Synthesis and optical properties of 4-(2-{[6-(1,1-dicyanoprop-1-en-2-yl)naphthalen-2-yl] (methyl)amino} ethoxy)-4-oxobutanoic acid fluorescent probe for β-amyloid

Article abstract of DOI:10.1007/s11164-013-1429-0

Abstract A novel 4-(2-{[6-(1,1-dicyanoprop-1-en-2-yl)naphthalen-2-yl](methyl)amino} ethoxy)-4-oxobutanoic acid (5) fluorescent probe for β-amyloids was synthesized by catalytic acylation using 4-dimethylaminopyridine between succinic anhydride and (1-{6-[(2-hydroxyethyl)(methyl) amino]-2-naphthyl}ethylidene)malononitrile (4). The structures of all compounds were identified by proton nuclear magnetic resonance spectroscopy, infrared spectroscopy, mass spectrometry, and ultraviolet-visible (UV-Vis) spectroscopy. The UV-Vis and fluorescence spectra of 1-{6-[(2-hydroxyethyl)(methyl) amino]-2-naphthyl}ethan-1-one (3), 4, and 5 in solvents with different polarities were investigated, and the effects of solvent polarity on the optical properties of the three compounds were studied. The objective product 5 showed high binding affinities toward Aβ(1-40) aggregates in vitro (K _d = 29.4 nmol/L) by fluorophotometry. This study provides a powerful fluorescent probe for the molecular diagnosis of Alzheimer's disease.

Full text of DOI:10.1007/s11164-013-1429-0

Res Chem Intermed
DOI 10.1007/s11164-013-1429-0
Synthesis and optical properties of 4-(2-{[6-(1,1-
dicyanoprop-1-en-2-yl)naphthalen-2-yl] (methyl)amino}
ethoxy)-4-oxobutanoic acid fluorescent probe for
b-amyloid
•
HuanBao Fa JingTing Zhou Dong Zhang Wei Yin
•
•
•
•
HaiFeng Zhang DanQun Huo ChangJun Hou
•
•
•
XiaoGang Luo YaLi Mao Jin Zhang
•
Received: 24 March 2013 / Accepted: 30 September 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract A novel 4-(2-{[6-(1,1-dicyanoprop-1-en-2-yl)naphthalen-2-yl](methyl)
amino} ethoxy)-4-oxobutanoic acid (5) fluorescent probe for b-amyloids was syn-
thesized by catalytic acylation using 4-dimethylaminopyridine between succinic
anhydride and (1-{6-[(2-hydroxyethyl)(methyl) amino]-2-naphthyl}ethylidene)mal-
ononitrile (4). The structures of all compounds were identified by proton nuclear
magnetic resonance spectroscopy, infrared spectroscopy, mass spectrometry, and
ultraviolet–visible (UV–Vis) spectroscopy. The UV–Vis and fluorescence spectra of
1-{6-[(2-hydroxyethyl)(methyl) amino]-2-naphthyl}ethan-1-one (3), 4, and 5 in sol-
vents with different polarities were investigated, and the effects of solvent polarity on
the optical properties of the three compounds were studied. The objective product 5
showed high binding affinities toward Ab(1–40) aggregates in vitro (Kd = 29.4 nmol/L)
by fluorophotometry. This study provides a powerful fluorescent probe for the molecular
diagnosis of Alzheimer’s disease.
H. Fa ꢀ J. Zhou ꢀ W. Yin ꢀ H. Zhang ꢀ Y. Mao ꢀ J. Zhang
College of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400030, China
e-mail: haifengzhang@cqu.edu.cn
H. Fa ꢀ D. Huo ꢀ C. Hou ꢀ X. Luo
Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering
College, Chongqing University, Chongqing 400030, China
H. Fa (&)
College of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, China
e-mail: huanbaofa@cqu.edu.cn
J. Zhou
Third Military Medical University, Chongqing 400038, China
D. Zhang
Department of Radiology, Third Military Medical University, Chongqing 400037, China
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H. Fa et al.
