Synthesis, photophysical properties, and DNA-binding of novel A-π-D-π-A’ two-photon absorption chromophores

Article abstract of DOI:10.1016/j.jphotochem.2018.05.038

Three novel two-photon absorption chromophores (1BI, 2TAZ, and 3PyB), which have trans-1,4-divinylbenzene π-bridge, methoxy donors, and different acceptors, were synthesized. Their structures were elucidated by means of hydrogen-1 nuclear magnetic resonan

Full text of DOI:10.1016/j.jphotochem.2018.05.038

Accepted Manuscript
Title: Synthesis, photophysical properties, and DNA-binding
of novel A-␲-D-␲-A’ two-photon absorption chromophores
Authors: Fei-Fei Ma, Zhi-Bin Cai, Sheng-Li Li, Yu-Peng Tian
PII:
S1010-6030(18)30192-8
DOI:
Reference:
https://doi.org/10.1016/j.jphotochem.2018.05.038
JPC 11308
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
11-2-2018
21-5-2018
22-5-2018
Please cite this article as: Ma F-Fei, Cai Z-Bin, Li S-Li, Tian Y-Peng, Synthesis,
photophysical properties, and DNA-binding of novel A-␲-D-␲-A’ two-photon
absorption chromophores, Journal of Photochemistry and Photobiology, A: Chemistry
(2018), https://doi.org/10.1016/j.jphotochem.2018.05.038
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Synthesis, photophysical properties, and DNA-binding of novel A-π-D-π-A'
two-photon absorption chromophores
a
a,*
b
b
Fei-Fei Ma , Zhi-Bin Cai , Sheng-Li Li , Yu-Peng Tian
a
College of Chemical Engineering, Zhejiang University of Technology, Hangzhou
10014, PR China
3
b
Department of Chemistry, Anhui Province Key Laboratory of Functional Inorganic
Materials, Anhui University, Hefei 230039, PR China
Graphical abstract
One of the novel A-π-D-π-A' compounds synthesized by us (3PyB) has good water
solubility, once bound to ctDNA, the two-photon action cross-section can be
increased to 45 GM, which may serve as a potential DNA fluorescent probe.
*
Corresponding author. Tel. : +86 571 88320891.
E-mail address: caizbmail@126.com (Z.-B. Cai).
Graphical abstract
1

Highlights
The highlights of this manuscript are as follows:
1
2
. Three novel A-π-D-π-A' two-photon absorption chromophores were synthesized.
. The photophysical properties in various solvents were systematically
investigated.
. The structure-property relationships were discussed aided with the DFT
calculations.
. The DNA-binding of 3PyB was systematically investigated.
3
4
Abstract
Three novel two-photon absorption chromophores (1BI, 2TAZ, and 3PyB), which
have trans-1,4-divinylbenzene π-bridge, methoxy donors, and different acceptors,
were synthesized. Their structures were elucidated by means of hydrogen-1 nuclear
magnetic resonance, carbon-13 nuclear magnetic resonance, Fourier transform
infrared spectroscopy, and high-resolution mass spectrometry. Their photophysical
properties, including linear absorption, single-photon excited fluorescence,
two-photon absorption, and two-photon excited fluorescence, were systematically
investigated in various solvents. The structure-property relationships were studied
aided with the density functional theory calculations. In addition, the absorption,
single- and two-photon fluorescence spectra of 3PyB-ctDNA were examined to
evaluate whether 3PyB could be used as a DNA stain. The possible interaction
2

mechanism of 3PyB-ctDNA was also studied by the NaCl titration and the viscosity
experiment. The results indicate that 3PyB may serve as a potential DNA fluorescent
probe.
Keywords: Two-photon absorption; DNA-binding; A-π-D-π-A' compound;
Structure-property relationship
1
. Introduction
In 1931, the concept of the two-photon absorption (TPA) process was first
[1]
proposed by Göppert-Mayer , which involves simultaneous absorption of two
photons of identical or different frequencies and leads to excitation of a molecule
from the ground state to the excited state. Since then, organic materials with TPA
properties have developed rapidly and been applied in various research areas, such as
[
2]
[3]
two-photon photodynamic therapy , three-dimensional optical data storage , optical
[4]
[5]
[6]
limiting , up-converted lasing , three-dimensional microfabrication , and
[
7]
especially two-photon fluorescence microscopy and imaging . Recently, two-photon
fluorescence microscopy and imaging has attracted significant interest as results of its
unique advantages over conventional single-photon fluorescence microscopy and
imaging, including near infrared photons as excitation source, imaging in the black
background, avoidance of photodamage and photobleaching, high transverse and
longitudinal resolutions, small absorption coefficient of light in tissue, lower tissue
[8]
autofluorescence, etc . To achieve this application, materials must have large
two-photon action cross-sections (Φ×δ). The fluorescence quantum yield (Φ) gives
the efficiency of the fluorescence process, which is defined as the ratio of the number
[9]
of photons emitted to the number of photons absorbed . The TPA cross-section (δ) is
defined as the probability for simultaneous absorption of two photons by an atom or a
[
1]
molecule to be excited from the ground state to an upper energy state . Φ and δ are
the important parameters to characterize the fluorescence and TPA properties of
materials. Consequently, it is significant and essential to develop TPA materials with
3

