and thus influenced the
excited-state dipole (Figure S2 and S3 in the SI).17,39
Furthermore, in the solvents of nonpolar dichloromethane and
polar PBS, which have significantly different polarities, each
probe displayed very similar fluorescent quantum yields (0.2−
1.8%, Table 1), indicating that the QYs of our probes are
nonsensitive to the microenvironment when in their free state.
This result is different from the QYs of DANIR 2c and 3c, which
have large increases (21- and 1000-fold, respectively, Table 1)
when the solvent transferred from PBS to DCM.11,17
3. RESULTS
3.1. Synthesis. The synthetic routes of the final products
(21a−c and 22a−c) are shown in Scheme 1. Briefly, the
arylacetylenes (7 and 8) were synthesized by the Sonogashira
cross-coupling from the corresponding iodinated compounds (3
and 4). In the following, Glaser and Hay couplings were used to
prolong the polyacetylene chain (n = 1−3) to obtain
corresponding compounds (9, 10, 17a−b, and 18a−b) in
moderate to high yields (33.9−88.6%). Then, the terminal
alcohols (13, 14, 17a−b, and 18a−b) were oxidized to
aldehydes (15, 16, 19a−b, and 20a−b) by Dess−Martin
periodinane reagent. The final probes (21a−c and 22a−c) were
prepared by Knoevenagel condensation from the aldehydes (15,
16, 19a−b, and 20a−b) and malononitrile with low to high yield
(9.2−95.7%) using basic Al2O3 as a base catalyst. All of the final
probes were purified by flash column chromatography and
recrystallization in the petroleum ether/CH2Cl2 system and fully
characterized by 1H NMR, 13C NMR, and high-resolution mass
spectrometry (HRMS), and the spectra were given in the SI.
Their purities were determined to be greater than 95% by
analytical HPLC (Figure S1 and Table S1 in the SI).
3.2. Fluorescence Properties. In general, D−π−A-based
molecules have typical ICT characteristics that the delocaliza-
tion effect of electrons could be enhanced by the extension of the
conjugated π system, which results in a higher electron density in
the electron acceptor moiety and a red shift of the fluorescent
emission wavelength.37,38 In this case, the chemical shifts of the
hydrogen in acceptor moiety (Figure 2, indicated by asterisks)
displayed gradual upfield shifts (from 7.09 to 6.96 ppm for 21a−
3.3. Fluorescent Responses upon Interaction with
Aβ1−42 Aggregates. An ICT probe usually displays obvious
fluorescent responses after binding to Aβ1−42 aggregates,
including blue shift of emission maximum and increase in
fluorescence intensity.11,17,21 As shown in Table 1, after the
incubation with Aβ1−42 aggregates, the emission maxima of our
probes increased with the extension of polyacetylene chain.
Among them, three of the probes, 21c, 22b, and 22c, displayed
long emission maxima of 649, 650, and 665 nm, respectively,
which are suitable for in vivo NIR imaging. Furthermore, a
significant increase in fluorescence intensity (45- to 360-fold,
Table 1 and Figure 3) was observed, which may be caused by
that the probe was forced to adapt an inflexible structure with
restricted rotational motion after inserted into the binding
pocket of Aβ1−42 aggregates, and thus the nonradiative decay was
forbade and the energy was depleted by fluorescence emitting.
Of note, unlike the irregular emission wavelength shift in free
status, an obvious red shift was observed with increasing number
of CC units (from 535 to 649 nm for benzene derivatives
21a−c, and from 603 to 665 nm for naphthalene derivatives
22a−c; Table 1) after incubating with Aβ1−42 aggregates. As
hindered by the binding pocket, the rotation of the new probes
was minimized, and these molecules were promoted a rigid
configuration, which makes the length of the π system a
dominant factor in regulating the fluorescence maxima. In
addition, the fluorescent response also suggested that no
interaction occurred between our probes and BSA (Figure 3).
1
c and 7.17 to 6.97 ppm for 22a−c) in H NMR spectra,
Figure 2. Chemical shifts (ppm) of the ethylenic hydrogens (indicated
1
by asterisks) of compounds 21a−c (A) and 22a−c (B) in H NMR
(600 MHz) spectra. All spectra were recorded in DMSO-d6 solution at
room temperature. The asterisks indicate the ethylenic hydrogens and
their corresponding peaks.
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Figure 3. Fluorescence emission spectra of 21a−c (A−C, respectively) and 22a−c (D−F, respectively) upon interaction with Aβ1−42 aggregates (10
μg/mL, black line) or BSA (10 μg/mL, red line). The spectra of the compound in PBS (50 nM, blue line) and PBS alone (pink line) were recorded in
the same condition.
Taken together, in free status, our new probes are insensitive to
the microenvironment polarity with faint fluorescent intensity
(low QYs <2%); however, after binding to Aβ1−42 aggregates,
these probes were efficiently turned on with a significant
increase in fluorescent intensity, which makes them a new class
of fluorescent probes for Aβ detection.
3.4. Binding Affinity. In vitro saturation binding assays were
performed to quantitatively explore the affinity of 21a−c and
22a−c to Aβ1−42 aggregates (Figure S4 in the SI). As shown in
Table 1, our probes were demonstrated to have a high to
moderate affinity (6.05−56.62 nM). Among these probes, 21b,
22a, and 22b displayed high affinities with Kd values of 6.05,
12.19, and 12.96 nM, respectively, whereas 21c and 22c with the
longest π-conjugated system displayed moderately decreased
affinity of 54.54 and 56.62 nM, respectively. In accordance with
our previous work, the torsion caused by too long polyacetylene
chain would block the access of our probes to Aβ fibrils, which
lead to a decreased affinity.17 In general, most of these probes
(21a, 21b, 22a, and 22b) displayed a higher affinity than that of
DANIR 2c (Kd = 26.9 nM) but lower than DANIR 3c (Kd = 1.9
nM).