Keywords Fluorescent probe ꢀ Synthesis ꢀ Optical property ꢀ b-Amyloid ꢀ
Alzheimer’s disease
Introduction
Alzheimer’s disease (AD) is the most common form of dementia affecting millions
of people around the world. The clinical symptoms of AD include cognitive decline,
irreversible memory loss, disorientation, language impairment, and so on. Major
neuropathology observations of postmortem AD brain show the presence of senile
plaques (SPs), neurofibrillary tangles (NFTs), and neurophil threads containing b-
amyloid aggregates and highly phosphorylated tau proteins. With the aging
population, AD is becoming one of the serious problems for human health and
living quality [1–3]. Pathogenesis studies showed that SPs and NFTs are hallmark
pathologies accompanying neurodegeneration. Furthermore, the growth of b-
amyloid plaques in the brain is a major cause of AD [4–7].
In recent years, with the development of medical technology, molecular imaging
technologies, such as positron emission tomography (PET), single-photon emission
computed tomography, and magnetic resonance imaging (MRI), have been widely
used for the early diagnosis of AD. Thus, the design of molecular imaging probes
for b-amyloid plaques in the brain has drawn increasing research attention [8, 9].
Several specific binding agents for Ab are derivatives of naphthalene, benzothi-
azole, stilbene, or other related heterocyclic derivatives with an electron-donating
group on the aromatic rings [10]. However, several studies suggested that analogs of
pathological staining dye, such as Chrysamine G and Congo Red [11–13], and
styrylbenzenes, such as 1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzene (X-
34) [14, 15], (E,E)1-bromo-2,5-bis- (3-hydroxyl carbonyl-4-hydroxy)styrylbenzene
(ISB) [16], (trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene
(BSB) [17], and (E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-methoxy)styryl-benzene
(IMSB) [18], are not suitable as imaging agents because of their low uptake in the
brain. To achieve high brain penetration, the use of neutral, small, and lipophilic
compounds is normally considered. 1,1-Dicyano-2-[6-(dimethylamino) naphtha-
lene-2-yl] propene (DDNP), a highly hydrophobic, viscosity-, and solvent-sensitive
fluorescent probe has been used previously to label SPs in the brains of AD patients
by PET [19, 20, 31].
Considering that the as-synthetic DDNP described by Jacobson et al. [21] had no
active group such as carboxyl to bind onto the surface of superparamagnetic iron oxide
nanoparticles (SPIONs), a novel MRI contrast agent of SPIONs coated with DDNP for
b-amyloid protein was needed. We synthesized a 4-(2-{[6-(1,1-dicyanoprop-1-en-2-
yl)naphthalen-2-yl] (methyl)amino} ethoxy)-4-oxobutanoic acid (5) fluorescent
probe with a –COOH group to target b-amyloid proteins and combine with SPIONs
through ligand exchange with oleic acid. Compound 5 was synthesized by treating (1-
{6-[(2-hydroxyethyl) (methyl) amino]-2-naphthyl}ethylidene)malononitrile (4) with
succinic anhydride through catalytic acylation using 4-dimethylamiopryidine
(DMAP). Compound 4 was prepared from 2-acetyl-6-methoxy-naphthalene (1) by
hydrolysis, Bucherer nucleophilic substitution, and Knoevenagel condensation
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Fluorescent probe for b-amyloid
reactions. The structures of all compounds were identified by proton nuclear magnetic
resonance spectroscopy (1H NMR), infrared spectroscopy (IR), magnetic spectrom-
etry (MS), and ultraviolet–visible (UV–Vis) spectroscopy. The UV–Vis and
fluorescence spectra of 1-{6-[(2-hydroxyethyl)(methyl)amino]-2-naphthyl}ethan-1-
1 (3), 4, and 5 in solvents of different polarities were investigated, and the effects of
solvent polarity on the optical properties of the three compounds were studied. The
objective product 5 showed high binding affinities toward Ab(1–40) aggregates
in vitro (Kd = 29.4 nmol/L) by fluorophotometry. This study provides a powerful
fluorescent probe for the molecular diagnosis of AD. The synthetic route of compound
5 is shown in Scheme 1.