large Φ×δ.
The structure of materials is a decisive factor for their TPA properties and
fluorescence properties. Only when the material can absorb light, it is possible to emit
fluorescence, so the structure of fluorescent materials usually contains strong
absorption groups with π-conjugated bonds[10]. In addition, a variety of researches
reveal that introduction of electron donors (D) and electron acceptors (A) to the
π-conjugated system can increase the degree of intramolecular charge transfer (ICT)
and improve the δ value[11]. Therefore, in order to obtain TPA materials with large
Φ×δ, it is critical to select the appropriate π-conjugated bridge, D, and A. The
trans-configuration of 1,4-divinylbenzene π-bridge has better planarity and
conjugation effect than the cis-configuration, which favors the intramolecular charge
delocalization. Methoxy, a relative strong electron-donating group, which has the lone
pair electrons supplied by the oxygen atom, can form p-π conjugation by connecting
with a benzene ring. The combination of the negative induction effect and the
negative conjugation effect makes the cyano group be a strong electron-withdrawing
group. Benzimidazole contains a benzene ring fused to an imidazole ring. The
imidazole ring is a noncentrosymmetric electron-deficient structure, which may link
to strong electron-donating groups and have excellent properties of electronic
transmission. s-Triazine is a six-membered N-heterocycle, which possesses good
coplanarity, good thermal stability, and strong electron affinity. Its derivatives exhibit
good application prospects in many aspects of organic optoelectronic functional
materials[12]. Owing to its positive charge, pyridinium cation not only has strong
electron-withdrawing ability but also can increase the water solubility of materials.
Numerous researches demonstrate that many chromophores with the above-mentioned
donors (methoxy[13]) or acceptors (cyano[14], benzimidazole[15], s-triazine[16]
,
pyridinium cation[17]) exhibit strong TPA and two-photon fluorescence properties.
Therefore, in order to obtain TPA materials with large Φ×δ, we designed and
synthesized three novel asymmetric TPA chromophores with A-π-D-π-A' structure
(
1BI, 2TAZ, and 3PyB) (Fig. 1), which have trans-1,4-divinylbenzene as
π-conjugated bridge, methoxy as D, cyano (benzimidazole, s-triazine, and pyridinium
4

1
13
cation) as A. Their structures were characterized by H NMR, C NMR, FT-IR, and
HRMS. Their linear and nonlinear photophysical properties in various solvents were
investigated. At the same time, the density functional theory (DFT) calculations were
utilized to survey their structure-property relationships.
Among the three materials, 3PyB has good water solubility, which is essential for
the realization of two-photon fluorescence microscopy and imaging in living cells.
Cells live in aqueous surroundings. The material that wants to cross the plasma
membrane into the cell must have good water solubility. DNA is the hereditary
information carrier for most living things, which plays an extremely important role in
the life process of breeding, inheritance, and variation of organisms. DNA detection
has become a hot topic for scientists of various subjects owing to its importance in
gene sequencing, biomedical research, diagnosis for the virus and so on. In this paper,
the linear absorption, single- and two-photon fluorescence spectra, and the interaction
mechanism of 3PyB-ctDNA were discussed to evaluate whether 3PyB could be used
as a fluorescent probe for DNA detection. The results show that 3PyB can bind to
DNA and has a moderate Φ×δ, which is promising to be an effective two-photon
DNA fluorescent probe.
5

Fig. 1. Molecular structures of the target compounds (1BI, 2TAZ, and 3PyB).
2
. Experimental
2
.1. Materials and instruments
Unless otherwise noted, all reagents and solvents were purchased from commercial
suppliers and used without further purification. DNA titration experiments were
-1
-1
performed in Tris-HCl buffer solution (10 mmol L Tris-HCl, 100 mmol L NaCl,
pH 7.2). The concentration of ctDNA was estimated by measuring the UV absorbance
-
1
-1
at 260 nm (ε = 6600 mol L cm ). All solutions were allowed to equilibrate for 5 min
before making measurements.
Melting points were measured on an X-4 micromelting point apparatus without
1
13
correction. H and C NMR spectra were recorded on a Bruker AVANCE III 500
spectrometer using DMSO-d or D O as a solvent and all shifts were referred to
6
2
tetramethylsilane (TMS). FT-IR spectra were recorded on a Thermo Nicolet 6700
spectrometer using a powder sample on a KBr plate. Mass spectra were performed on
a Thermo LCQ TM Deca XP plus ion trap mass spectrometry instrument.
The linear absorption spectra were measured on a Shimadzu UV-2550 UV-visible
spectrophotometer. The single-photon excited fluorescence (SPEF) spectra
measurements were performed using a RF-5301PC fluorescence spectrophotometer
with the maximum absorption wavelengths as the excitation wavelengths. The
two-photon excited fluorescence (TPEF) spectra were measured using a femtosecond
Ti : sapphire laser system (Chameleon UltraⅡ, 680-1080 nm, 80 MHz, 140 fs).
2
.2. Physical measurements
The Φ was determined using fluorescein in 0.1 mol L sodium hydroxide aqueous
-1
solution (Φ = 0.9[18]) as the standard according to the following equation:
6

