1, 3, 5-Triazine-cored derivatives dyes containing triphenylamine based two-photon absorption: Synthesis, optical characterization and bioimaging

Article abstract of DOI:10.1016/j.dyepig.2012.03.017

A series of new one, two and three-branched two-photon absorption triazine dyes containing triphenylamine with a π-bond and a σ-electron pair as a bridge, and different electron-donating groups, have been synthesized and their photophysical properties have been systematically investigated. These dyes showed obvious solvatochromic effects, i.e., significant bathochromic shifting of the emission spectra and larger Stokes shifts were observed in more polar solvents mainly due to intramolecular charge-transfer (ICT). The two-photon absorption (2PA) cross section values were determined by two-photon excited fluorescence (TPEF) measurements in DMF. This result further proved that a σ-electron pair as a bridge is an efficient way to transfer charge as well as a π bridge to provide a large 2PA cross section, and that their 2PA cross section values (δ) increase with increasing electron-donating strength of the end group and branch number. In addition, two-photon fluorescence cell imaging of dye 7a in HeLa and MCF-7 cancer cells was demonstrated.

Full text of DOI:10.1016/j.dyepig.2012.03.017

Dyes and Pigments 94 (2012) 570e582
Contents lists available at SciVerse ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
1, 3, 5-Triazine-cored derivatives dyes containing triphenylamine based
two-photon absorption: Synthesis, optical characterization and bioimaging
,
a
a
a
a
a
Hongping Zhou a , Zheng Zheng , Guoyi Xu , Zhipeng Yu , Xiaofei Yang , Longhuai Cheng ,
*
Xiaohe Tian b, Lin Kong a, Jieying Wu a, Yupeng Tian a
,c,d
a Department of Chemistry, Anhui University, Key Laboratory of Functional Inorganic Materials Chemistry of Anhui Province, 230039 Hefei, PR China
b Department of Biomedical Science, University of Sheffield, Sheffield, UK
c State Key Laboratory of Crystal Materials, Shandong University, 250100 Jinan, PR China
d State Key Laboratory of Coordination Chemistry, Nanjing University, 210093 Nanjing, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new one, two and three-branched two-photon absorption triazine dyes containing triphe-
Received 3 January 2012
Received in revised form
13 March 2012
Accepted 14 March 2012
Available online 23 March 2012
nylamine with a
p-bond and a s-electron pair as a bridge, and different electron-donating groups, have
been synthesized and their photophysical properties have been systematically investigated. These dyes
showed obvious solvatochromic effects, i.e., significant bathochromic shifting of the emission spectra and
larger Stokes shifts were observed in more polar solvents mainly due to intramolecular charge-transfer
(ICT). The two-photon absorption (2PA) cross section values were determined by two-photon excited
fluorescence (TPEF) measurements in DMF. This result further proved that a
an efficient way to transfer charge as well as a bridge to provide a large 2PA cross section, and that their
2PA cross section values ( ) increase with increasing electron-donating strength of the end group and
s-electron pair as a bridge is
Keywords:
Triazine dyes
Triphenylamine
p
d
branch number. In addition, two-photon fluorescence cell imaging of dye 7a in HeLa and MCF-7 cancer
cells was demonstrated.
s
-Electron pair as a bridge
Photophysical properties
2PA cross section
Two-photon fluorescence cell imaging
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
However, most of the currently used one-photon excited bio-
logical fluorophores show low 2PA cross sections (d) and are thus
The development of materials displaying large two-photon
absorption (2PA) [1,2] has attracted great interest in past decades
due to a variety of potential applications in photonics and opto-
electronics, such as three-dimensional optical data storage, fluo-
rescence imaging, optical limiting, up-converted lasing and
microfabrication [3]. The big advantage of these materials is that
excitation in the lower-energy near-IR region can lead to inherent
higher-energy photophysical properties of the chromophores. It is
important to prevent damage to the materials as a result of higher-
energy photons. Besides, the quadratic dependence of two-photon
absorption on excitation intensity causes photochemistry to occur
in a small focal region, allowing for more control in micro-
fabrication and imaging applications [4]. In the early 1990s, two-
photon fluorescence (2PF) imaging was pioneered by Webb et al.
[5]. Since then it has become the technique of choice for noninva-
sive biological imaging in thick tissue and living animals, due to the
above merits it provides.
limited their use in 2PF imaging. Therefore, there is a pressing need
to synthesize materials with large 2PA cross sections for in vivo
imaging. Some design strategies for the construction of molecules
with large two-photon absorption cross sections (d) have been
studied from both experimental [6,7,14] and theoretical [8,9]
perspectives. The accumulated knowledge and experience from
many research groups to understand the connections between
molecular structure and 2PA properties by testing and modeling
various dye systems since the mid-1990s have revealed that
molecular 2PA is related to a combination of several structural
parameters, such as intramolecular charge-transfer (ICT) efficiency
and/or the effective size of the
p-conjugation domain within
a molecule, i.e., the conjugation length and strength of the donor
and acceptor [10,11]. Besides, designing a multi-branched backbone
molecular structure is one of the effective strategies [11,12] to
obtain a large 2PA cross section, as the branched architecture not
only provides a way to incorporate several 2PA-enhancing param-
eters into a single system, but also allows material chemists to
optimize a dye molecule by combining various desired character-
istics together for specific purposes. Cho et al [13e16] reported that
* Corresponding author. Tel.: þ86 551 5108151; fax: þ86 551 5107342.
E-mail address: zhpzhp@263.net (H. Zhou).
