New conjugated organic dyes with various electron donors: One- and two-photon excited fluorescence, and bioimaging

Article abstract of DOI:10.1016/j.dyepig.2014.04.032

Six new fluorescent donor-π bridge-acceptor (D-π-A) structural molecules with various electron donors were synthesized and fully characterized. Influence of electron-donating strength on optical properties was investigated by the systematic alteration of the electron donors. The optical properties of the dyes were successfully tuned by choosing different electron donors. All the dyes in solution show strong one-photon excited fluorescence (OPEF) and high quantum yield. With increasing electron-donating strength of donors, dye 5 with diphenylamine and dye 6 with (4,4′-diethoxyphenyl)amino substituents, exhibit strong two-photon excited fluorescence (TPEF), and the large two-photon absorption (TPA) cross-section values are 1378 GM for 5 and 1654 GM for 6. The structure-property relationships were detailed through quantum chemical calculations and X-ray crystallography. The results of living cell imaging experiments show the values of them in one- or two-photon fluorescence microscopy bioimaging applications.

Full text of DOI:10.1016/j.dyepig.2014.04.032

Accepted Manuscript
New conjugated organic dyes with various electron donors: one- and two-photon
excited fluorescence, and bioimaging
Feng Jin , Dong-Ling Xu , Hui-Zhi Zhu , Yan Yan , Jun Zheng , Jun Zhang , Hong-
Ping Zhou , Jie-Ying Wu , Yu-Peng Tian
PII:
S0143-7208(14)00160-0
10.1016/j.dyepig.2014.04.032
DYPI 4360
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 16 October 2013
Revised Date: 23 February 2014
Accepted Date: 22 April 2014
Please cite this article as: Jin F, Xu D-L, Zhu H-Z, Yan Y, Zheng J, Zhang J, Zhou H-P, Wu J-Y,
Tian Y-P, New conjugated organic dyes with various electron donors: one- and two-photon excited
fluorescence, and bioimaging, Dyes and Pigments (2014), doi: 10.1016/j.dyepig.2014.04.032.
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ACCEPTED MANUSCRIPT
Graphical abstract
One- and two-photon fluorescence of six dyes, being promising in bioimaging
applications, were successfully tuned through varying electron donors.

ACCEPTED MANUSCRIPT
New conjugated organic dyes with various electron donors: one-
and two-photon excited fluorescence, and bioimaging
a, b
a
a
a
c
a
Feng Jin , Dong-Ling Xu , Hui-Zhi Zhu , Yan Yan , Jun Zheng , Jun Zhang ,
a,
a
a
Hong-Ping Zhou, ∗ Jie-Ying Wu , Yu-Peng Tian
a
College of Chemistry and Chemical Engineering, Anhui University, Hefei 230039, P.
R. China
b
Department of Chemistry, Fuyang Normal College, Fuyang 236041, P. R. China
c
Center of Modern Experimental Technology, Anhui University, Hefei 230039, P. R.
China
*
Corresponding author. Fax: +86-551-5107342; Tel: +86-551-5108151
E-mail address: zhpzhp@263.net
ABSTRACT
Six new fluorescent donor-π bridge-acceptor (D-π-A) structural molecules with
various electron donors were synthesized and fully characterized. Influence of
electron-donating strength on optical properties was investigated by the systematic
alteration of the electron donors. The optical properties of the dyes were successfully
tuned by choosing different electron donors. All the dyes in solution show strong
one-photon excited fluorescence (OPEF) and high quantum yield. With increasing
electron-donating strength of donors, dye 5 with diphenylamine and dye 6 with
(4,4′-diethoxyphenyl)amino substituents, exhibit strong two-photon excited
fluorescence (TPEF), and the large two-photon absorption (TPA) cross-section values
1

ACCEPTED MANUSCRIPT
are 1378 GM for 5 and 1654 GM for 6. The structure-property relationships were
detailed through quantum chemical calculations and X-ray crystallography. The
results of living cell imaging experiments show the values of them in one- or
two-photon fluorescence microscopy bioimaging applications.
Keywords: electron donor, one-photon excited fluorescence, two-photon excited
fluorescence, two-photon absorption, structure-property relationship, bioimaging
1
. Introduction
Conjugated organic molecules with large delocalized π-electron systems
continue to be the subject of very active research due to their potential applications
as one- or two- photon optical materials in several areas such as fluorescence
imaging, photodynamic therapy, photoswitching devices and 3-D data storage
[
1-14]. Organic molecules that can be used for such applications are expected to
have high fluorescence quantum yield and large two-photon absorption
cross-section values. However, the relatively low quantum yield and small
two-photon absorption cross-section values of the materials often limit their
widespread utility [15-18]. Therefore, extensive efforts have been concentrated on
the synthesis of dyes with high fluorescence quantum yield and large two-photon
cross-sections. The two-photon absorbing organic materials with large two-photon
absorption are particularly attractive due to the advantages of two-photon
absorption processes [19]. It is well known that TPA cross-section can be
increased either by an enhancement of the conjugation length as well as the
coplanarity of the π-center, or by an appropriate combination of electron donors
2

ACCEPTED MANUSCRIPT
and acceptors. The molecules with D-π-A structure often have two-photon
fluorescence property. It has been well-established that increasing the
electron-donating strength of terminal groups and extending conjugation, can exert
significant influence on enhancement of TPA cross-section due to the highly
extended π-conjugation [20]. Recently, remarkable progress in the design and
synthesis of organic dyes with very high TPA cross sections (>1000 GM) has been
reported [21].
Dipyridyl has been widely used as electron acceptor group to design optical
materials, due to its electron-deficient nature of the heterocycle, excellent optical
property, good thermal and chemical stabilities, as well as excellent metal cation
coordinationability [22]. Our group has reported a series of TPA pyridine or
bipyridine derivatives and the corresponding complexes [23]. As continuous
efforts to explore new molecules with high fluorescence quantum yield and large
TPA cross section values, 2,2-bipyridyl group as electron acceptor moiety is
incorporated into various electron donors through a π-conjugating spacer, we
obtained six dyes with excellent optical properties. The adopted electron donors
are imidazole (D1), triazole (D2), carbazole (D3), anthracene (D4), diphenylamine
(
D5), and (4,4′-diethoxyphenyl)amino (D6), whose electron-donating strength
increases in the order of D6 > D5 > D3 > D4 > D1 > D2. By using the different
electron donors, the one- and two-photon fluorescence properties of the dyes have
been successfully tuned. All the compounds have high fluorescence quantum yield.
With increasing electron-donating strength of donors, 5 and 6 exhibit excellent
3