2
A n F
r
s
s
Φ  Φr
(1)
s
2
A n F
s
r
r
where the subscripts s and r designate the sample and the reference, respectively. A is
the absorbance at the excitation wavelength, n is the refractive index of the relevant
solution, and F is the integrated area under the corrected emission spectrum.
The δ was obtained by comparing the TPEF intensity of the sample with that of a
reference compound by the following equation[19]
:
F Φ n c
r
s
r
r
s

r
(2)
F Φ n c
s
r
s
s
where the subscripts s and r denote the sample and the reference, respectively. F and
Φ represent the TPEF integral intensity and the fluorescence quantum yield. n and c
are the refractive index and the concentration of the solution.
The binding constant (k) was obtained by the single-photon fluorescence titration
method according to the Scatchard equation[20]
:
r

kn kr
(3)
cf
where k and n refer to the binding constant and the binding sites, respectively. c
f
is the
concentration of the free dye. r is a ratio of c
b
to cDNA. c
b
is the concentration of the
bound dye, and c
F  F0
b
is calculated by the following equation[21]
:
cb 
ct
(4)
Fmax  F0
where c
t
is the total dye concentration. F and F are the fluorescence integral intensity
0
of the free dye and the dye-DNA complex, respectively. Fmax is the saturated
fluorescence integral intensity in DNA titration.
Viscosity experiment was carried out using an Ubbelohde viscometer maintained at
a constant temperature of 25 ºC in a thermostatic waterbath. Data were presented as (η
1
/3
/
η
0
) versus the concentration of 3PyB-ctDNA solution, where η is the viscosity of
DNA in the presence of the dye, η is the viscosity of DNA alone. η was calculated
from the relation η = (t – t ) / t , where t is the observed flow time of 3PyB-ctDNA
solution, t
is the flow time of buffer[22]
0
0
0
0
.
7

2
2
.3. Synthesis
.3.1. 4-[(1E)-2-(4-Formyl-2,5-dimethoxyphenyl)ethenyl]benzonitrile (4b)
Trimethyl orthoformate (0.58 g, 5.5 mmol) was added slowly to the mixture of
,5-dimethoxy-1,4-benzenedicarboxaldehyde (1b) (0.97 g, 5 mmol) and methanol (30
2
mL). Then the mixture was stirred at room temperature for 16 h. The solvent was
evaporated under reduced pressure. The residue was purified by silica gel column
chromatography eluted with petroleum ether / ethyl acetate (10 : 1, V / V) to give
4
-(dimethoxymethyl)-2,5-dimethoxybenzaldehyde (1d) as a colorless oily liquid (0.87
1
g). Yield: 72.5%. H NMR (DMSO-d
6
, 500 MHz) δ: 10.34 (s, 1H), 7.25 (s, 1H), 7.21
(
s, 1H), 5.57 (s, 1H), 3.89 (s, 3H), 3.81 (s, 3H), 3.31 (s, 6H).
d (1.44 g, 6 mmol), [(4-cyanophenyl)methyl]phosphonic acid diethyl ester (4a)
1.52 g, 6 mmol), and potassium tert-butoxide (0.81 g, 7.2 mmol) in dry THF (25 mL)
1
(
were added into a dry flask under a nitrogen atmosphere with ice bath cooling. The
reaction mixture was stirred at room temperature for 16 h (monitored by TLC). Then
acetic acid (3.60 g, 60 mmol) in water was added and stirred at room temperature for
6
h. After neutralization with saturated aqueous sodium hydrogen carbonate solution
and extraction with CH Cl (2 × 30 mL), the combined organic layers were washed
2
2
with water (20 mL) and brine (20 mL), dried with anhydrous sodium sulfate, and then
filtered. The filtrate was concentrated under vacuum. The residue was purified by
silica gel column chromatography eluted with petroleum ether / ethyl acetate (15 : 1,
V / V) to give 4b as bright yellow needle crystals (1.24 g). Yield: 70.5%. m.p.
1
1
7
3
1
2
2
2
59-160 ºC; H NMR (DMSO-d
6
, 500 MHz) δ: 10.34 (s, 1H), 7.87 (d, J = 8.5 Hz, 2H),
.82 (d, J = 8.5Hz, 2H), 7.63 (s, 2H), 7.58 (s, 1H), 7.28 (s, 1H), 3.98 (s, 3H), 3.89 (s,
H); FT-IR (KBr) ν: 3060, 2947, 2846, 2224, 1670, 1601, 1488, 1468, 1410, 1320,
-1
268, 1212, 1036, 969, 890, 689, 550 cm ; HRMS (ESI): m/z calcd for C18
H
16NO :
3
+
94.1130 [M+H] ; found: 294.1119.
.3.2.
-[[4-[(1E)-2-(1H-Benzimidazol-2-yl)ethenyl]-2,5-dimethoxyphenyl]methylene]
propanedinitrile (1BI)
8