Experimental
Apparatus
1H NMR spectra were obtained using a Brucker 500MHZ spectrometer. IR spectra
were obtained using
a
Nicolet Shimadzu Fourier transform-infrared
O
O
HO
N
HCl
H
HO
O
Na2S2O5 H2O
2
1
N
N
C
C
O
NC
CN
HO
HO
N
N
pyridine
4
3
N
N
C
C
O
O
O
O
triethylamine DMAP
O
HO
N
O
5
Scheme 1 Synthetic routes of compound 5
123

H. Fa et al.
spectrophotometer. UV–Vis spectra were recorded on an Analytik Jena SPECORD
200 spectrophotometer. Fluorescence spectra were determined using a Shimadzu
RF-5301 spectrophotometer. MS spectra were obtained with a VG Auto Spec-3000
spectrophotometer. The melting points were measured using an SGW X-4
microscopic melting point apparatus.
Materials
DMAP, succinic anhydride, triethylamine, EtOH, and ethyl acetate were purchased
from Aladdin, Shanghai, China. Compound 1 was obtained from Tokyo Chemical
Industry, Japan. The in vitro binding experiment was carried out at 37 °C in a pH
7.4 phosphate-buffered medium. Sodium dihydrogen phosphate and disodium
hydrogen phosphate were purchased from Xiya, Chengdu. b-amyloid was obtained
from Merck, Taiwan. All other chemicals were of analytical grade.
Synthesis of compound 5
1-(6-Hydroxy-2-naphthyl)-1-ethanone (2)
Compound 2 was prepared in accordance to a previous method [22]. A solution of
350 mL of HCl (d = 1.16) was stirred in a three-neck round-bottom flask and
heated to boiling. Subsequently, a solution of 1.212 g (6.06 mmol) of 1-(6-
methoxy-2-naphthyl)-1-ethanone (1) in a minimum amount of dichloromethane was
added, and the mixture was stirred and heated at reflux for 2 h. The hot solution was
filtered through a mineral wool plug to remove the oily residue. The solid that
separated after cooling was filtered through a glass frit and dissolved in 26 mL of
ethyl acetate. The solution was washed with brine, dried over anhydrous magnesium
sulfate, and evaporated to yield a product of 1 g (84 %). After recrystallization from
triethylamine, the sample was melted at 174–177 °C (Ref. [22]: 173.5–177 °C);
1
UV–Vis (EtOH) kmax 317 nm; H NMR (DMSO-d6, 500 MHZ) d 2.650 (s, 3H,
COCH3), 7.173 (d, J = 8.5 Hz, 1H, H–C7), 7.193 (d, 2.5 Hz, 1H, H–C5), 7.750 (d,
J = 9 Hz, 1H, H–C4), 7.864 (dd, J = 8.5 and 2.5 Hz, 1H, H–C8), 7.971 (d,
J = 9 Hz, 1H, H–C3), 8.537 (s, 1H, H–C1), 10.212 (s, 1H, OH); IR (KBr) m/cm-1
3,362.0, 3,073.7, 2,999.7, 2,926.0, 1,661.7, 1,627.5, 1,582.9, 1,570.4, and 1,207.1.
Compound 3
Compound 3 was prepared by Bucherer nucleophilic substitution reaction as
described previously [23]. Briefly, a mixture of compound 2 (744 mg, 3.92 mmol),
sodium metabisulfite (3.5 g, 18 mmol), 2-ethylaminoethanol (5.2 mL, 64 mmol),
and water (34 mL) was heated in an autoclave at 125–130 °C for 64 h. After
cooling, the mixture was evaporated to dryness. The residue was treated with
CH2Cl2/CH3OH (8:2) and filtered. The filtrate was evaporated and purified by
chromatography on silica gel and eluted stepwise with CH2Cl2, CH2Cl2/EtOAc
(98:2), and CH2Cl2/EtOAc (96:4) to yield a product of 671 mg (Yield, 67.2 %).
M.p. 99.5–105 °C (Ref. [23]: 105.5–106 °C); thin-layer chromatography (TLC): Rf
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Fluorescent probe for b-amyloid
0.4 (CH2Cl2/EtOAc V:V = 98:2); TLC: Rf 0.4 (CH2Cl2/EtOAc V:V = 98:2); UV–
1
Vis (EtOH) kmax 373 nm; H NMR (DMSO-d6, 500 MHZ) d 2.602 (s, 3H, CH3),
3.071 (s, 3H, NCH3), 3.573 (t, J = 6 Hz, 2H, NCH2), 3.828 (t, J = 6 Hz, 2H,
OCH2), 7.115 (d, J = 2.3 Hz, 1H, H–C5), 7.178 (dd, J = 8.6 and 2.3 Hz, 1H,
H–C7), 7.556 (d, J = 8.2 Hz, 1H, H–C4), 7.719 (d, J = 8.6 Hz, 1H, H–C8), 7.846
(d, J = 8.2 HZ, 1H, H–C3), 8.243 (s, 1H, H–C1); IR (KBr) m/cm-1 3,458.9, 3,063.6,
2,933.8, 2,881.3, 1,669.0, 1,651.0, 1,621.2, 1,508.2, 1,490.7, and 1,204.7.