0143-7208/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2012.03.017

H. Zhou et al. / Dyes and Pigments 94 (2012) 570e582
571
the
p-conjugated core played a major role in the branched octu-
2. Experimental section
polar molecules’ 2PA properties, and the three-arm octupolar
structure showed significant improvement of the 2PA response in
comparison with singly or doubly branched counterparts [17,18].
On the other hand, the fluorescence quantum yields of
a majority of 2PA materials are found to decrease at high concen-
tration due to the aggregation of molecules, which generally leads
to fluorescence quenching in the solid state. Consequently a special
molecular design for a 2PA material is required not only to ensure
a large two-photon activity, but more importantly, to overcome
fluorescence quenching at high concentration. Very recently, Tian
et al. [19] reported two new AIE (aggregation-induced emission)-
active multi-branched molecules with a 1, 3, 5-triazine core,
which exhibit aggregation-induced emission and a large two-
photon absorption cross section (8629 GM). Hua et al. [20] repor-
ted a series of new multi-branched triazine chromophores con-
taining triphenylamine derivatives and demonstrated that multi-
branched triazine chromophores are a highly suitable class of
two-photon absorbing materials. To date, most of studies about the
two-photon materials have been focused on employing C]C bonds
as the linkage to combine a 1, 3, 5-triazine core with an electron-
donating end group while very little attention has been paid to
the alteration of the acceptoredonor linkage. Recently, spectral,
and photophysical characteristics of some N-triazinyl derivatives
has been reported [21], but 2PA and applications of such materials
are studied rarely. Wang et al. [22] reported a new 2PA-active
compound employing a nitrogen atom as the linkage to combine
a 1, 3, 5-triazine core with substituents. This new strategy can not
only increase the solubility in common organic solutions, but can
also offer the advantages of increasing internal rotation of the
chromophores in the monomer form without causing aggregation
in media, and electron delocalization is favoured. Based on that, we
were motivated to design a series of dyes containing a 1, 3, 5-
triazine unit and investigate their structureeproperty relation-
ships as 2PA materials.
2.1. Materials and physical measurements
IR spectra were recorded with a Nicolet FT-IR NEXUS 870
spectrometer (KBr discs) in the 4000e400 cmꢀ1 region. 1H and 13
C
NMR spectra were recorded on a 400 or 500 MHz NMR instrument
using CDCl3 or (CD3)2CO as 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). Mass
spectra were obtained on a Micromass GCT-MS Spectrometer.
MALDI-TOF mass spectra were recorded on a time-of-flight (TOF)
mass spectrometer using a 337 nm nitrogen laser with alpha-
cyano-4-hydroxycinnamic acid as matrix.
2.2. Optical measurements
The one-photon absorption (OPA) spectra were recorded on a UV-
265 spectrophotometer. The one-photon excited fluorescence (OPEF)
spectra measurements were performed using a Hitachi F-7000 fluo-
rescence spectrophotometer. OPA and OPEF of dyes 5ae7a, 5be7b
were measured in five organic solvents of different polarities with
the concentration of 1.0 ꢁ 10ꢀ5 mol Lꢀ1. The quartz cuvettes used are
of 1 cm path length. The fluorescence quantum yields (F) were
determined by using coumarin 307 as the reference according to the
literature method [26]. Quantum yields were corrected as follows:
ꢀ
ꢁ
A
A
rh2s Ds
sh2r Dr
Fs
¼
Fr
where the s and r indices designate the sample and reference
samples, respectively, A is the absorbance at lexc is the average
refractive index of the appropriate solution, and D is the integrated
,
h
area under the corrected emission spectrum [27].
On the basis of these interesting findings, we intend to connect
two electron-donating groups diphenyl and diethoxyphenyl to one
nitrogen of 1, 2-bis(4-aminophenyl)ethylene, which is connected
Two-photon absorption (2PA) cross sections (d) of the samples
were obtained by two-photon excited fluorescence (TPEF) method
[28] at femtosecond laser pulse and Ti: sapphire system
(680e1080 nm, 80 MHz, 140 fs) as the light source. The sample was
dissolved in different solvents at a concentration of 1.0 ꢁ 10ꢀ3
mol Lꢀ1. The intensities of TPEF spectra of the reference and the
sample were determined at their excitation wavelength. Thus, 2PA
to the electron-withdrawing 1, 3, 5-triazine core through a
s-
electron pair on the N atom, to build up new octupolar 2PA dyes.
This idea is also based on the following considerations: (1) 1, 3, 5-
triazine-based compounds show good optical and electrical prop-
erties due to high electron affinity and symmetrical structure [23].
In particular, octupolar molecules consisting of a strong triazine
electron-accepting center and an electron-donating end group
cross section (d) of samples was determined by Eq.:
Fref cref nref
F
d
¼
dref
F
c
n Fref
linked through a p-conjugated bridge have proved to be excellent
2PA materials, because of high coplanarity of the conjugated
system, strong intramolecular charge-transfer (ICT), and additional
cooperative enhancement between the branches [24]. (2) Triphe-
nylamine has been widely used in opto- and electroactive mate-
rials, due to their good electron-donating and transporting
capabilities, as well as their special propeller starburst molecular
Where the ref subscripts stand for the reference molecule (here
coumarin 307 in ethanol solution at concentration of 1.0 ꢁ 10ꢀ3
mol Lꢀ1 was used as reference).
d is the 2PA cross-sectional value, c is
the concentration of the solution, n is the refractive index of the
solution, F is the TPEF integral intensities of the solution emitted at
the exciting wavelength, and
F is the fluorescence quantum yield.
structure [25]. (3) A s-electron pair as a bridge can be used to not
only extend the molecular conjugated system and promote solu-
The dref value of reference was taken from the literature [29].
bility, but also to avoid the aggregation-induced fluorescence
quenching of the strong
-systems.