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two-photon excited fluorescence with the maximum TPA cross-section values of
1
378 GM for 5 and 1654 GM for 6, which are relatively large compared to those
reported and the commercial fluorophores, such as rhodamine B [24].
The molecular structures for the dyes (1-6) are shown in Scheme 1. All the
compounds can be obtained in high yield and exhibit a simple preparation and
purification at lower cost. In addition, the simple modification of molecular structure
by introducing a benzene ring into the six position of pyridyl may produce a steric
hindrance, which is expected to suppress molecular internal rotation to some extent
and prevent the intermolecular πꢀꢀꢀπ interactions between the coplanar conjugated
backbones. Finally, dyes 1-6 are N^N^C-cyclometalated ligands, which are suitable
for preparing cyclometalated platinum(II) and ruthenium(II) complexes for use in
dye-sensitized solar cells or luminophores [25], and the related studies are currently
underway in our group.
Here, we report synthesis and optical properties (linear and nonlinear) of the
six organic compounds, together with quantum-chemical calculation and X-ray
crystallography for discussion on the correlation between molecular structures and
spectral properties. One- and two-photon fluorescence cell imaging experiment
indicated the suitability of them for this potential application.
2
. Experimental section
2
.1. General procedures
4

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All commercially available chemicals are of analytical grade. Every solvent was
purified as conventional methods beforehand. Elemental analyses were carried out on
Perkin-Elmer 240 analyzer. IR spectra were recorded with a Nicolet FT-IR NEXUS
-1
1
13
8
70 spectrometer (KBr discs) in the 4000-400 cm region. H and C NMR spectra
were recorded on a Bruker AV 400 spectrometer using CDCl or (CD ) SO as
3
3 2
solvent. Chemical shifts were reported in parts per million (ppm) relative to internal
TMS (0 ppm) and coupling constants in Hz. Splitting patterns were described as
singlet (s), doublet (d), triplet (t), quartet (q), or multiplet (m). The mass spectra were
obtained on a Bruker Autoflex III smartbeam mass spectrometer and a LTQ-Orbitrap
XL mass spectrometer. The X-ray diffraction measurements were performed on a
CCD area detector using graphite monochromated MoKα radiation (λ = 0.71069 Å) at
2
98 (2) K. Intensity data were collected in the variable ω-scan mode. The structures
were solved by direct methods and difference Fourier syntheses. The non-hydrogen
atoms were refined anisotropically and hydrogen atoms were introduced
geometrically. Calculations were performed with SHELXTL-97 program package.
Crystallographic data for the structures reported in this paper have been deposited
with the Cambridge Crystallographic Data Centre as supplementary publication no.
CCDC: 931717(for 2), 938586(for 3), 921209(for 4), 928376(for 5).
2
.2. Optical measurements
The one-photon absorption (OPA) spectra were recorded on a SPECORD S600
spectrophotometer. The one-photon excited fluorescence spectra measurements
were performed using a Hitachi F-7000 fluorescence spectrophotometer. In the
5

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measurements of emission and excitation spectra, the pass width is 5 nm for all
compounds. OPA and OPEF of compounds 1–6 were measured in five organic
-
5
-1
solvents of different polarities with the concentration of 1.0 × 10 mol L . The
quartz cuvettes used are of 1 cm path length. The absolute fluorescence quantum
yield (Φ) values were determined using an integrating sphere. For time-resolved
fluorescence measurements, the fluorescence signals were collimated and focused
onto the entrance slit of a monochromator with the output plane equipped with a
photomultiplier tube (HORIBA HuoroMax-4P). The decays were analyzed by
‘
least-squares’. The quality of the exponential fits was evaluated by the goodness
of fit ( 2).
χ
Two-photon absorption cross-sections (δ) of the samples were obtained by
two-photon excited fluorescence method [26] at femtosecond laser pulse and Ti:
sapphire system (680–1080 nm, 80 MHz, 140 fs) as the light source. The sample
-
4
-1
was dissolved in different solvents at a concentration of 5.0 × 10 mol L . The
intensities of TPEF spectra of the reference and the sample were determined at
their excitation wavelength. Thus, TPA cross-section of samples was determined
by Eq.:
Φref cref nref
F
δ
= δref
Φ
c
n Fref
Where the ref subscripts stand for the reference molecule (here fluorescein in the
-
1
-3
-1
aqueous NaOH solution (1 mol L ) at concentration of 1.0 × 10 mol L was used
as reference). δ is the TPA cross-sectional value, c is the concentration of the
solution, n is the refractive index of the solution, F is the TPEF integral intensities
6

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of the solution emitted at the exciting wavelength, and Φ is the fluorescence
quantum yield. The δ value of reference was taken from the literature [27].
ref
2
.3. Cytotoxicity assays in cells
To ascertain the cytotoxic
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was
effect
of
the
compounds,
the
3
performed. HepG2 cells were trypsinized and plated to ~70% confluence in
6-well plates 24 h before treatment. Prior to the compounds' treatment, the
9
DMEM was removed and replaced with fresh DMEM, and aliquots of the
compound stock solutions (1 mM DMSO) were added to obtain final
concentrations of 5, 10, 20 and 40 ꢁM. The treated cells were incubated for 24 h at
ο
3
7 C and under 5% CO . Subsequently, the cells were treated with 5 mg/mL MTT
2
ο
(
40 ꢁL/well) and incubated for an additional 4 h (37 C, 5% CO ). Then, DMEM
2
was removed, the formazan crystals were dissolved in DMSO (150 ꢁL/well), and
the absorbance at 490 nm was recorded. The cell viability (%) was calculated
according to the following equation: cell viability % = OD490 (sample)/OD490
(
control) × 100, where OD490 (sample) represents the optical density of the wells
treated with various concentration of the compounds and OD490 (control)
represents that of the wells treated with DMEM + 10% FCS. Three independent
trials were conducted, and the averages and standard deviations are reported. The
reported percent cell survival values are relative to untreated control cells.
2
.4. Cell culture and incubation
7