Compound 4
A solution of compound 3 (421 mg, 1.73 mmol) and malononitrile (4.5 mL,
6.93 mmol) in pyridine (10 mL) was stirred and heated at 105–110 °C under a
slow stream of argon for 24 h. TLC indicated full consumption of the starting
material. The reaction mixture was evaporated and then coevaporated at least
twice with toluene to remove pyridine. The crude reaction mixture was purified by
column chromatography on silica gel and eluted stepwise with CH2Cl2, CH2Cl2/
EtOAc (98:2), and CH2Cl2/EtOAc (95:5) to give a product of 411 mg, (Yield,
82.6 %). TLC: Rf 0.42 (CH2Cl2/EtOAc 95:5); M.p. 126–129.5 °C (Ref. [23]:
1
128–131 °C); UV–Vis (EtOH) kmax 438 nm; H NMR (DMSO-d6, 500 MHZ) d
2.688 (s, 3H, CH3), 3.091 (s, 3H, NCH3), 3.571 (t, J = 4.5 Hz, 2H, NCH2), 3.610
(t, J = 4.5 Hz, 2H, OCH2), 4.752 (t, J = 4.5 Hz, 1H, OH), 6.955 (s, 1H, H–C5),
7.295 (d, J = 9 Hz, 1H, H–C7), 7.626 (d, J = 8.5 Hz, 1H, H–C3), 7.704 (d,
J = 8.5 Hz, 1H, H–C4), 7.817 (d, J = 9 Hz, 1H, H–C8), 8.168 (s, 1H, H–C1); IR
(KBr) m/cm-1 3,512.4, 3,074.8, 2,923.4, 2,223.8, 1,620.5, 1,546.2, 1,504.4, and
1,188.9.
Compound 5
A mixture of succinic anhydride (125 mg, 1.25 mmol), DMAP (124 mg, 1 mmol),
and water-free CH2Cl2 (10 mL) was magnetically stirred at room temperature
under nitrogen for 30 min. Subsequently, 291 mg (1 mmol) of compound 4 and
140 lL of water-free triethylamine were added. The reaction mixture was
continuously stirred overnight at room temperature. The solution was then
evaporated to dryness. The residue was purified by chromatography on silica gel
and eluted stepwise with CH2Cl2, CH2Cl2/CH3OH (99:1), and CH2Cl2/CH3OH
(98:2) to give a product of 211 mg (Yield, 54.1 %). M.p. 135.8–137.2 °C; UV–
1
Vis (CH2Cl2) kmax 432 nm; H NMR (DMSO-d6, 500 MHZ) d 2.425 (s, 4H, CO–
CH2–CH2–CO), 2.699 (s, 3H, C=CCH3), 3.090 (s, 3H, N–CH3), 3.773 (t,
J = 5.5 Hz, 2H, N–CH2), 4.244 (t, J = 5.5 Hz, 2H, O–CH2), 7.013 (s, 1H, H–C5,
7.322 (dd, J = 2.5 and 9 Hz, 1H, H–C4), 7.644 (dd, J = 2.5 and 9 Hz, 1H, H–
C8), 7.738 (d, J = 9 Hz, 1H, H–C7), 7.850 (d, J = 9 Hz, 1H, H–C3), 8.190 (s, 1H,
H–C1), 12.234 (s, 1H, COOH); IR (KBr) m/cm-1 3,438.4, 3,013.1, 2,959.8,
2,924.9, 2,215.6, 1,728.1, 1,705.5, 1,618.1, 1,532.9, 1,505.3, and 1,167.7; MS
(70 eV) m/z %: 390.1 (M?).
123

H. Fa et al.
Solution preparation
Spectral analysis
Compounds 1–5 were dissolved in EtOH to obtain a final concentration of
2 9 10-5 mol dm-3 and then the UV–Vis and fluorescence spectra were studied.