Here we have strategically designed and presented a similar one-
step method to prepare of a series of new pull-push type octupolar
triazine dyes (7ae7b) and their one-and two-branched dyes
(5ae5b, 6ae6b) by changing the ratio of RNH2 and 2, 4, 6-trichloro-
1, 3, 5-triazine, upon incorporating different electron-donating
groups with 1, 3, 5-triazine. Linear photophysical characterization
and investigation of 2PA properties are presented as a basis for
potential applications in two-photon fluorescence imaging.
p
e
p
stacking interaction in these large flat
2.3. Materials and synthesis
p
Triphenylamine, 4-iodophenol, and cyanuric chloride were
available commercially, and the solvents were purified as conven-
tional methods before use. These compounds were characterized
by 1H NMR, 13C NMR, FT-IR, and MS spectrometry.
2.3.1. Preparation of 3a
0.6 g (5.4 mmol) t-BuOK was placed into a dry mortar and well
milled into powder, then 1.0 g (2.2 mmol) 1 and 0.5 g (1.8 mmol) 2a

572
H. Zhou et al. / Dyes and Pigments 94 (2012) 570e582
were added and mixed. The mixture was milled vigorously and
monitored by TLC. The mixture became sticky and was continu-
ously milled for another 10 min. After completion of the reaction,
the mixture was dissolved in 100 mL CH2Cl2. The mixture was
filtered and concentrated. The product was recrystallized from
anhydrous ethanol, to give 0.33 g red arborescent solid. Yield:
(400 Hz, (CD3)2SO),
d
(ppm): 7.31 (d, J ¼ 8.0 Hz, 2H), 7.22 (d,
J ¼ 8.0 Hz, 2H), 6.98 (d, J ¼ 8.8 Hz, 4H), 6.86 (t, 5H), 6.78 (d,
J ¼ 16.8 Hz, 1H), 6.74 (d, J ¼ 8.0 Hz, 2H), 6.55 (d, J ¼ 8.4 Hz, 2H), 5.24
(s, 2H), 3.99 (q, J ¼ 6.9 Hz, 4H), 1.32 (t, J ¼ 6.8 Hz, 6H). 13C NMR
(125 MHz, CDCl3):
d
(ppm) ¼ 155.3, 148.0, 143.3, 140.9, 140.8, 130.2,
127.5, 127.2, 127.0, 126.6, 126.0, 125.8, 125.5, 120.9, 120.7, 120.5,
116.7, 115.4, 63.8, 15.1. IR (KBr, cmꢀ1): 3452, 3374, 3028, 2979, 2925,
1607, 1503, 1476, 1392, 1283, 1238, 1046, 962, 827. HRMS (GCT-MS)
Calcd for C30H30N2O2, 450.23; Found, 450.2336.
47.0%. 1H NMR: (400 Hz, (CD3)2CO),
d
(ppm): 8.23 (d, J ¼ 8.0 Hz, 2H),
7.85 (d, J ¼ 8.4 Hz, 2H), 7.60 (d, J ¼ 8.0 Hz, 2H), 7.51 (d, J ¼ 16.4 Hz,
1H), 7.35 (d, J ¼ 7.8 Hz, 4H), 7.38 (d, J ¼ 16.4, 1H), 7.13 (d, J ¼ 7.2 Hz,
6H), 7.03 (d, J ¼ 8.0 Hz, 2H). 13C NMR (125 MHz, CDCl3):
d
(ppm) ¼ 148.7, 147.8, 147.4, 146.5, 145.0, 144.5, 133.5, 133.0, 130.0,
2.3.5. Preparation of 5a
129.7, 129.5, 129.4, 128.2, 126.7, 126.5, 125.1, 124.8, 124.3, 123.8,
123.6, 122.8, 122.3. IR (KBr, cmꢀ1): 3055, 1680, 1588,1511, 1484,
1439, 1334, 1311, 995, 860, 753, 696. HRMS (GCT-MS) Calcd for
C26H20N2O2, 392.15; Found, 392.1487.
A suspension of 1.0 g (5.5 mmol) of cyanuric chloride, 4.8 mL of
N, N-Diisopropylethylamine (DIPEA) dissolved in 20 mL of dry THF
was added into a round-bottom flask equipped with a magnetic
stirrer in an ice-salt bath, and a solution of 2 g (5.5 mmol) of 4a
dissolved in 10 mL dry THF was added dropwise with stirring for
1 h. The reaction was monitored by TLC. After the completion of the
reaction, the solvent was removed under reduced pressure and the
residue was purified by column chromatography with petroleum
(b.p. 60-90 ꢂC)/ethyl acetate (20:1 by volume) to give 1.2 g of yellow
2.3.2. Preparation of 3b
Black oil 3b was obtained using 2b instead of 2a, with only one
silica gel column chromatography step with petroleum b.p.