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5
HepG2 cells were seeded in 6 well plates at a density of 2 × 10 cells per well
and grown for 96 hours. The cell cultures were incubated with the dyes (10% PBS:
9
0% cell media) at concentrations 2.5 ꢁM and maintained at 37 °C in an
atmosphere of 5% CO and 95% air for incubation times ranging for 2 hours. The
2
cells were then washed with PBS (3 × 3 mL per well) and 3 mL of PBS was added
to each well.
2
.5. Fluorescence imaging
HepG2 cells were luminescently imaged on a Zeiss LSM 710 META upright
confocal laser scanning microscope using magnification 40 × and 100 ×
water-dipping lenses for monolayer cultures. Image data acquisition and processing
was performed using Zeiss LSM Image Browser, Zeiss LSM Image Expert and Image
J.
2
6
.6. Synthesis
-phenyl-4´-(4-[4-(1H-imidazolyl)styryl]phenyl)-2,
2´-bipyridine
(1).
All
compounds were prepared by
a
similar procedure. 4-(6-Phenyl-2,
´-bipyridine-4´)-benzyl triphenyl phosphonium bromide (E) (6.63 g, 10 mmol),
-(1H-imidazol-1-yl)benzaldehyde (1.72 g, 10 mmol) and t-BuOK (5.60 g, 50
2
4
mmol) were placed in a dry mortar. The mixture milled vigorously for about 20
min. It became sticky, and then tetrahydrofuran (5 mL) was added. It was
continuously milled for 10 min. After completion of the reaction (monitored by
Thin Layer Chromatography (TLC)), the mixture was poured into distilled water
8

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(
500 mL). The product was extracted twice with CH Cl , and the organic layer was
2
2
dried overnight over anhydrous MgSO . The solvent was removed with a rotary
4
evaporator to give the crude product. It was purified by recrystallization from the
o
mixture of CH Cl and methanol. Yield: 80%. mp: 268.8 C. Anal. Calcd. (%) for
2
2
C H N : C, 83.17; H, 5.08; N, 11.76. Found (%): C, 83.51; H, 4.75; N, 11.50.
3
3
24
4
-
1
FT-IR (KBr, cm ): 3131, 3033, 1600, 1583, 1565, 1523, 1476, 1390, 1302, 1258,
1
1
112, 1050, 970, 833, 792, 730, 689, 539. H NMR ((CD ) SO, 400 MHz): δ 7.13
3
2
(
s, 1H), 7.46 (s, 2H), 7.50-7.55 (m, 2H), 7.59 (t, J = 8.4 Hz, 2H), 7.72 (d, J = 8.4
Hz, 2H), 7.81-7.85 (m, 5H), 8.02-8.08 (m, 3H), 8.34-8.40 (m, 4H), 8.66, (t, J = 5.6
1
3
Hz, 2H), 8.78 (d, J = 4.4 Hz, 1H). C NMR (100 MHz, CDCl ):
δ
(ppm) = 117.2,
18.2, 121.6, 121.7, 123.9, 127.1, 127.3, 127.6, 127.7, 127.9, 127.8, 128.8, 129.2,
29.9, 136.3, 136.8, 137.0, 137.6, 138.2, 139.5, 149.0, 149.5, 156.3, 156.3, 157.3.
3
1
1
+
+
MS (ESI) (m/z): Calc. for C H N : 477.20 [M] ; Found: 477.21 [M] .
3
3
24
4
6
-Phenyl-4´-(4-[4-(1H-1,2,4-triazolyl)styryl]phenyl)-2,2´-bipyridine
(2).
The
reaction was carried out using the same method used for 1, except that 4-(1H-1, 2,
4
4
-triazol-1-yl)benzaldehyde (1.73 g, 10 mmol) was used instead of
o
-(1H-imidazol-1-yl)benzaldehyde. Yield: 82%. mp: 266.5 C. Anal. Calcd. (%)
for C H N : C, 80.48; H, 4.85; N, 14.66. Found (%): C, 80.16; H, 4.49; N, 14.98.
3
2
23
5
-
1
FT-IR (KBr, cm ): 3130, 3032, 1600, 1583, 1565, 1542, 1522, 1473, 1276, 1214,
1
1
2
6
045, 976, 837, 792, 732, 690, 670, 539. H NMR (CDCl , 400 MHz): δ 7.21 (s,
3
H), 7.36 (s, 1H), 7.47 (t, J = 6.8 Hz, 1H), 7.55 (t, J = 7.2 Hz, 2H), 7.66-7.70 (m,
H), 7.86-7.91 (m, 3H), 8.01 (s, 1H), 8.13 (s, 1H), 8.22 (d, J = 7.2 Hz, 2H), 8.58
9