Solvent effects
Compounds 3, 4, and 5 were dissolved in nine solvents including cyclohexane, toluene,
CH2Cl2, CHCl3, CH3COOC2H5, EtOH, CH3OH, PBS, (pH 7.4), and AcOH to obtain a
final concentration of 2 9 10-5 mol dm-3. In the phosphate buffer solution (PBS) and
cyclohexane solutions, compounds 3, 4, and 5 were initially dissolved in a minimum
amount of methanol and diluted by PBS and cyclohexane to obtain the desired
concentration. The spectral shift will be less than 0.5 nm and could be negligible when the
methanol concentration is less than 2 % [24]. The UV–Vis and fluorescence spectra of the
above solutions were determined.
Binding of compound 5 to Ab(1–40) fibril in vitro
Ab(1–40) fibril formation
Ab(1–40) fibrils were prepared as described previously [25]. Briefly, 0.5 mg of Ab(1–40)
was dissolved in 1 mL of PBS (pH 7.4) and mixed with a magnetic stir bar for 3 days at
37 °C in a bed temperature incubator. The solution became visibly cloudy. The fibrils
were used immediately after their production was confirmed.
Binding of compound 5 to Ab(1–40) fibril
Fresh solutions of compound 5 in EtOH were appropriately diluted with PBS (pH
7.4) to obtain the following series of concentrations: 0.1, 0.5, 1.0, 1.5, 2.0, 2.5,
3.0, 3.5, and 4.0 9 10-7 mol/L. Each solution was treated as follows: first,
300 lL of compound 5 was pipetted into a 500-lL cuvette; second, 5 lL of Ab
solution and 300 lL of compound 5 solution were pipetted into another 500-lL
cuvette; finally, the two solutions were placed in a bed temperature incubator at
37 °C and 300 r/min for 15 min. The fluorescence intensity of each sample was
measured (integrated peak area) three times with three parallels at kex = 440 nm
using a fluorescence spectrophotometer. The binding of compound 5 to Ab(1–40)
was illustrated by a double-reciprocal plot, with the compound 5 concentration as
the abscissa and the reciprocal of fluorescence intensity difference between the
two solutions as the vertical axis.
123

Fluorescent probe for b-amyloid
Results and discussion
Recrystallization of compound 2
Previous studies indicated that the recrystallization solvent of compound 2 is ethyl
acetate [22]. In our study, triethylamine was used as the recrystallization solvent for
compound 2, which is weakly acidic and easily soluble in alkaline triethylamine,
and the neutral impurities are insoluble in triethylamine. Compound 2 was easily
separated by a suction filter, and the filtrate was neutralized with diluted HCl. The
white crystal 2 could be obtained from the filtrate. The purification result was better
than that reported previously [22].
IR spectra
The IR spectra of compounds 4 and 5 in Fig. 1 show a broad absorption band at
3,550 cm-1(mO–H) to 3,390 cm-1(mO–H), which implied the presence of a hydroxyl
group in the compound. The broad peak from 2,850 to 2,930 cm-1 represented the
stretching vibration of C–H, and the peak from 2,230 to 2,210 cm-1 was assigned to
the stretching vibration of C:N. The peaks at 1,504, 1,533, and 1,618 cm-1 were
characteristic absorption peaks of the aromatic rings. The C–O stretching vibration
at 1,188.9 and 866 cm-1 was assigned to the bending vibration of C–H in the
aromatic ring plane. Compared with compound 4, compound 5 had an anhydride
group, which indicated that their IR spectra were basically the same. The
characteristic absorption bands of the anhydride group at 1,728.1 cm-1 (mC=O in
carboxyl group) and 1,705.5 cm-1(mC=O in ester group) were found in the IR spectra
of compound 5. The bands at 1,100–1,120 cm-1 were attributed to the bending
vibration of C–O and C–N.
1H NMR of compound 5
The chemical structure of compound 5 was characterized by 1H NMR in deuterated
dimethyl sulfoxide (DMSO-d6) solution, and the results are shown in Fig. 2. The
hydrogen atoms of compound 5 were marked as 1, 2, 3, 4, 5, 6, and 7. As shown in
the figure, the signals at 2.425 and 12.234 ppm were attributed to the signals of 1
proton of –COCH2CH2CO– and 7 protons of –COOH, respectively. The singlet
peaks at 2.699 and 3.090 ppm were ascribed to =CCH3 and –NCH3, respectively.