60e90 ꢂC/ethyl acetate (20:1 by volume). Yield: 51.0%. 1H NMR:
(400 Hz, (CD3)2SO),
d
(ppm): 8.19 (d, J ¼ 8.8 Hz, 2H), 7.78 (d,
solid. Yield: 43.0%. 1H NMR: (400 Hz, (CD3)2CO),
d (ppm): 9.89 (s,
J ¼ 8.8 Hz, 2H), 7.47 (d, J ¼ 8.4 Hz, 2H), 7.04 (t, 5H), 6.91 (t, 5H), 6.74
1H), 7.68 (d, J ¼ 8.4 Hz, 2H), 7.38 (d, J ¼ 8.4 Hz, 2H), 7.31 (t, J ¼ 7.8 Hz,
4H), 7.22 (d, J ¼ 8.8 Hz, 2H), 7.07 (q, J ¼ 6.3 Hz, 6H), 6.91 (d,
J ¼ 8.8 Hz, 2H), 6.61 (d, J ¼ 12.0 Hz, 1H), 6.57 (d, J ¼ 12.0 Hz, 1H). 13C
(d, J ¼ 8.80 Hz, 2H), 4.00 (q, J ¼ 7.1 Hz, 4H),1.32 (t, J ¼ 6.9 Hz, 6H). 13C
NMR (100 MHz, CDCl3):
d
(ppm) ¼ 155.7, 149.5, 146.2, 144.6, 140.1,
133.2, 127.9, 127.8, 127.1, 126.3, 124.2, 123.1, 119.5, 115.4, 63.7, 14.9. IR
(KBr, cmꢀ1): 2978, 2924, 1580, 1504, 1476, 1391, 1334, 1239, 954,
834. HRMS (GCT-MS) Calcd for C30H28N2O4, 480.20; Found,
480.2026.
NMR (125 MHz, CO(CD3)2):
d
(ppm) ¼ 169.6, 164.3, 147.6, 147.4,
136.1, 134.8, 131.7, 129.4, 127.9, 127.5, 126.8, 126.2, 124.4, 123.3,
123.2, 121.6. IR (KBr, cmꢀ1): 3285, 3028, 2924, 1591, 1555, 1510,
1492, 1388, 1280, 1228, 1168, 1021, 961, 836, 753, 696. HRMS (GCT-
MS) Calcd for C29H21Cl2N5, 509.12; Found, 509.1193.
2.3.3. Preparation of 4a
A solution of 3.0 g (10 mmol) of 3a dissolved in 150 mL ethanol
was added into a round-bottom flask equipped with a magnetic
stirrer and heated at 80 ꢂC. Then 0.3 g of Pd/C catalyst was added
into the preceding reaction system and a solution of 4.9 mL of 85%
hydrazine hydrate was added dropwise for about 0.5 h. The reac-
tion was monitored by TLC. After the completion of the reaction, the
reaction mixture was filtered immediately and the solution was
cooled to room temperature to give a cream solid. 2.56 g of 4b was
obtained after filtration and drying in vacuo.Yield: 75.0%. 1H NMR:
2.3.6. Preparation of 5b
A suspension of 1.0 g (5.4 mmol) of cyanuric chloride, 4.7 mL of
DIPEA dissolved in 10 mL of dry THF was added into a round-
bottom flask equipped with a magnetic stirrer in an ice-salt bath,
and a solution of 2.42 g (5.4 mmol) of 4b dissolved in 10 mL dry THF
was added dropwise for 1 h. The reaction was monitored by TLC.
After the completion of the reaction, the solvent was removed
under reduced pressure and the residue was purified by column
chromatography with petroleum (b.p. 60e90 ꢂC)/ethyl acetate
(20:1 by volume) to give 1.03 g of yellowish-brown solid. Yield:
(400 Hz, (CD3)2CO),
7.05 (m, 10H), 6.93 (d, J ¼ 16.4 Hz, 1H), 6.68 (d, J ¼ 8.4 Hz, 2H), 4.78
(s, 2H). 13C NMR (100 MHz, CDCl3):
d
(ppm): 7.45 (d, J ¼ 8.8 Hz, 2H), 7.30 (m, 6H),
32.0%. 1H NMR: (400 Hz, (CD3)2CO),
d (ppm): 9.90 (s, 1H), 7.73 (d,
d
(ppm) ¼ 147.54, 147.50, 147.3,
J ¼ 8.4 Hz, 2H), 7.61 (d, J ¼ 8.4 Hz, 2H), 7.41 (d, J ¼ 8.4 Hz,2H), 7.17 (d,
J ¼ 16.4 Hz, 1H), 7.06 (m, 5H), 6.90 (d, J ¼ 8.8 Hz, 4H), 6.84 (d,
J ¼ 8.4 Hz, 2H), 4.04 (q, J ¼ 6.8 Hz, 4H), 1.37 (t, J ¼ 6.8 Hz, 6H). 13C
134.2, 131.3, 129.3, 127.6, 127.5, 127.3, 126.0, 124.5, 123.5, 123.0,
120.1. IR (KBr, cmꢀ1): 3449, 3371, 3023, 1616, 1586, 1515, 1489, 1330,
965, 834, 750, 697. HRMS (GCT-MS) Calcd for C26H22N2, 362.18;
Found, 362.1777.
NMR (100 MHz, CDCl3):
d
(ppm) ¼ 170.5, 165.2, 156.7, 149.5, 141.3,
136.7, 136.0, 130.3, 129.2, 128.2, 127.7, 127.5, 125.8, 122.5, 120.6,
116.2, 64.2, 15.2. IR (KBr, cmꢀ1): 3382, 2977, 2926, 1599, 1545, 1503,
1477, 1390, 1319, 1288, 1046, 1017, 961, 829, 795. HRMS (GCT-MS)
Calcd for C33H29Cl2N5O2, 597.17; Found, 597.1743.