ACCEPTED MANUSCRIPT
1
3
(
s, 1H), 8.69, 8.73 (m, 3H). C NMR (100 MHz, CDCl ):
δ
(ppm) = 117.2, 118.2,
20.2, 121.7, 124.0, 127.1, 127.3, 127.6, 127.8, 127.9, 128.8, 129.2, 136.1, 137.2,
37.6, 138.1, 139.4, 140.7, 148.9, 149.5, 152.6, 156.2, 157.3. MS (ESI) (m/z):
3
1
1
+
+
Calc. for C H N : 478.20 [M] ; Found: 478.20 [M] .
3
2
23
5
6
-Phenyl-4´-(4-[4-(1H-carbazolyl)styryl]phenyl)-2,2´-bipyridine (3). The reaction
was carried out using the same method used for 1, except that
4
4
-(carbazol-9-yl)benzaldehyde (2.71 g, 10 mmol) was used instead of
o
-(1H-imidazol-1-yl)benzaldehyde. Yield: 82%. mp: 172.6 C. Anal. Calcd. (%)
for C H N : C, 87.62; H, 5.08; N, 7.30. Found (%): C, 87.29; H, 5.39; N, 7.65.
4
2
29
3
-
1
FT-IR (KBr, cm ): 3035, 1599, 1582, 1564, 1540, 1513, 1475, 1449, 1390, 1361,
1
1
333, 1313, 1228, 1116, 969, 832, 791, 771, 746, 721, 689, 620, 537. H NMR
(
CDCl , 400 MHz): δ 6.73 (s, 2H), 7.25-7.28 (m, 1H), 7.36-7.52 (m, 15H), 7.80
3
(
d, J = 7.6 Hz, 2H), 7.89 (t, J = 6.4 Hz, 1H), 7.99 (s, 1H), 8.12 (d, J = 8.0 Hz, 2H),
1
3
8
.18 (d, J = 7.2 Hz, 2H), 8.69-8.74 (m, 3H). C NMR (100 MHz, CDCl ):
δ
(ppm)
3
=
109.8, 117.6, 118.4, 120.0, 120.3, 122.0, 123.4, 124.0, 125.9, 126.7, 127.0,
1
1
27.2, 128.7, 129.2, 129.6, 130.1, 130.2, 130.3, 136.0, 136.6, 137.2, 138.0, 139.2,
40.6, 148.0, 149.7, 155.5, 157.3. MS (ESI) (m/z): Calc. for C H N : 576.24
4
2
29
3
+
+
[
M] ; Found: 576.24 [M] .
6
-Phenyl-4´-(4-[(9-anthryl)vinyl]phenyl)-2, 2´-bipyridine (4). Compound 4 was
prepared according to a similar procedure of 1 using 9-anthracene aldehyde (2.06
g, 10 mmol) instead of 4-(1H-imidazol-1-yl) benzaldehyde. Yield: 78%. mp: 207.8
o
C. Anal. Calcd. (%) for C H N : C, 89.38; H, 5.13; N, 5.49. Found (%): C,
3
8
26
2
1
0

ACCEPTED MANUSCRIPT
-
1
8
9.69; H, 4.78; N, 5.52. FT-IR (KBr, cm ): 3050, 2922, 1600, 1581, 1566, 1539,
1
513, 1469, 1449, 1412, 1389, 896, 879, 837, 793, 777, 735, 693, 638, 614, 520.
1
H NMR (CDCl , 400 MHz): δ 7.04 (d, J = 16.4 Hz, 1H), 7.40 (s, 1H), 7.50-7.51
3
(
m, 5H), 7.56 (t, J = 7.2 Hz, 2H), 7.83 (d, J = 7.2 Hz, 2H), 7.91 (d, J = 7.2 Hz,
1
8
H), 7.96 (d, J = 7.6 Hz, 2H), 8.03-8.07 (m, 4H), 8.24 (d, J = 7.6 Hz, 2H),
1
3
.38-8.40 (m, 2H), 8.44 (s, 1H), 8.26 (s, 1H), 8.76 (s, 2H). C NMR (100 MHz,
(ppm) = 125.2, 125.4, 125.6, 125.7, 125.8, 126.0, 126.7, 127.1, 127.3,
27.6, 128.0, 128.1, 128.7, 128.9, 129.0, 129.7, 130.0, 131.5, 133.4, 134.3, 135.0,
36.5, 137.9, 138.4, 141.6. MALDI-TOF MS Calc for C H N , 510.21; Found,
CDCl ):
δ
3
1
1
5
6
5
3
8
26
2
10.50.
-Phenyl-4´-(4-[4-(diphenylamino)styryl]phenyl)-2, 2´-bipyridine (5). Compound
was prepared according to a similar procedure of 1 using 4-diphenylamino
benzaldehyde (2.73 g, 10 mmol) instead of 4-(1H-imidazol-1-yl) benzaldehyde.
o
Yield: 75%. mp: 182.0 C. Anal. Calcd. (%) for C H N : C, 87.32; H, 5.41; N,
4
2
31
3
-
1
7
1
6
2
8
7
.27. Found (%): C, 87.68; H, 5.15; N, 7.05. FT-IR (KBr, cm ): 3030, 2955, 2923,
585, 1513, 1489, 1465, 1378, 1329, 1313, 1275, 1173, 1023, 969, 834, 753, 734,
1
94, 617, 537, 502. H NMR (CD ) SO, 400 MHz) δ (ppm): 6.98 (d, J = 8.0 Hz,
3
2
H), 7.03-7.10 (m, J = 7.3 Hz, 6H), 7.24 (d, J = 16.4 Hz, 1H), 7.30-7.38 (m, J =
.4 Hz, 5H), 7.49-7.60 (m, J = 6.0 Hz, 6H), 7.78 (d, J = 8.0 Hz, 2H), 8.03 (d, J =
.2 Hz, 3H), 8.38 (t, J = 9.6 Hz, 3H), 8.65 (d, J = 11.2 Hz, 2H), 8.77 (d, J = 4.8
1
3
Hz, 1H). C NMR (100 MHz, CDCl ):
δ
(ppm) = 117.2, 118.2, 121.6, 123.1,
3
1
23.4, 123.9, 124.6, 126.1, 126.9, 127.1, 127.5, 128.8, 129.1, 129.1, 129.3, 131.2,
1
1