The three sharp peaks at 3.763–3.784 and 4.246–4.266 ppm were attributed to the
methylene protons of –NCH2 and –OCH2, respectively. The peaks of the 6 protons
of the aromatic ring were found at 7.013–8.190 ppm.
UV–Vis spectra
Figure 3 shows the UV–Vis spectra of compounds 1–5 in EtOH solution. The
absorption bands at 210–250, 255–270, and 270–550 nm were attributed to the E1,
E2, and B characteristic bands of the naphthalene ring, respectively. The B band
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H. Fa et al.
Fig. 1 IR spectra of compound 5 (a) and compound 4 (b)
originally possessed a vibrational and rotational fine structure. However, consid-
ering that the naphthalene derivatives were affected by polar solvent molecules and
could not rotate freely, the fine structure of the B absorption band of the five
compounds disappeared completely and became a broad absorption band [26]. In
addition, the 6-position of the naphthalene ring was replaced by the auxochromic
group –OR, –OH, or –NR2 whose lone electron pairs could form p-p conjugated
systems. The conjugation effect led to the red shift of the B absorption band.
As shown in Fig. 3, the maximum absorption wavelengths of the B absorption
band of the five compounds were 308, 317, 373, 438, and 432 nm. The 6-position of
the naphthalene ring of compound 3 was replaced by –NR2. 2-Hydroxyethyl
methylamino is a strong electron donor group and can increase the electron cloud
density of the naphthalene ring. Thus, compared with compound 2, the B absorption
band of compound 3 showed a red shift of 56 nm. However, the B absorption band
of compound 4 showed a red shift of 65 nm compared with compound 3 after
introduction of the chromophore –C=C(CN)2 to the 2-position of the naphthalene
ring by Knoevenagel condensation reaction between compound 3 and malononitrile,
which enhanced the conjugated system of compound 4. Ester and carboxyl groups
were introduced to compound 5 by acylation between succinic anhydride and
compound 4. Compared with compound 4, compound 5 showed a blue shift of 6 nm
in the B absorption band. The electron-withdrawing C=O groups reduced the
conjugation effect of –NR2 when naphthalene was reduced.
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Fluorescent probe for b-amyloid
Fig. 2 1H NMR of compound 5
Different solvents have different impacts on the absorption wavelength and
intensity of the UV–Vis spectra [27]. To investigate the behavior of compounds 3, 4,
and 5 in different solutions, their UV–Vis spectra were measured in nine solvents of
various polarities, including cyclohexane, toluene, ethyl acetate, methylene
chloride, chloroform, acetic acid, EtOH, methanol, and PBS. The maximum
absorption wavelength (kmax) and molar extinction coefficient of the three
compounds are shown in Table 1. The results indicated that the UV–Vis spectra
of the three compounds were highly dependent on the nature of the solvent. A
stronger solvent polarity resulted in a greater red-shift degree of the maximum
absorption wavelength. With enhanced solvent polarity, the decrease in the p*
molecular orbital energies of compounds 3, 4, and 5 was larger than the ground state
p orbital energy. Thus the p ? p* transition energy change DE was reduced and
k
max was red-shifted [27]. As shown in Table 1, the kmax range of compounds 3, 4,
and 5 in the nine solvents were 343–381, 423–441, and 421–437 nm, respectively.
The molar extinction coefficient data indicated that compounds 3 and 4 possessed
maximum absorbance in methanol and minimum absorbance in dichloromethane,
whereas compound 5 possessed maximum absorbance in EtOH and minimum
absorbance in PBS and acetic acid.