The mixture of
4
-[(1E)-2-(1H-benzimidazol-2-yl)ethenyl]-2,5-dimethoxybenzaldehyde (1c) (1.23 g, 4
mmol), propanedinitrile (0.29 g, 4.4 mmol), piperidine (0.34 g, 4 mmol), and
methanol (30 mL) was refluxed for 6 h. After cooling to room temperature, the
reaction mixture was filtered. The filter cake was recrystallized from DMF / water to
1
give 1BI as red needle crystals (1.04 g). Yield: 73.1%. m.p. 161-163 ºC; H NMR
(
DMSO-d
6
, 500MHz) δ: 12.97 (s, 1H), 8.42 (s, 1H), 7.92 (d, J = 16.6 Hz, 1H), 7.72 (s,
1
4
1
5
1
H), 7.60 (d, J = 16.6 Hz, 1H), 7.58 (s, 1H), 7.56-7.58 (m, 2H), 7.20-7.23 (m, 2H),
1
3
.00 (s, 3H), 3.91 (s, 3H); C NMR (DMSO-d , 125 MHz) δ: 154.24, 153.59, 150.64,
6
38.30, 132.76, 127.24, 122.46, 120.11, 114.68, 113.80, 110.61, 110.36, 80.20, 56.64,
6.11; FT-IR (KBr) ν: 3356, 3050, 2937, 2834, 2227, 1607, 1563, 1495, 1469, 1404,
-1
361, 1291, 1228, 1035, 966, 848, 742, 611 cm ; HRMS (ESI): m/z calcd for
+
C
21
H
17
N
4
O
2
: 357.1352 [M+H] ; found: 357.1364.
2
4
.3.3.
-[(1E)-2-[4-[(1E)-2-(4,6-Dimethyl-1,3,5-triazin-2-yl)ethenyl]-2,5-dimethoxyphenyl]
ethenyl]benzonitrile (2TAZ)
b (0.88 g, 3 mmol) dissolved in methanol (30 mL) was added dropwise to the
mixture of 2,4,6-trimethyl-s-triazine (2a) (0.55 g, 4.5 mmol), potassium hydroxide
0.3 g), and methanol (30 mL). The reaction mixture was refluxed for 12 h. After the
4
(
solvent was removed under reduced pressure, the residue was purified by silica gel
column chromatography eluted with petroleum ether / ethyl acetate (8 : 1, V / V) to
1
give 2TAZ as yellow crystalline powder (0.78 g). Yield: 65.3%. m.p. 186-188 ºC; H
NMR (DMSO-d
6
, 500MHz) δ: 8.42 (d, J = 16.1 Hz, 1H), 7.84 (d, J = 8.4 Hz, 2H),
.79 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 16.5 Hz, 1H), 7.51 (d, J = 16.5 Hz, 1H), 7.49 (s,
H), 7.43 (s, 1H), 7.31 (d, J = 16.1 Hz, 1H), 3.96 (s, 3H), 3.93 (s, 3H), 2.55 (s, 6H);
, 125 MHz) δ: 175.55, 170.60, 152.21, 151.28, 141.99, 135.42,
32.63, 128.66, 127.77, 127.15, 126.16, 126.08, 124.15, 118.95, 110.82, 109.82,
09.57, 56.25, 56.18, 25.16; FT-IR (KBr) ν: 3053, 2922, 2852, 2221, 1628, 1602,
7
1
13C NMR (DMSO-d
6
1
1
1
-1
531, 1464, 1411, 1375, 1211, 1042, 968, 872, 675, 551 cm ; HRMS (ESI): m/z calcd
+
for C24
H
23
N
4
O
2
: 399.1821 [M+H] ; found: 399.1833.
9