2.3.4. Preparation of 4b
A solution of 1.09 g (2.27 mmol) of 3b dissolved in 25 mL of
ethanol was added into a round-bottom flask equipped with
a magnetic stirrer and heated at 80 ꢂC. Then 0.12 g of Pd/C catalyst
was added into the preceding reaction system and a solution of
1.1 mL of 85% hydrazine hydrate dissolved in 25 mL ethanol was
added dropwise for 0.5 h. The reaction was monitored by TLC. After
the completion of the reaction, the solvent was removed under
reduced pressure. The mixture was washed with water and
extracted with ethyl acetate. The organic phase was dried over
MgSO4, filtered and concentrated to provide light red oil. Purifica-
tion was carried out by silica gel column chromatography using
petroleum (b.p. 60e90 ꢂC)/ethyl acetate (10:1 by volume) to afford
0.61 g of yellow powder. Yield: 60.0%. 1H NMR(cis-): (400 Hz,
2.3.7. Preparation of 6a
A suspension of 0.12 g (0.65 mmol) of cyanuric chloride, 1.2 mL
of DIPEA, 0.25 g (0.65 mmol) of 4a and 10 mL of dry THF was added
into a round-bottom flask equipped with a magnetic stirrer .The
mixture was stirred at room temperature for 4 h and refluxed at
70 ꢂC for 8 h. Another molar amount of 4a (0.25 g, 0.65 mmol) was
added into the preceding reaction system. The reaction was
refluxed and monitored by TLC. After the completion of the reac-
tion, appropriate amount of CH2Cl2 was added and the solution was
washed with water. The organic phase was dried with anhydrous
MgSO4, filtered, concentrated and recrystallized with ethanol to
give 0.17 g of yellow solid. Yield: 52.7%. 1H NMR: (400 Hz,
(CD3)2SO),
d
(ppm): 7.10 (d, J ¼ 8.4, 2H), 6.98 (t, J ¼ 7.0 Hz, 6H), 6.88
(d, J ¼ 8.8 Hz, 4H), 6.63 (d, J ¼ 8.4 Hz, 2H), 6.44 (d, J ¼ 8.0 Hz, 2H),
6.28 (d, J ¼ 12.4 Hz, 1H), 6.19 (d, J ¼ 12.4, 1H), 5.18 (s, 1H), 3.99 (q,
(CD3)2SO),
d
(ppm): 9.93 (s, 1H), 7.64 (d, J ¼ 8.0 Hz, 2H), 7.52 (d,
J ¼ 8.0 Hz, 2H), 7.32 (t, J ¼ 7.4 Hz, 4H), 7.22 (d, J ¼ 16.4 Hz, 1H), 7.15
J ¼ 6.9 Hz, 4H),
d
(ppm): 1.31 (t, J ¼ 7.0 Hz, 6H); 1H NMR(trans-):
(d, J ¼ 16.4 Hz,1H), 7.08 (t, J ¼ 8.8 Hz, 6H), 7.01 (d, J ¼ 8.0 Hz, 2H). 13C

H. Zhou et al. / Dyes and Pigments 94 (2012) 570e582
573
NMR (125 MHz, CDCl3):
d
(ppm) ¼ 168.4, 164.1, 148.3, 136.6, 135.2,
7.34 (d, J ¼ 8.0 Hz, 4H), 6.95 (d, J ¼ 8.4 Hz, 12H), 6.85 (d, J ¼ 8.4 Hz,
8H), 6.71 (d, J ¼ 8.0 Hz, 4H), 3.95 (q, J ¼ 6.7 Hz, 8H), 1.28 (t,
132.1, 130.1, 128.7, 128.1, 127.5, 126.8, 125.2, 124.2, 123.8, 122.2. IR
(KBr, cmꢀ1): 3385, 3025,1591,1570,1493, 1414,1278, 1075, 961, 831,
753, 695. MALDI-TOF Calcd for C55H42ClN7, 835.32; Found, 836.1.
J ¼ 6.8 Hz, 12H). 13C NMR (125 MHz, CDCl3):
(ppm) ¼ 165.1, 156.2,
d
148.8, 141.0, 140.2, 132.5, 130.5, 128.3, 127.8, 127.4, 126.7, 121.5,
120.5, 116.5, 64.3, 15.9. IR (KBr, cmꢀ1): 3397, 3026, 2977, 2925, 1599,
1569, 1504, 1413, 1317, 1238, 1046, 961, 828. MALDI-TOF Calcd for
C93H87N9O6, 1425.68; Found, 1426.17.