ACCEPTED MANUSCRIPT
1
37.1, 137.2, 138.5, 139.5, 147.5, 147.6, 148.9, 149.7, 156.2, 157.2. MALDI-TOF
MS Calc for C H N , 577.25; Found, 577.36.
4
2
31
3
6
-Phenyl-4´-(4-[4-(4,4'-diethoxyphenyl)amino)styryl]phenyl)-2,2´-bipyridine (6).
Compound 6 was prepared according to a similar procedure of 1 using
4
4
-[(4,4′-diethoxyphenyl)amino]benzaldehyde (3.61 g, 10 mmol) instead of
o
-(1H-imidazol-1-yl)benzaldehyde. Yield: 69%. mp: 153.8 C. Anal. Calcd. (%)
for C H N O : C, 82.98; H, 5.90; N, 6.31. Found (%): C, 82.65; H, 5.69; N, 6.68.
4
6
39
3
2
-
1
FT-IR (KBr, cm ): 3031, 2976, 2925, 1590, 1502, 1474, 1389, 1319, 1281, 1236,
1
1
1
6
165, 1111, 1044, 961, 826, 794, 692, 579, 530. H NMR (CDCl , 400 MHz): δ
3
.42 (t, J = 7.0 Hz, 6H), 4.00-4.05 (q, J = 7.0 Hz, 4H), 6.84 (d, J = 7.2 Hz, 4H),
.92 (d, J =8.4 Hz, 2H), 6.98 (d, J = 16.4 Hz, 1H), 7.07 (d, J = 9.2 Hz, 4H), 7.14
(
d, J = 16.4 Hz, 1H), 7.35 (d, J = 8.4 Hz, 2H), 7.51-7.58 (m, 3H), 7.67 (d, J = 7.2
Hz, 2H), 7.90 (s, 1H), 8.10 (d, J = 6.8 Hz, 2H), 8.18 (s, 3H), 8.51 (s, 1H),
1
3
9
6
1
1
.02-9.07 (m, 2H), 9.41 (s, 1H). C NMR (100 MHz, (CD ) SO): δ (ppm) = 16.5,
3 2
3.1, 115.4, 116.0, 117.6, 118.8, 120.8, 124.4, 124.5, 126.8, 126.8, 127.0, 127.3,
27.6, 128.5, 128.7, 129.3, 135.7, 138.5, 138.7, 139.6, 148.2, 148.9, 149.2, 155.2,
55.6, 156.4. MALDI-TOF MS Calc for C H N O , 665.30; Found, 665.01.
4
6
39
3
2
3
. Results and discussion
3
.1 Synthesis
1
2

ACCEPTED MANUSCRIPT
Scheme 1
Synthetic routes of compounds 1-6 are depicted in Scheme 1. The syntheses of
intermediates were described in the Supporting Information, and all kinds of
substituted benzaldehydes, namely, 4-(1H-imidazol-1-yl)benzaldehyde, 4-(1H-1,
2
4
,
4-triazol-1-yl)benzaldehyde,
4-(carbazol-9-yl)benzaldehyde,
-diphenylaminobenzaldehyde, and 4-[(4,4′-diethoxyphenyl)amino]benzaldehyde
were prepared according to the literature procedures [28,29]. Substituted
benzaldehydes and 4-(6-phenyl-2, 2´-bipyridine-4´)benzyl triphenyl phosphonium
bromide were coupled by the solvent free Wittig reaction, compounds 1-6 were
obtained in high yields. All compounds exhibit a simple preparation and
purification at lower cost. In our experiments, we ground the reaction mixture in a
mortar, using t-BuOK as base. The reaction could be accomplished within half an
hour and the product could be handled easily and the yield was above 70%.
Therefore, solvent free Wittig reaction is an inexpensive and convenient method to
synthesize alkene. Single crystals of 2-5 were obtained by slow evaporation from
methanol/CH Cl mixture solution.
2
2
3
.2. Crystal structures
Fig. 1
The structures of compounds 2-5 were confirmed by X-ray crystallography.
The molecular structures have been depicted in Fig. 2–5a. The structural features
1
3

ACCEPTED MANUSCRIPT
are discussed from the following three aspects. First, in the electronic acceptor
moiety, as shown in Fig. 1 and Table S2, the P0 ring adjoins the P1, P2, and P3
rings with the dihedral angles being 8.78/8.10, 8.45/8.69, and 27.12/28.01° for 2,
1
.55, 2.35, and 24.59° for 3, 7.22, 9.63, and 27.90° for 4, as well as 6.27, 13.19,
and 32.42° for 5. These angles indicate that the acceptor moieties are all good
planar, meaning there exist good π-conjugated system in the electronic acceptor
moieties of compounds 2-5. Second, at the electronic donor moiety, the smaller
dihedral angle (1.34°) between P4 and P5 in 2 indicates the higher π-conjugated
degree. However, the dihedral angles between the carbazolyl ring (P5) and its
neighboring phenyl plane P4 in 3, as well as the anthryl ring and P3 in 4 are 62.47
and 62.16°, respectively, indicating the highly twisted conformations. This highly
twisted conformation will reduce the degree of π conjugation, and further decrease
the charge transfer from donor to acceptor. For 5, at the triphenylamine moiety,
three phenyl rings around the central nitrogen are arrayed in a propeller-like
fashion and they are twisted from the trigonal NC plane with the torsion angles of
3
4
5.94, 34.68 and 42.85°, respectively. The N atom in the triphenylamine group has
2
an sp geometry favoring intramolecular p-π interactions between nitrogen and its
adjacent phenyl rings. Third, in the conjugated bridge section, as shown in Tables
S2, S3, the linkage between P3 and P4 is fine π-conjugated, and the bond lengths
of the C−C bonds between P3 and P4 in 2-5 are in the range of 1.264(7) and
1
.53(1) Å. And other linkage bond lengths between two neighboring aromatic
rings are all quite conjugated and are shorter than the normal C−C single bond
1
4