2
.3.4. 4-[(1E)-2-[4-[(1E)-2-(4-Cyanophenyl)ethenyl]-2,5-dimethoxyphenyl]ethenyl]
-
1-ethylpyridinium bromide (3PyB)
The mixture of 1-ethyl-4-methylpyridinium bromide (3a) (0.61 g, 3 mmol), 4b
0.88 g, 3 mmol), piperidine (0.26 g, 3 mmol), and methanol (30 mL) was refluxed for
(
1
0 h. After the solvent was removed under reduced pressure, the residue was purified
by silica gel column chromatography eluted with dichloromethane / methanol (15 : 1,
V / V) to give 3PyB as red crystalline powder (0.87 g). Yield: 60.8%. m.p. 225-227
1
ºC; H NMR (D
2
O, 500MHz) δ: 8.37 (d, J = 6.6 Hz, 2H), 7.56 (d, J = 6.6 Hz, 2H),
7
.28 (d, J = 8.5 Hz, 2H), 7.24 (d, J = 8.5 Hz, 2H), 7.21 (d, J = 16.4 Hz, 1H), 6.79 (d, J
=
16.5 Hz, 1H), 6.75 (s, 1H), 6.70 (s, 1H), 6.60 (d, J = 16.4 Hz, 1H), 6.54 (d, J = 16.5
1
3
Hz, 1H), 4.24 (q, 2H), 3.82 (s, 3H), 3.80 (s, 3H), 1.44 (t, J = 7.4 Hz, 3H); C NMR
DMSO-d , 125 MHz) δ: 152.99, 152.52, 151.28, 143.96, 141.94, 134.92, 132.70,
29.14, 128.29, 127.25, 125.94, 124.42, 124.18, 123.74, 118.97, 111.01, 110.11,
09.70, 56.36, 56.31, 55.17, 16.16; FT-IR (KBr) ν: 3055, 2927, 2856, 2218, 1641,
(
6
1
1
1
-1
598, 1495, 1468, 1413, 1323, 1212, 1174, 1041, 965, 861, 686, 550 cm ; HRMS
+
(
ESI): m/z calcd for C26
H
25
N
2
O
2
: 397.1916 [M-Br] ; found: 397.1907.
3
. Results and discussion
3
.1. Synthesis and characterization
The synthetic routes of 1BI, 2TAZ, and 3PyB are detailedly showed in scheme 1.
1 [23], 1b[24], 1c[25], 2a[26], 3a[27], and 4a[28]were prepared with reference to the
a
reported methods. The important intermediate 4b was reported here for the first time,
which was synthesized by the Horner-Wadsworth-Emmons (HWE) reaction of 4a
with 1d, followed by hydrolysis with aqueous acetic acid. 1BI was obtained by the
Knoevenagel condensation of 1c with propanedinitrile. 2TAZ and 3PyB were
synthesized in a similar way as 1BI.
1
In the H NMR spectra of 1BI, 2TAZ, and 3PyB, the trans-configurations of the
C-C double bonds are evidenced by the coupling constants J = 16.1-16.6 Hz for the
olefinic protons. The chemical shifts (δ) at 3.91 and 4.00 ppm (1BI), 3.93 and 3.96
ppm (2TAZ), 3.80 and 3.82 ppm (3PyB) correspond to the methoxy protons. For 1BI,
1
0

the singlet at 12.97 ppm can be attributed to the NH proton. In the FT-IR spectra of
-1
1
BI, 2TAZ, and 3PyB, the characteristic absorption peaks at 2218-2227 cm indicate
-1
the presence of CN group. The peaks at 1211-1228 cm are attributed to the C-O-C
-1
asymmetric stretching vibration, and the peaks at 1035-1042 cm are attributed to the
-1
C-O-C symmetric stretching vibration. The peaks at 965-968 cm correspond to the
C-H deformation vibration which belongs to the trans-configurations of the C-C
double bonds.
=
1
1

Scheme 1. Synthesis of the target compounds (1BI, 2TAZ, and 3PyB).
3
.2. Linear and nonlinear photophysical properties
The linear and nonlinear photophysical properties of the target compounds are
summarized in Tables 1 and 2, respectively. The spectra of linear absorption and
SPEF in CH Cl are depicted in Figs. 2 and 3. The Φ×δ spectra of the target
2
2
compounds are depicted in Figs. 4 and 5.
As shown in Fig. 2 and Table 1, the target compounds exhibit two absorption bands.
The band occurring in the 335-389 nm region ascribed to the π-π* transition of the
trans-1,4-divinylbenzene moiety, while the other band, namely the maximum linear
absorption peak, occurring in the 411-470 nm region is assigned to the ICT transition.
abs
λ
The maximum absorption peak positions ( max) of 1BI and 2TAZ change slightly
(
±5 nm) as the solvent polarity increases from THF to C
2
5
H OH, which indicates that
the surrounding solvent molecules have little effect on the transition energy of the
abs
λ
target compounds. Whereas the maxof 3PyB changes considerably in different
solvents and exhibits the minimum value in water. This phenomenon is attributed to a
relatively high polar mesomeric form, which is predominant in the ground state. As a
result, the hydrogen bond donating solvent decreases the energy of the ground state,
1
2