2.3.8. Preparation of 6b
A suspension of 0.61 g (3.33 mmol) of cyanuric chloride, 2.9 mL
of N,N-Diisopropylethylamine, 1.5 g (3.33 mmol) of 4b and 25 mL of
dry THF was added into a round-bottom flask equipped with
a magnetic stirrer .The mixture was stirred at room temperature for
4 h. Another molar amount of 4b (1.5 g, 3.33 mmol) was added into
the preceding reaction system. Then the reaction mixture was
refluxed at 70 ꢂC for 8 h and monitored by TLC. After the completion
of the reaction, appropriate amount of ethanol was added with
stirring and the solution was filtered to give a yellow solid. The
residue was purified by column chromatography with petroleum
(b.p. 60e90 ꢂC)/ethyl acetate (6:1 by volume) to give 1.7 g of yellow
2.3.11. Cell culture and incubation
MCF-7 (human breast cancer cell line) cells were seed and
plated on
(SigmaeAldrich) supplemented with 10% fetal bovine serum
(Sigma),1%
-glutamine, 1% penicillin, and 1% streptomycin, at 37 ꢂC
a T75 Flask and cultured in RPMI-1640 Media
L
in a humid atmosphere with 5% CO2 and 95% air for 2 h incubation,
and then MCF-7 cells were seeded in BD FalconÔ Clear 96-well
MicrotestÔ Plate at a density of 1 ꢁ 104 cells per well and incu-
bated at the same conditions as indicated above until 80e90%
confluency was reached. Stock solutions of dye 7a dissolved in
solid. Yield: 51.4%. 1H NMR: (400 Hz, (CD3)2SO),
d (ppm): 10.33 (s,
1H), 7.62 (d, J ¼ 8.4 Hz, 2H), 7.51 (d, J ¼ 8.0 Hz, 2H), 7.37 (d, J ¼ 7.2 Hz,
2H), 7.13 (d, J ¼ 16.4 Hz, 1H), 7.00 (m, 5H), 6.89 (d, J ¼ 8.4 Hz, 4H),
6.74 (d, J ¼ 7.6 Hz, 2H), 3.99 (q, J ¼ 6.7 Hz, 4H),1.32 (t, J ¼ 7.0 Hz, 6H).
DMSO were prepared at concentrations of 1 mM in PBS. For live cell
imaging, cell cultures were incubated with complex dye 7a solution
at and maintained at 37 ꢂC in an atmosphere of 5% CO2 and 95% air
for 2 h incubation. The cells were washed with PBS buffer
13C NMR (125 MHz, CDCl3):
d
(ppm) ¼ 169.1, 164.9, 156.2, 148.9,
140.9, 138.4, 134.2, 130.6, 130.2, 129.7, 128.4, 127.8, 127.5, 127.0,
126.3, 122.5, 120.4, 116.5, 64.3, 15.9. IR (KBr, cmꢀ1): 3383, 3028,
2976, 2925, 1599, 1559, 1503, 1413, 1391, 1237, 1045, 827, 723.
MALDI-TOF Calcd for C63H58ClN7O4, 1011.42; Found, 1011.7.
(2 ꢁ 200 ul per well). Costaining was performed using 2
m
M SYTO9Ò
M PI (lex ¼ 543,
(
lex ¼ 488, lem ¼ 500e530 nm) and 10
m
lem ¼ 560e580 nm) for 10 min in PBS then cells were washed with
PBS buffer (2 ꢁ 200 ul per well) and 200 ul of PBS was added to each
well, and the cells were taken to a two-photon microscope for
imaging without fixation.
2.3.9. Preparation of 7a
A suspension of 0.35 g (1.9 mmol) of cyanuric chloride, 2.08 g
(5.7 mmol) of intermediate 4a, 1.6 mL of DIPEA and 10 mL of THF
under nitrogen was added into a three-necked flask equipped with
a magnetic stirrer, a reflux condenser, and a nitrogen input tube. The
mixture was heated to 70 ꢂC and 1.0 g (9.5 mmol) of Na2CO3 and
5 mL of DMF were added. The reaction was heated to 140 ꢂC after
30 min and then 0.06 g (5 mol %) Pd(PPh3)4 was added into the flask.
The reaction mixture was refluxed under nitrogen about 30 h and
monitored by TLC. Then appropriate amount of CH2Cl2 was added
and the solution was washed with water. The organic phase was
dried with anhydrous MgSO4, filtered and concentrated. The yellow
product was purified by column chromatography with petroleum
b.p. 60e90 ꢂC/ethyl acetate (8:1 by volume) as eluent to yield 0.89 g
2.3.12. Two-photon fluorescence imaging
The cells were imaged in a tissue culture chamber (5% CO2,
37 ꢂC) and through a Zeiss 510 LSM (upright configuration) confocal
microscope with a femtosecond-pulsed Ti:Sapphire laser. The
excitation beam produced by the fs laser, was tunable from 720 to
900 nm, (lex ¼ 720 nm, w1 mW), which was passed through an
LSM 510 microscope with HFT 650 dichroic and focused onto the
cover-slip adherent cells using a 40ꢁ water immersion objective.
2.3.13. Cytotoxicity tests [MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
tetrazolium bromide) assays]
To ascertain the cytotoxic effect of dyes 6ae7a, 6be7b treatment
over a 24 h period, the MTT assay was performed. MCF-7 cells were
passed and plated to 80 %e90% confluence in 24-well plates 24 h
before treatment. Prior to dyes 6ae7a, 6be7b treatment, the RPMI
was removed and replaced with fresh media, and then aliquots of
of yellow solid. Yield:41.2%. 1H NMR: (400 Hz, (CD3)2SO),
d (ppm):
9.40 (s,1H), 7.83 (s, 4H), 7.51 (t, J ¼ 7.8 Hz, 8H), 7.32 (t, J ¼ 8.2 Hz, 8H),
7.7 (m, 16H), 6.96 (d, J ¼ 8.4 Hz, 4H). 13C NMR (125 MHz, CDCl3):
d
(ppm) ¼ 163.5, 148.2, 147.6, 132.8, 130.8, 128.6, 128.4, 128.1, 127.7,
127.1, 126.6, 125.2, 125.0, 124.3, 121.9, 120.8. IR (KBr, cmꢀ1): 3393,
3025, 1685, 1590, 1513, 1489, 1414, 1279, 1075, 961, 833, 753, 696.