123

H. Fa et al.
Fig. 3 UV–Vis spectra of compounds 1–5, final concentration in ethanol 5 9 10-6 mol/L
Table 1 kmax and e for compounds 3–5 in different solvents
Solvent
PBS MeOH EtOH AcOH CHCl3 CH2Cl2 CH3COOC2H5 PhCH3 CyH
ET(30)25 63.1 55.4
kmax (nm)
51.9
51.7
41.1
39.1
38.1
33.9
30.9
3
4
5
381
441
437
374
439
434
373
438
432
376
433
428
357
437
431
354
435
431
352
432
426
350
432
422
343
423
421
e(1 9 10-4) (dm3 mol-1 cm-1
)
3
4
5
1.7
1.8
1.7
2.2
1.9
1.8
1.8
1.7
1.9
1.8
1.8
1.7
1.8
1.7
1.8
1.6
1.6
1.8
2.0
1.7
1.8
1.8
1.7
1.8
1.7
1.8
1.8
PBS phosphate-buffered saline (pH 7.4), ET(30)25 empirical parameters of solvent polarity, the higher
value of ET(30)25, the greater solvent polarity
Fluorescence spectrum
Compound 5 was developed as a lipophilic probe for fluorescence spectroscopy to
target b-amyloid [28]. Figures 4 and 5 show excitation spectra and emission spectra
of compounds 1–5 in 10-6 mol/L EtOH. From Fig. 4, the excitation maxima of
compounds 1–5 were 324, 334, 379, 458, and 462 nm respectively. Considering the
highly conjugated aromatic systems and rigid plane structures of compounds 1–5,
123

Fluorescent probe for b-amyloid
their lowest singlet excited states (S1) were p ? p* transition, and they all had
excellent fluorescence performance [21]. The emission maxima of compounds 1–5
were observed at 421, 427, 483, 575, and 566 nm, respectively. The hydroxyl group
at the 6-position of compound 2 was substituted by –NR2. –NR2 is an electron-
donating group and its electron cloud is parallel to the p orbits of the aromatic ring
to form p–p conjugation, which resulted in the approximately 56-nm red shift of the
maximum emission peak and the increase in the fluorescence intensity of compound
3. The introduction of the chromophore –C=C(CN)2 group increased the conjugate
system and conjugate degree of compound 4. Thus, the non-localized p electrons
were easily excited, and the fluorescence spectrum changed obviously with a 92-nm
red shift. Compared with compound 4, the maximum emission peak of compound 5
was blue-shifted by 9 nm, and the fluorescence intensity was decreased. These
spectral changes were attributed to the ester and carboxyl groups of compound 5.
The C=O in compound 5 is an electron-withdrawing group that reduced the electron
density of –NR2, which weakened the conjugate interaction between –NR2 and the
aromatic ring.
The physical constants (dipole moment, dielectric constant, and refractive index)
of the nine solvents and the fluorescence excitation wavelength (kex), emission
wavelength (kem), and Stokes shift of compounds 3, 4, and 5 in the nine different-
polarity solvents are shown in Table 2. The results indicated that the kex and kem of
compounds 3, 4, and 5 showed a significant red shift when the solvent polarity was
enhanced. The fluorescence maximum emission ranges of the three compounds
were 395–492, 474–585, and 465–575 nm. Compound 5 possessed maximum kem in
Fig. 4 The excitation spectra of compounds 1–5, final concentration in ethanol 5 9 10-6 mol/L
123

H. Fa et al.
Fig. 5 The emission spectra of compounds 1–5, final concentration in ethanol 5 9 10-6 mol/L
EtOH and minimum kem in cyclohexane. The solvent effect indicated that
compounds 3, 4, and 5 were intramolecular electron-transfer complexes, the
electrons of which transfer from the electron donor –NR2 to the electron acceptor
naphthalene ring. Compounds 3, 4, and 5 were neutral molecules in the ground state,
but their polarities were increased when their p electrons were excited. The
stabilization role of solvents in the excited states was stronger than in the ground
state in the polar solvent. Thus, the transition energy was reduced with the increase
in solvent polarity, and the maximum emission peak was red-shifted [28].
The dipole moment change in compound 5 at ground state and excited state could
be calculated by the Lippert equation:
ꢀ
ꢁ
2 þ k;
ð1Þ
2
e ꢁ 1
n2 ꢁ 1 ðlà ꢁ lÞ
ta ꢁ tf ffi
ꢁ
hc 2e þ 1 2n2 þ 1
a3
where ma and mf are the absorption and emission wave numbers in the same solvent,
respectively, (ma - mf) is the Stokes shift, h is the Planck constant, c is the speed of
light, e and n are the dielectric constant and refractive index of the solvent,
respectively, l* and l are the electronic dipole moment of the compound at excited
state and ground state, respectively, and a is the ball volume radius of the solute
molecules in solution. According to the crystallography data of naphthalene
derivatives, the distance from N to O of compound 5 is approximately 0.84 nm [29].