ACCEPTED MANUSCRIPT
(
1.54 Å) or C−N single bond (1.47 Å). It is worth noting that, for 5, the dihedral
angle between P3 and P4 is only 9.33°, and it is much smaller than those in 3 and 4
18.36° and 62.16°, respectively). The result clearly shows the more planar
(
structure of 5, which is expected to facilitate TPA.
Fig. 2
In short, the structural features suggest that all non-hydrogen atoms of molecules
2
-5 show different conjugate degree, which combined with the different
electron-donating strength of terminal moieties will exert very important influence on
the fluorescence properties.
Fig. 3
The molecules are held together in the crystal by the weak C−H···π stacking
and/or C−HꢀꢀꢀN hydrogen bonding interactions, and no π-π stacking interaction
was found. As shown in Fig. 2b, the neighboring unites of 2 are linked each other
through
C(5)−H(5)ꢀꢀꢀN(4),
C(5)−H(5)ꢀꢀꢀN(4)
hydrogen
bonding
and
C(27)−H(27)ꢀꢀꢀπ interactions to form one-dimensional structure along a-axis. As
shown in Fig. 3-5b, in the structures of 3-5, only C−Hꢀꢀꢀπ interactions were
detected, and the weak interactions link the molecules to generate one-dimensional
(
for 4 and 5) or two-dimensional (for 3) structures.
Fig. 4
1
5

ACCEPTED MANUSCRIPT
Fig. 5
3
.3 TDDFT calculations
To further investigate the influence of different electron donors on the characteristic
of charge transition of the compounds, the quantum chemical calculations based on
time-dependent density functional theory (TDDFT) were performed on 1-6 using
Gaussian 03 program. The B3LYP/6-31G basis sets were used for the calculations.
The TDDFT calculations were performed on the optimized structure. The isosurfaces
of the highest occupied and the lowest unoccupied molecular orbitals (HOMO and
LUMO) are shown in Fig. 6. For 1 and 2, the electron-cloud is delocalized throughout
the entire backbones, and the HOMO and LUMO diagrams clearly show the π–π*
transitions characteristics upon photoexcitation. This result suggests that, on the one
hand, the optical transition from LUMO to HOMO is a direct one, which offers a high
fluorescent efficiency, on the other hand, the molecules have no intramolecular charge
transfer (ICT) process. In sharp contrast, with the enhancement of the
electron-donating ability, the HOMO and LUMO of 3-6 exhibit the obvious
intramolecular charge transfer characteristics, which may be favorable for the TPA of
the compounds.
Fig. 6
3
.4. One-photon absorption and emission properties
The one-photon absorption and one-photon excited fluorescence were
–
5
measured in five solvents of different polarities at the concentration of 1×10
mol/L. There is no linear absorption beyond 450 nm for the molecules. The linear
and nonlinear optical properties for 1–6 in different polar solvents are listed in
1
6

ACCEPTED MANUSCRIPT
Table 1. In order to investigate the spectra relationship of the different compounds,
the OPA and the OPEF data of 1–6 in ethyl acetate were just used as examples to
discussed in detail. As expected, the absorption maxima of the compounds mainly
depend on the nature of the different electron-donating edge substituents, and the
linear absorption maxima for these dyes are tuned from 332 to 404 nm by using
different electron donors.
Fig. 7
As shown in Fig. 7 and Fig. S21, S22, compounds 1, 2 and 4 exhibit one
absorption band. The absorption maxima are located at 337, 335 and 386 nm in
ethyl acetate, respectively, which correspond to the π–π* transitions of the
backbones of 1 and 2, as well as the intramolecular charge-transfer for 4 on the
basis of density functional calculations [30]. The shoulder peak at 366 nm of 4
originates from a locally excited (LE) state of anthracene. Compounds 3, 5 and 6
are featured by two distinct absorption bands. The bands in the high energy region
are assigned to the π–π* transition, and those at longer wavelengths originates
from intramolecular charge-transfer interaction. In short, the red-shifted linear
absorption maximum with the order of 2 < 1 < 3 < 5 < 6 is consistent with the
trend of the increasing electron-donating ability and can be primarily explained by
the significant stabilization of the LUMO leading to a smaller HOMO-LUMO
energy gap [31].
Fig. 8
1
7

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From Fig. 7, Table 1, and Fig. S21, S22, one can see that the linear absorption
spectra of compounds 1-6 were slightly red-shifted with increasing solvent polarity
(
except in dichloromethane) and all of them show definite solvatochromic
behavior. This positive solvatochromism is indicative of a larger stabilization of
the excited state as compared to the ground state by a polar solvent, which
suggests that a significant charge redistribution takes place upon excitation [22a].
Table 1
As shown in Fig. 7 and Fig. S20, S21, all of the compounds have strong
one-photon fluorescence. The fluorescence spectra exhibit a single peak, which
indicates that the emission occurs from the lowest excited state with the largest
oscillator strength. Compounds 1-6 all have a trend of red-shift with increasing the
solvent polarity (except in dichloromethane). Accompanying this bathochromic
shift is a weakening of the emission intensity. The Stokes shifts also show a
tendency to increase with increasing the solvent polarity. This can be explained by
the fact that the excited state possess a higher polarity than the ground state, and
the increased dipole–dipole interaction between the solute and solvent leads to a
lowering of the energy level of the excited state [32]. Additionally, with increasing
the solvent polarity, the red-shifts of 3, 5 and 6 are more obvious than those of 1, 2
and 4, which may be due to the more strength of the donors in 3, 5 and 6, as well
as different charge-transfer mechanism of them. It is noted that the one-photon
fluorescence emission of 6 highly quenched in ethanol, acetonitrile and DMF, and
1
8