MALDI-TOF Calcd for C81H63N9, 1161.52; Found, 1162.3.
dyes 6ae7a, 6be7b stock solutions (500
to obtain final concentrations of 1, 5 and 10
m
M in DMSO) were added
m
M. The treated cells
were incubated for 24 h at 37 ꢂC and under 5% CO2. Subsequently,
the cells were treated with 5 mg/mL MTT (1 mL/well) and incubated
for an additional 1 h (37 ꢂC, 5% CO2). Then DMEM was removed, the
cells were dissolved in DMSO (400 mL/well), and the excitation
wavelengths were recorded. The cell viability (%) was calculated
2.3.10. Preparation of 7b
A suspension of 0.12 g (0.66 mmol) of cyanuric chloride, 1.2 mL
of DIPEA, 0.89 g (1.98 mmol) of 4b and 10 mL of THF under nitrogen
was added into a three-necked flask equipped with a magnetic
stirrer, a reflux condenser, and a nitrogen input tube. The mixture
was heated to 70 ꢂC and 0.7 g (9.5 mmol) of Na2CO3 and 10 mL of
DMF were added. The reaction was heated to 140 ꢂC after 30 min
and then a catalytic amount of Pd(PPh3)4 was added into the flask.
The reaction mixture was refluxed under nitrogen about 30 h and
monitored by TLC. Appropriate amount of ethyl acetate was added
and the solution was washed with water. The organic phase was
dried with anhydrous MgSO4, filtered and concentrated. The crude
product was purified by column chromatography with petroleum
(b.p. 60e90 ꢂC)/ethyl acetate (10:1 by volume) as eluent to yield
0.21 g of yellow solid. Yield: 22.7%. 1H NMR: (400 Hz, (CD3)2SO),
according to the following equation: Cell viability % OD570(sample)
/
OD570(control) ꢁ 100, where OD570(sample) represents the optical
density of the wells treated with various concentration of dyes
6ae7a, 6be7b and OD570(control) represents that of the wells treated
with DMEM þ 10% FCS. Three independent trials were conducted,
and the average and standard deviations are reported. The reported
percent cell survival values are relative to untreated control cells.
3. Results and discussion
The targeted dyes (5ae5b, 6ae6b and 7ae7b) were synthesized
according to Scheme 1. On the one hand, the ethyoxyl chains on the
triphenylamine moieties in these model chromophores were
d
(ppm): 9.30 (s, 2H), 7.75 (d, J ¼ 5.2 Hz, 4H), 7.44 (d, J ¼ 8.0 Hz, 4H),

574
H. Zhou et al. / Dyes and Pigments 94 (2012) 570e582
Scheme 1. Synthetic routes to target dyes 5ae5b, 6ae6b and 7ae7b.

H. Zhou et al. / Dyes and Pigments 94 (2012) 570e582
575
Table 1
The linear and Two-photon absorption properties of dyes 5ae5b, 6ae6b and 7ae7b.
max/MWc
lmax
Dne
Ff
lmax
sh
s
/MWi
a
b
d
g
Solvents
lmax
3
3
max
5a
6a
7a
5b
6b
7b
Benzene
THF
Ethanol
Acetonitrile
DMF
Benzene
THF
Ethanol
Acetonitrile
DMF
Benzene
THF
Ethanol
Acetonitrile
DMF
Benzene
THF
Ethanol
Acetonitrile
DMF
Benzene
THF
Ethanol
Acetonitrile
DMF
Benzene
THF
Ethanol
Acetonitrile
DMF
382
379
377
374
376
379
376
373
372
378
378
376
373
372
377
394
389
386
384
384
388
386
382
383
386
390
388
383
382
389
4.15
4.55
4.51
4.32
4.44 (1.0)
9.77
10.64
10.63
9.41
10.96(2.5)
10.04
11.59
7.67
5.56
11.80 (2.7)
3.99
4.45
4.14
3.87
4.17(1.0)
8.08
8.97
7.70
429
437
437
453
448
428
438
442
453
448
429
439
443
453
450
466
479
491
513
503
464
482
497
514
505
459
471
491
508
500
2868
3502
3642
4663
4274
3021
3765
4185
4807
4134
3145
3817
4236
4807
4303
3921
4830
5540
6548
6161
4221
5160
6057
6654
6105
3855
4542
5743
6493
5707
0.071
0.132
0.092
0.056
0.558 (5.3)
0.310
0.294
0.078
0.114
0.134 (1.3)
0.171
0.221
0.090
0.148
0.106(1.0)
0.109
0.163
0.066
0.027
0.233 (2.6)
0.270
0.226
0.104
0.020
0.127 (1.4)
0.178
0.152
0.100
0.067
0.0087 (1.0)
0.0131 (1.5)
0.0102 (1.2)
0.0070 (1.0)
0.0081 (1.2)
0.0095 (1.4)
490
484
490
530
530
523
34
0.0668 (1.0)
0.1713 (2.6)
0.1601 (2.4)
0.1725 (1.0)
0.2828 (1.6)
0.7140 (4.2)
143
186
103
4.54
8.24(2.0)
12.32
13.36
12.77
12.84
13.60 (3.3)
286
0.091(1.0)
1018
a
Absorption peak position in nm (1 ꢁ 10ꢀ5 mol Lꢀ1).
b
c
d
e
f
Maximum molar absorbance in 104 molꢀ1 L cmꢀ1
.
The reduced
3
max, i.e. the value of
divided by the molecular weight, the numbers in parentheses are relative values by assigning that of 5a and 5b as 1.