abs
leading to a blue shift of the absorption band. The
λ
maxvalues can be sequenced as
1
BI > 3PyB > 2TAZ (except in CH Cl ). This might be attributed to that the number
2
2
of cyano acceptors increases (1BI cf. 2TAZ and 3PyB) and pyridinium cation has
stronger electron-withdrawing ability than s-triazine (3PyB cf. 2TAZ).
SPEF
As shown in Fig. 3 and Table 1, the SPEF maxima ( λmax ) of 1BI and 2TAZ
SPEF
undergo red shifts with increasing the solvent polarity. For example, the λmax of 1BI
(
2TAZ) is located at 526 (481) nm in THF and red-shifted to 544 (519) nm in
OH. One reasonable interpretation is that the polarity of the excited state is larger
C
2
H
5
than that of the ground state, and the enhanced dipole-dipole interactions caused by
increasing the polarity of the solutes and / or solvents will lead to a more significant
energy level decrease for the excited state[29]. On the contrary, the λmax of 3PyB
SPEF
changes slightly with increasing the solvent polarity, which is always located in the
orange light region (570-578 nm). Analyzing the data collected in Table 1, it follows
that the Φ of 1BI is very low and almost independent of the solvent polarity, the Φ of
2
TAZ significantly decreases with increasing the solvent polarity, and the Φ of 3PyB
in the medium polar solvents (THF, CH Cl ) is larger than that in the highly polar
solvents (DMF, CH CN, C OH, and H O), especially in water (Φ = 0.04). The Φ of
2
2
3
2
H
5
2
target compounds can be sequenced as 2TAZ > 3PyB > 1BI. The low Φ values
indicate that the emitting state of 1BI might facilitate the nonradiative pathways[30]
.
1
3

Table 1. Linear photophysical properties of the target compounds (1BI, 2TAZ, and
PyB).
3
a
b
c
d
e
abs
4
SPEF
max
λ
10 
λ
Φ
max
ν
-1
-1
(cm-1)
2823
2836
3165
3446
3596
3306
3713
3935
4291
4771
4908
Compound Solvent
(nm)
458
460
459
456
(mol L cm )
(nm)
526
529
537
541
544
481
492
496
499
519
571
1
2
3
BI
THF
CH Cl
DMF
CH CN
3.64
0.08
0.07
0.05
0.05
0.06
0.72
0.68
0.53
0.41
0.28
0.37
2
2
4.12
3.96
3
3.23
C
2
H
5
OH 455
415
3.74
TAZ
THF
CH
DMF
CH CN
7.36
2
Cl
2
416
415
411
8.42
3.44
3
7.39
C
2
H
5
OH 416
446
4.41
PyB
THF
3.97
1
4

CH
2
Cl
2
470
449
443
3.55
4.93
5.60
4.02
3.82
578
574
574
574
3976
4850
5152
4653
5498
0.31
0.12
0.11
0.13
0.04
DMF
CH CN
3
C
2
H
5
OH 453
434
H
2
O
570
a
b
c
d
e
-5
-1
Maximum linear absorption wavelength, c = 1×10 mol L .
Maximum molar absorption coefficient.
-6
-1
Maximum single-photon excited fluorescence wavelength, c = 1×10 mol L .
Stokes shift.
-1
Fluorescence quantum yield, measured using fluorescein in 0.1 mol L sodium
hydroxide aqueous solution as the standard.
Fig. 2. Linear absorption spectra of the target compounds (1BI, 2TAZ, and 3PyB) in
-5
-1
CH
2
Cl with a concentration of 1×10 mol L .
2
1
5

Fig. 3. Single-photon excited fluorescence spectra of the target compounds (1BI,
-6
-1
2
TAZ, and 3PyB) in CH
2
Cl with a concentration of 1×10 mol L .
2
There is no linear absorption in the wavelength range of 600-920 nm for 1BI,
TAZ, and 3PyB, which indicates that there are no energy levels corresponding to an
2
electron transition in this spectral range. Therefore, if frequency up-converted
fluorescence appears excited by a tunable laser in this spectral range, it should be
attributed to TPEF. Meanwhile, the log-log plot of the excited fluorescence signal
versus the exciting powers is depicted in Fig. 6b (with a slope of 2.01 for 3PyB),
which provides the direct evidence for a two-photon excitation process.
As shown in Table 2, the maximum δ values of 1BI, 2TAZ, and 3PyB are 607
(
CH
2
Cl
2
), 264 (THF), and 713 GM (CH
2
Cl ), respectively, which follows the order:
2
3
PyB > 1BI > TAZ. In addition, it is found that the peak positions of TPEF are
red-shifted by 28-38 nm compared with those of SPEF, which can be explained by the
effect of reabsorption in highly concentrated solutions[31]. As shown in Table 2, Figs.
4
and 5, for 1BI, 2TAZ and 3PyB, their maximum Φ×δ are 42 (CH
and 221 GM (CH Cl ), respectively. Meanwhile, due to its good water solubility, the
Φ×δ of 3PyB in H O is 11 GM, which is much larger than that of the commercial
DNA stain DAPI (0.16 GM)[32]
In order to better understand the structure-property relationships of the target
2
Cl ), 190 (THF),
2
2
2
2
.
1
6