ACCEPTED MANUSCRIPT
the same case occurs in the two-photon fluorescence emission, which may be due
to the poor solubility of 6 in those solvents. As shown in Fig. 8, the maximum
emission of 1-6 in ethyl acetate locates at 408, 403, 436, 488, 487 and 524 nm,
respectively. The order of emission wavelength for dyes 1, 2, 3, 5, and 6 was
consistent with the strength of the donors: (4,4′-diethoxyphenyl)amino >
diphenylamine > carbazole > imidazole > triazole. The emission wavelengths for
3
-6 are much longer than those for 1 and 2, which can be explained by the fact that
the donor groups in 3-6 have stronger electron-donating strength than the
imidazolyl and triazolyl groups. Correspondingly, the charge transfer within the
molecules of 3-6 is much stronger and the band gaps are smaller, which can be
confirmed by the DFT calculation [33]. Although the electron donating ability of
carbazolyl is stronger than that of anthryl, the emission band of dye 4 is red-shift
about 52 nm compared to that of 3, which may be explained that the shorter
conjugation bridge of 4 enhances the intramolecular charge-transfer interaction.
The fluorescence quantum yield values were determined using an integrating
sphere. As shown in Table 1, all the compounds have high fluorescence quantum
yields, proving the validity of our molecular design strategy for
fluorescence-based solution application. The fluorescence quantum yields of 1 and
2
are similar in the same solvent, which may be due to the similar structure
character of the molecules. Compared with the others, 4 shows the lower
fluorescence quantum yield, which might be caused by its twisted structure.
Molecules with a large torsional distortion in the ground state go through fast
1
9

ACCEPTED MANUSCRIPT
nonradiative decay and show small fluorescence quantum yield. The fluorescence
quantum yield of 6 is slightly lower than that of 5, which suggests that a
significant non-radiative transition might occur in 6 due to its flexible
(
4,4′-diethoxyphenyl)amino chains [34]. The same result was obtained in our
previous work [35]. Therefore, we draw a conclusion that the compounds with
diphenylamine as the donor moiety appear to have high fluorescence quantum
yield compared with the dyes with (4,4′-diethoxyphenyl)amino as the donor
moiety. Additionally, concurrently with the spectral red-shift and intensity
reduction, the fluorescence quantum yields of all compounds are gradually reduced
with increasing solvent polarity.
To further characterize the photophysical properties of the synthesized
compounds, time-resolved fluorescence measurements were performed by using the
single-photon-counting method with picosecond laser excitation. The fluorescence
lifetimes (τ) of compounds 1-6 in ethyl acetate are 0.89 ns, 0.82 ns, 1.38 ns, 2.68, 1.84
ns and 2.97 ns, respectively (Table 1 and Fig. 9). The result further indicates that
introducing strong electronic donor group may greatly increase the ICT strength and
reduce the energy of the excited state. Furthermore, the fluorescence lifetimes almost
show a tendency to increase with increasing polarity of the solvents.
Fig. 9
Additionally, the fluorescence lifetimes of 1-6 in solution was calculated by
multiplying the corresponding quantum yield on natural lifetime, which can be
2
0

ACCEPTED MANUSCRIPT
easily calculated from the known Strickler-Berg equation (eqn. (1)) [36].
~
~
I(
ν
)d
ν
~
1
∫
ε(ν )
~
ν
−
9
2
(1)
=
2.88×10 n
dν
−
3
~
∫
~
~
~
τ 0
I(
ν
)
ν
dν
∫
in which n is the refractive index, I is the fluorescence emission, ε is the extinction
~
coefficient, and
ν
is the wave-number.The natural radiative lifetime τ0 and the
are related through the quantum yield Φ by
fluorescence lifetime
τ
τ
(2)
Φ =
τ 0
The calculated fluorescence lifetimes are showed in Table 1, which also show a
tendency to increase with increasing polarity of the solvents, according with the
experimental values.
As discussed above, the fluorescence spectra show large Stokes shifts
depending on the solvent polarity. The Lippert-Mataga equation is the most widely
used equation to evaluate the dipole moment changes of the compounds with
photoexcitation [37]:
2
∆f
2
(1)
∆ν
=
(µe − µg ) + b
3
4
πε0hca
2
ε
−1
n −1
∆f =
−
(2)
2
2ε
+1 2n +1
in which ∆ν = νabs - νem stands for Stokes shift, νabs and νem are absorption and
-
1
emission (cm ),
h
is the Planck’s constant, c is the velocity of light in vacuum, a
is the Onsager radius and b is a constant. ∆f is the orientation polarizability, µ and
e
µ are the dipole moments of the emissive and ground states, respectively, and ε is
g
0
2
the permittivity of the vacuum. (µ -µ ) is proportional to the slope of the Lippert
e
g
2
1

ACCEPTED MANUSCRIPT
-
Mataga plot.
Fig. 10
As shown in Fig. 10, the Lippert-Mataga plots of compounds 3 and 5 give
much larger slop than those of other dyes, which indicates larger dipole moment
changes for 3 and 5 with photoexcitation [37]. The slope of the best-fit line is
related to the dipole moment change between the ground and excited states (µ -µ ).
e
g
-
1
The slopes of the lines are 6547, 3771, 10721, 3228 and 11604 cm for
compounds 1-5, respectively. So the values of µ -µ were calculated as 12.6 D for
e
g
1
, 8.4 D for 2, 17.0 D for 3, 8.9 D for 4, and 17.5 D for 5. The large values of 3
and 5 indicate that the molecule in the excited state has an extremely polar
structure, which is the typical characterization of ICT [38]. The result accords with
their linear and nonlinear optical properties.
3
.5. Two-photon excited fluorescence spectra and TPA cross-sections
The detail experiments indicate that compounds 5 and 6 show strong
two-photon excited fluorescence, and the fluorescence spectra data are listed in
Table 1. However, 3 only exhibits very weak TPEF (Fig. S28), and 1, 2 as well as
4
show no TPEF. This fact clearly showed that careful selection of the
electron-donating group can have a significant impact on the TPEF behavior of
dyes. They have no linear absorption beyond 450 nm, which indicates that there
are no energy levels corresponding to an electron transition in this spectral range.
Therefore, if frequency upconverted fluorescence appears upon excitation with a
2
2