In Eq. 1, the directional polarization rate Df is as follows:
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Fluorescent probe for b-amyloid
123

H. Fa et al.
Fig. 6 Stokes shift of compound 5 versus Df values
Fig. 7 Fluorescence enhancement of compound
4 9 10-7 mol/L in PBS
5
binding with Ab(1–40), final concentration
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Fluorescent probe for b-amyloid
Fig. 8 Double reciprocal illustration of compound 5 band with Ab(1–40)
e ꢁ 1 n2 ꢁ 1
Df ¼
ꢁ
:
ð2Þ
2eþ1 2n2 þ 1
As shown in Fig. 6, the straight line represents the mapping of the Stokes shifts
of compound 5 in different solvents with Df. The slope of the line was 8,921.5,
R2 = 0.9215. The dipole moment change of compound 5 at ground state and excited
state was 7.25 D, which was calculated using the line slope and the Lippert
equation. The intramolecular electron transfer in compound 5 was induced after
excitation, which resulted in intramolecular electron rearrangement. Such rear-
rangement increased the dipole moment difference between the excited state and the
ground state. Electronic rearrangement in the solvent molecules around compound 5
was likewise induced to adapt to the new charge distribution of compound 5. This
result narrowed the energy gap between the excited state and the ground state. Thus,
the increase in solvent polarity caused the red shift in the maximum emission peak.
Binding of compound 5 to Ab(1–40) fibrils in vitro
Figure 7 shows the fluorescence spectrum of compound 5 with 4 9 10-7 mol/L in
PBS binding to Ab(1–40). An enhanced fluorescence emission was observed upon
binding of the Ab(1–40) fibrils to compound 5. The strong viscosity of Ab(1–40)
fibrils limited the intramolecular rotational relaxation of compound 5 and induced
compound 5 to form a rigid plane structure that could improve the fluorescence
quantum yield and enhance the fluorescence integral intensity of compound 5 [30].
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H. Fa et al.
The double reciprocal illustration of compound 5 binding to Ab(1–40) was used
to study the combination of Kd in Fig. 8, with the concentration of compound 5 as
the abscissa and the reciprocal of fluorescence intensity difference between the two
solutions as the vertical axis. As shown in Fig. 8, the intersection between the
straight lines and the x-axis was -1/Kd = -3.39 and Kd = 29.4 nmol/L. Kd was
inversely proportional to the affinity of the molecules. The results showed that
compound 5 has potential for the early detection and monitoring of the progression
of AD.
Conclusions
We designed and synthesized a 4-(2-{[6-(1,1-dicyanoprop-1-en-2-yl) naphthalen-2-
yl](methyl)amino}ethoxy)-4-oxobutanoic acid fluorescent probe for b-amyloid that
can be used for the early detection of AD. The structures of all compounds were
confirmed by 1H NMR, IR, MS, and UV–Vis techniques. The UV–Vis and
fluorescence spectra of compounds 3, 4, and 5 in the nine solvents with various
polarities were investigated, and the effects of solvent polarity on the optical
properties of the three compounds were studied. The solvent effect indicated that
k
max, kex, and kem were red-shifted with the increase in solvent polarity. According
to the Lippert equation, the dipole moment change of compound 5 between the
ground state and excited state was 7.25 D. This result caused the electrons of the
solvent molecules around compound 5 to undergo electronic rearrangement. Such
rearrangement narrowed the energy gap between the excited state and ground state
and caused the red shift in the maximum emission peak with the increase in solvent
polarity. Compound 5 showed high binding affinity toward Ab(1–40) aggregates
in vitro (Kd = 29.4 nmol/L) by fluorophotometry. These results suggest that
compound 5 can be used for the early detection and monitoring of the progression of
AD. The conjugation of compound 5 with SPIONs to obtain an MRI contrast agent
shall be further studied.
Acknowledgments We gratefully acknowledge the support of this research by National natural science
funds (NSFC, No. 30970799, 31101284), Natural Science Foundation Project of CQ CSTC (No. CSTC,
2008BB5285, 2010BB1209), Central College Operational costs of basic research (No. CQDXWL-2012-
034, CQDXWL-2012-035).
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