Figure 4. 1H NMR (600 MHz) spectra of KLVFFA alone (black) and
upon incubation with 21b (blue) and 22b (red). All spectra were
recorded in DMSO-d6 solution at 37 °C. The green boxes indicate the
peaks with changes in shape.
incubation with 21b and 22b, significant changes in the peak
shape of KLVFFA hydrogens were observed: (1) the overlap
degree of the F and V hydrogens (indicated by the green dashed
box) was decreased and (2) the broad peak of leucine hydrogen
(indicated by the green solid box) transformed into a spike,
which indicated the interaction between our probes (21b and
22b) and the Aβ16−21 segment.
3.5. In Vitro Fluorescent Staining. To explore the
feasibility of our probes in the detection of Aβ plaques, in vitro
fluorescent staining was performed in brain slices from a
transgenic (Tg) mouse (APPswe/PSEN1, 13 months old, male)
and an AD patient (95 years old, female). As shown in Figures 5
Accordingly, peptide fragment Aβ16−21 (KLVFFA) with high
hydrophobicity is widely accepted as the vital segment for the Aβ
aggregation process and serves as the main binding site for small-
molecule targeting Aβ plaques.36,40 To further verify the
interaction mechanism between our probes and Aβ plaques,
21b and 22b were selected to incubate with segment Aβ16−21
(KLVFFA), and the result was obtained with NMR spectros-
1
copy. As shown by the H NMR spectra in Figure 4, upon
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Figure 5. Histological fluorescent staining results in brain sections of Tg mouse (A−F; APPswe/PSEN1, 13 months old, male; magnification, 5×) and
AD patients (G−L; 64 years old, female; magnification, 20×) with 21b (A, G) and 22b (D, J). These staining results were confirmed by Th-S (middle;
B, E, H, K); merge images of the double fluorescent staining results (right: C, F, I, L); and merged senile plaques were indicated by white arrows.
and S5−S7 in the SI, probes 21a, 21b, 22a, and 22b, which have
a higher affinity (<20 nM) and intense fluorescent response
toward Aβ1−42 aggregates, could precisely identify Aβ plaques in
brain slices from both the Tg mouse and AD patient, and these
fluorescent patterns were consistent with the staining results of
thioflavin-S (Th-S, a traditional fluorescent dye for Aβ plaques).
However, 21c and 22c with moderate affinities (Kd = 51.54 and
56.62 nM, respectively) displayed undesirable staining patterns:
21c only showed poor staining performance and 22c failed to
identify any plaques in human brain slices.
moderate to high affinity and could be turned on with a
significant increase in fluorescence intensity (45- to 360-fold).
The in vitro fluorescent staining results revealed that our probes
with high affinity (21a, 21b, 22a, and 22b) could efficiently label
the Aβ plaques in brain sections from both Tg mouse and AD
patient. Of note, compound 22b with a high affinity (Kd = 12.96
nM) showed remarkable fluorescent responses to Aβ1−42
aggregates with an emission maximum at 650 nm, which
suggests its potential application in in vivo imaging. In summary,
we designed and synthesized a series of new Aβ probes that
contain polyacetylene chains as a π-conjugated system and
successfully used them for in vitro detection of Aβ plaques. Our
research provides new guidelines for developing smarter and
more activatable NIR probes that target Aβ.
4. CONCLUSIONS
In this report, six new D−π−A probes with polyacetylene chains
of various lengths were designed, synthesized, and evaluated for
the first time as fluorescent probes for Aβ plaques in the brain.
Compared with the probes with polyene chains, our new probes
displayed irregular emission wavelengths that were not increased
with the extension of the π-conjugated system. Furthermore, the
QYs of these probes were insensitive to the solvent polarity.
Upon interaction with Aβ1−42 aggregates, these probes showed a
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jpcb.0c08845.
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thioflavin-S; TLC, thin-layer chromatography; TMEDA,
tetramethylethylenediamine
Experimental methods; purity determination; optical
spectra; binding inhibition studies; in vitro fluorescent
1
staining; and H NMR, 13C NMR, and HRMS spectra
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jpcb.0c08845
Author Contributions
M.C., H.F., and L.Z. conceived and designed the experiment.
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properties were determined by L.Z. In vitro saturation binding
assays were performed by L.Z. In vitro fluorescent staining was
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ACKNOWLEDGMENTS
This work was supported by the National Natural Science
Foundation of China (nos. U1967221 and 22022601)
■
ABBREVIATIONS USED
■
AD, Alzheimer’s disease; Aβ, β-amyloid; BSA, bovine serum
albumin; DCM, dichloromethane; DMP, Dess−Martin period-
inane reagent; DMSO, dimethyl sulfoxide; Et3N, triethylamine;
LUMO, lowest unoccupied molecular orbital; HOMO, highest
occupied molecular orbital; HPLC, high-performance liquid
chromatography; HRMS, high-resolution mass spectrometry;
ICT, intramolecular charge transfer; MS, mass spectrometry;
NIR, near-infrared; NMR, nuclear magnetic resonance; PBS,
phosphate-buffered saline; QY, quantum yield; rt, room
temperature; SI, Supporting Information; TBAF, tetrabutylam-
monium fluoride; Tg, transgenic; THF, tetrahydrofuran; Th-S,
505
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The Journal of Physical Chemistry B
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