compounds, DFT calculations were performed using the Gaussian 09 program and
B3LYP 6-31G (d, p) basis sets were used for the calculations. The optimized
geometrical structures, HOMO and LUMO distributions of the target compounds are
shown in Fig. 7. The HOMO-LUMO energy gaps (∆E) are calculated and listed in
Table 3. For 1BI and 3PyB, the electron clouds of the HOMO are mainly localized on
the methoxy donor, the 1,4-divinylbenzene π-bridge, and the benzimidazole (or
benzonitrile) acceptor, whereas in the LUMO, most of the electrons transfer from the
benzimidazole (or benzonitrile) acceptor to the more strongly electron-withdrawing
cyano (pyridinium cation) acceptor. However, for 2TAZ, the electron clouds of the
HOMO are mainly distributed on the methoxy donor and the 1,4-divinylbenzene
π-bridge, whereas in the LUMO, the electron cloud density of the terminal acceptors
(
s-triazine and benzonitrile) increases significantly. The δ value shows negative
correlation with △E[33]. The △E of 1BI, 2TAZ, and 3PyB are 2.69, 2.99, and 2.15 eV,
respectively, which is consistent with the sequence of δ (3PyB > 1BI > 2TAZ).
Table 2. Nonlinear photophysical properties of the target compounds (1BI, 2TAZ,
and 3PyB).
a
b
c
TPEF
λmax
max
Φmax
Compound
Solvent
THF
(nm)
564
565
518
524
600
606
594
(GM)
459
607
264
194
530
713
285
(GM)
37
1
2
3
BI
CH
THF
CH Cl
THF
2
Cl
2
42
TAZ
PyB
190
132
196
221
2
2
CH
2
Cl
2
H
2
O
11
a
b
c
-3
-1
Maximum two-photon excited fluorescence wavelength, c = 1×10 mol L .
Two-photon absorption cross-section, 1 GM = 1×10-50 cm s photon .
4
-1
Two-photon action cross-section.
1
7

Fig. 4. Two-photon action cross-sections of 1BI, 3PyB in CH
2
Cl and 2TAZ in THF
2
in the 690-910 nm range.
Fig. 5. Two-photon action cross-sections of 3PyB in different solvents in the 690-910
nm range.
(a)
1
8

(b)
Fig. 6. (a) Two-photon excited fluorescence spectra of 3PyB in CH
2
Cl
(c = 1×10-3
2
-1
mol L ) at 850 nm under different input powers. (b) Logarithmic plot of the output
fluorescence integral (Iout) of 3PyB versus the input laser powers (Iin).
1
9

1
BI
2TAZ
3PyB
Top view
Front view
HOMO
LUMO
Fig. 7. Optimized geometrical structures, HOMO and LUMO distributions of the
target compounds (1BI, 2TAZ, and 3PyB).
Table 3. Calculated HOMO, LOMO energy and HOMO-LOMO energy gaps of the
target compounds (1BI, 2TAZ, and 3PyB).
HOMO
eV)
LUMO
(eV)
HOMO-LUMO energy gaps
(eV)
Compound
(
1
2
3
BI
-5.64
-5.47
-7.83
-2.95
-2.48
-5.68
2.69
2.99
2.15
TAZ
PyB
3
.3. DNA-binding studies
In order to check the possibility of 3PyB as a DNA stain, the changes in the
absorption, single- and two-photon fluorescence spectra with the addition of ctDNA
were investigated. The results are shown in Figs. 8, 9, and 10. With the addition of
ctDNA, the absorbance values decrease when the ratio of cctDNA to c3PyB is lower than
1
0, then increase and level off after the ratio of 65 : 1. Meanwhile, the maximum
2
0