ACCEPTED MANUSCRIPT
tunable laser in this spectral range, it should be attributed to multi-photon
absorption excited fluorescence. Because of very weak TPEF of 3, here, only
two-photon excited fluorescence of 5 and 6 are discussed in detail. Two-photon
fluorescence spectra of compounds 5 and 6 in ethyl acetate pumped by
femtosecond laser pulses at 300 mW at different excitation wavelengths are
presented in Fig. 11. The insert figures show TPF spectra under different pump
intensities and logarithmic plots of the fluorescence integral versus pumped powers
with a slope of 1.95 for 5 and 1.92 for 6 when the input laser power is increasing,
suggesting a two-photon excitation mechanism. As shown in Fig. 11, and Fig.
S25-S27, compounds 5 and 6 display good TPA activity in the range of 680-900
and 680-960 nm, respectively, and the optimal excitation wavelengths are 800 and
8
20 nm, respectively, which are slightly higher than twice the corresponding linear
absorption maximum (390 and 400 nm for 5 and 6, respectively). From Table 1,
one can see that the peak positions of TPEF show red shift compared to those of
SPEF. This can be explained by the effect of reabsorption in the high concentration
solution [39].
Fig. 11
From Fig. S29, we can clearly find that the maxima of the two-photon excited
fluorescence also exhibits red-shift with increasing solvent polarity, and the TPEF
emission of 6 highly quenched in ethanol, acetonitrile and DMF, which is similar
to one-photon excited fluorescence. This observation indicates that both of the
2
3

ACCEPTED MANUSCRIPT
one-photon excited fluorescence and two-photon excited fluorescence are
produced from the same excited state even though two different excitation
processes are used. Two-photon absorption cross-sections have been measured
using TPEF. As shown in Table 1 and Fig. 12, the maximum absorption
cross-sections are 1378 GM for 5 and 1654 GM for 6. Obviously, the result
indicates that the TPA cross sections of the linear conjugated molecules with a
D-π-A structure strongly depend on the electron donating ability of the donor
moiety and the coplanarity of backbone. A stronger donor as well as good
planarity of backbone will generally increase charge transfer and enhance the
transition dipole moments, contributing to a larger value of δTPA because of
stronger ICT processes [40].
Fig. 12
3
.6. Cytotoxicity assays
Cytotoxicity is a potential side effect of dyes that must be controlled when dealing
with living cells or tissues. Considering their application in cell imaging, the MTT
assay was performed to ascertain the cytotoxic effect of compounds 1-6 against
HepG2 cells over a period of 24 h. Fig. S30 shows the cell viability for HepG2 cells
treated with 1-6 at different concentrations for 24 h. The results clearly indicated that
HepG2 cells incubated with concentration of 5 ꢁM of compounds 1-6 remained 89%,
8
8%, 80%, 70%, 89% and 86%, respectively viable after feeding time of 24 h,
demonstrating the biocompatibility of all compounds. Additionally, it was found that
2
4

ACCEPTED MANUSCRIPT
high concentration only leads to a gradual decrease of viable cells as shown in Fig.
S31. As a result, cytotoxicity tests definitely indicate that the low-micromolar
concentrations of compounds 1, 2, 5 and 6 have small toxic effects on living cells
over a period of 24 h, and they have great potentials for further biological studies.
3
.7. One- and two- photon fluorescence microscopy cell imaging
To further demonstrate the potential of the compounds in one- and two-photon
fluorescence microscopy imaging applications in living cells, we carried out imaging
studies with live HepG2 cells stained with compounds 1-6 by both one- and
two-photon microscopy. In one-photon microscopy imaging, the excitation
wavelength was fixed at 405 nm. As shown in Fig. 13, 14a, and Fig. S32-35a, bright
blue fluorescence from the cells indicates that compounds 1-6 could be effectively
internalized by HepG2 cells. In contrast, except for 4, the images of HepG2 cells
stained with other compounds show brighter fluorescence signal, which is due to
the higher fluorescence quantum yields and stronger emission abilities of them.
The bright-field image of each cell was taken immediately prior to the
fluorescence microscopy imaging. The fluorescent images and the merged images
confirmed that incubation with HepG2 cells for 2 hours (Fig. 13, 14, and Fig.
S31-34), compounds 1-6 all went through the membrane and localized uniformly
in the cytoplasm, suggesting that only the cell cytoplasm can be labelled by the
dyes.
Fig. 13
2
5

ACCEPTED MANUSCRIPT
Two-photon fluorescence microscopy provides key advantages over
one-photon fluorescence imaging, namely, increased penetration depth, lower
tissue autofluorescence and self-absorption, and reduced photodamage and
photobleaching [41]. In two-photon microscopy imaging, the optimal excitation
wavelength of each compound was used. As shown in Fig. 14b and Fig. S34b, the
TPEF image of HepG2 cells shows bright green fluorescence upon excitation.
These results highlight the potential of 5 and 6 for both one- and two-photon
fluorescence microscopy imaging applications. The image of HepG2 cells stained
with 3 shows weak fluorescence (Fig. S33b), which may be due to the weak
fluorescence emission and smaller TPA cross-section of 3.
Fig. 14
4
. Conclusion
In conclusion, the fluorescence properties of compounds 1-6 were successfully
tuned from OPEF to TPEF through increasing electron-pushing abilities of donors.
As a result, compounds 5 and 6 exhibit two-photon excited fluorescence
properties, due to the enhanced intramolecular charge transfer caused by the
stronger electron donors and more planar structures. The corresponding
fluorescence property, low toxicity on living cells, as well as cell permeability
enable them to be very promising in one- or two-photon fluorescence cell imaging.
Acknowledgements
2
6

ACCEPTED MANUSCRIPT
This work was supported by Program for New Century Excellent Talents in
University (China), Doctoral Program Foundation of Ministry of Education of
China (20113401110004), National Natural Science Foundation of China
(
21271003, 21271004, 21201005), the 211 Project of Anhui University, Doctoral
Startup Foundation of Anhui University (33190077), the Anhui University
Students Research Training Program (kyxl2012020), the Graduate Students
Innovative Program of Anhui University (10117700063, 10117700066), Natural
Science Foundation of Anhui Province (1308085QB33), and Natural Science
Foundation of Education Committee of Anhui Province (KJ2012A024,
KJ2013B201).
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