absorption peaks are marginally red-shifted (from 432 nm to 441 nm). In general, the
hypochromism and red shift are associated with the intercalative binding of the dye to
DNA, due to strong stacking interaction between the dye and the base pairs of DNA,
while the hyperchromism and red shift can be attributed to external contact (surface
binding) with DNA[34]
.
As shown in Fig. 9, the fluorescence intensity increases sharply (about 10 fold),
then levels off after the ratio of 85 : 1. However, the maximum emission peaks remain
steady at around 567 nm. Moreover, the binding constant between 3PyB and ctDNA
was confirmed on the basis of the single-photon fluorescence titration according to the
5
-1
Scatchard equation. The result shows that the binding constant is 3.05×10 mol L .
As shown in Fig. 10, the two-photon fluorescence intensity first increases quickly,
then levels off after the ratio of 10 : 1. This trend is similar to that of the single-photon
fluorescence titration. The following factor can explain the fluorescence enhancement.
When the dye is in a twisted intramolecular charge transfer (TICT) state, the
nonradiative process quenches the fluorescence. Inversely, the dye will become highly
luminescent, for the formation of a TICT state is restricted. For 3PyB, the TICT can
occur between the electron donor and the electron acceptors. In water, the dye in the
excited state interacts with the highly polar solvent more strongly than that in the
ground state, resulting in the larger dipole moment in the excited state, which can lead
to charge separation and finally results in the formation of a TICT state. However, the
formation of a TICT state is restricted when 3PyB is protected by DNA, and the
fluorescence of 3PyB will be restored[35]. Moreover, 3PyB has a low Φ×δ value in
Tris-HCl buffer solution (6.6 GM), but once bound to ctDNA, the Φ×δ value can be
increased to 45 GM, which is far higher than the commerical DNA stain DAPI (2.18
GM)[36]. These demonstrate that 3PyB is a potential DNA fluorescent probe.
(a)
2
1

(b)
2
2

Fig. 8. (a) Linear absorption spectra of 3PyB with the addition of ctDNA. (b) Change
-5
-1
of absorbance (●) and peak position (■). c3PyB = 1×10 mol L , Ratio: cctDNA / c3PyB
.
(a)
(b)
2
3

Fig. 9. (a) Single-photon excited fluorescence spectra of 3PyB with the addition of
-5
ctDNA. (b) Change of SPEF intensity (●) and peak position (■). c3PyB = 1×10 mol
-1
L , Ratio: cctDNA / c3PyB
.
(a)
(b)
2
4

Fig. 10. (a) Two-photon excited fluorescence spectra of 3PyB with the addition of
-5
-1
ctDNA. (b) Two-photon fluorescence enhancement curves. c3PyB = 5×10 mol L .
Ratio: cctDNA / c3PyB
.
In order to further explore the possible interaction mechanism of 3PyB-ctDNA, the
fluorescence of 3PyB-ctDNA under different NaCl concentrations and the viscosity
-5
experiment were investigated. The concentrations of 3PyB and ctDNA are 1×10 mol
-1
-5
-1
L and 5×10 mol L , respectively. The concentrations of NaCl vary from 0.1 to 0.6
-1
mol L . The changes in fluorescence intensity with adding NaCl are shown in Fig. 11.
+
Generally, Na can be partially close to phosphate of the nucleotide skeleton. If the
dye-DNA interaction is an electrostatic mode, the changes in fluorescence intensity
+
will be disturbed with the increase of Na concentrations. As we can see, the
fluorescence intensity of 3PyB-ctDNA changes slightly with the addition of NaCl,
which indicates that the electrostatic interaction does not play a major role.
The hydrodynamic measurements that are sensitive to the length change are, in the
absence of crystallographic data, regarded as the most critical tests of a DNA-binding
mode. The intercalation mode is expected to lengthen the DNA helix as the base pairs
are pushed apart to accommodate the bound dye, leading to an increase in the
viscosity of DNA. In contrast, the partial, non-classical intercalation mode can bend
2
5

(or kink) the DNA helix and reduce its effective length and, concomitantly, its
viscosity. When the outside binding mode occurs, the viscosity of DNA would not
change basically[37]. As shown in Fig. 12, the viscosity of ctDNA increases
remarkably with increasing the concentration of 3PyB, as expected for the
lengthening of the DNA double helix resulting from intercalation. The result is
consistent with the hypochromism and red shift in the absorption titration and
fluorescence enhancement in the fluorescence titration. Based on the above analysis,
it can be inferred that the interaction of 3PyB-ctDNA should mainly be the
intercalation binding at a low ratio of cctDNA to c3PyB and the groove binding at a high
ratio of cctDNA to c3PyB
.
−5
−1
-5
Fig. 11. Effect of NaCl concentration. c3PyB = 1×10 mol L , cctDNA = 5×10 mol
-
1
L .
2
6

Fig. 12. Effect of increasing amounts of 3PyB on the relative viscosity of ctDNA.
-4
-1
c
ctDNA = 1 × 10 mol L , Ratio: c3PyB / cctDNA.
Conclusions
In summary, three novel A-π-D-π-A' compounds (1BI, 2TAZ, and 3PyB) were
synthesized and characterized. Their linear and nonlinear photophysical properties in
different solvents have been systematically studied. Meanwhile, the spectral
properties of 3PyB in the DNA titratiion experiments were investigated. The possible
interaction mechanism of 3PyB-ctDNA was also studied by the NaCl titration and the
viscosity experiment. The results show that 3PyB may serve as a potential fluorescent
probe for DNA detection.
Acknowledgements
We are grateful to the financial supports from the National Natural Science
Foundation of China (Grant No. 21103151) and the Natural Science Foundation of
Zhejiang province, China (Grant No. LY15B030006).
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