Peak position of SPEF in nm (1.0 ꢁ 3 m10axꢀ5 mol Lꢀ1), excited at the absorption maximum.
Stokes shift in cmꢀ1
.
Quantun yields determined by using coumarin 307 (1.0 ꢁ 10ꢀ5 mol Lꢀ1) as the standard.
TPEF peak position in nm pumped by femtosecond laser pulses at 800 mw at their maximum excitation wavelength.
2PA cross section in GM.
g
h
i
Reduced cross section, i.e., 2PA cross section divided by molecular weight. The numbers in parentheses are relative values.
expected to enhance their solubility in common organic solvents,
which is another important parameter to be considered in the
molecular design of dyes for use in experimental studies and in
excited fluorescence (OPEF) were measured in five solvents of
different polarities at a concentration c ¼ 1 ꢁ 10ꢀ5 mol Lꢀ1. To
further investigate the spectra relationship of the compounds, the
one-photon absorption and the one-photon excited fluorescence
data of dyes 5ae5b, 6ae6b and 7ae7b are restricted to DMF.
Representative spectra of dye 7b are shown in Fig. 1, while the
others are included in the Supporting Information (Fig. S1).
potential. On the other hand, generally,
pep aggregation in the
solid state could quench the emission of organic luminophores due
to the formation of detrimental excimers, i.e., the degree of fluo-
rescence quenching would increase with the stronger
of the luminophores molecules. To avoid fluorescence quenching,
we adopt conjugated systems of a fused and -electron moieties
-conjugated bridge.
pep stacking
s
p
3.2.1. The linear optical properties of dyes 5ae5b, 6ae6b, 7ae7b in
various solvents
as bridges to replace the
p
As shown in Fig. 1 and Fig. S1, dyes 5ae5b, 6ae6b and 7ae7b
exhibit two major absorption bands: the peak at about 305 nm is
attributed to the absorption of the triphenylamine moiety and
remains almost unchanged for all the one-, two-, and three-
branched dyes, while the other peak at about 372e394 nm,
3.1. Synthesis
Synthetic routes of these series of dyes 5ae5b, 6ae6b and 7ae7b
and their intermediates are depicted in Scheme 1. The intermediate
1, aldehyde 2a and 2b were synthesized efficiently according to the
literature [30,31]. The yield of 2a was 92%, while it is 40% for 2b.
Similarly, 3a and 3b were produced by the Wittig reaction between
the Wittig reagent and the corresponding aldehyde in the solid
phase with t-BuOK as base. 4a and 4b were prepared by the
hydrazine hydrate reduction method in the presence of Pd/C catalyst
in ethanol medium. The targeted dyes (5ae5b, 6ae6b and 7ae7b)
were obtained by the palladium-catalyzed CeN coupling strategy.
indicating
considerable
charge-transfer
character.
Upon
increasing the solvent polarity, an obvious blue shift in absorption
of dyes 5ae5b, 6ae6b and 7ae7b was observed while the OPEF
peak position lmax showed an increasing tendency for the six
dyes as the solvent polarity increased following the order:
benzene < THF < ethanol < acetonitrile, except in DMF. There may
be great changes in the molecular geometry of the excited states
before fluorescence emission. The fluorescence deviation in solu-
tion can be attributed to the interaction of the solute with the local
molecular environment including solvent molecules and other
surrounding solute molecules. The Stokes shift also showed
monotonically increasing tendency with the increase of solvent
3.2. One-photon absorption and emission properties
The photophysical data of dyes 5ae5b, 6ae6b and 7ae7b are
polarity except in DMF. One can see that the quantum yields (F) of
listed in Table 1. The one-photon absorption (OPA) and one-photon

576
H. Zhou et al. / Dyes and Pigments 94 (2012) 570e582
Fig. 1. Linear absorption and fluorescence of dye 7b in five organic solvents of different polarities with a concentration of 1 ꢁ 10ꢀ5 mol Lꢀ1
.
dyes 6be7b decreased consistently and significantly as the polarity
of solvent increases except in DMF, and were especially highly
quenched in acetonitrile (Fig. 1 and Fig. S1). These can be explained
by the stronger solute/solvent interaction at the excited state
comparing with that at the ground state, which indicates that the
increasing polarity of the excited state increases. The energy level
can be lowered greatly by increasing dipoleedipole interaction
between the solute and solvent. These photophysical data indicate
that dyes can exhibit ICT phenomena between the fluorophore
center and the amino or ethoxylated group.
The OPEF maxima were obtained at their maxima excitation
wavelengths in DMF. From dye 5a to dye 7a, OPEF spectra show
regular red shifts as the branch number increases (see Fig. 2). These
red shifts may be ascribed to charge redistribution and extended
delocalization. However, as indicated by Table 1, Fig. 2, a slight red
shift in OPEF spectra is apparent in comparing dye 5b to dye 6b,
while it is found to be obviously blue-shifted for dye 7b relative to
dyes 5b and 6b, respectively. It is interesting that the Stokes shift of
each system shows the same increasing tendency with the OPEF as
the branch number increases. This may be due to the electron-
donating ability of 4-ethoxy-N-(4-ethoxyphenyl)-N-phenylaniline
is higher than triphenylamine. Besides, the ICT effect should be
taken into account considering the existence of an electron acceptor
and chromophore served by 1, 3, 5-triazine and styryl, respectively,
including the above-mentioned electron donors. The ICT state of
3.2.2. The linear optical properties of different branches
As listed in Table 1 and Fig. 2, from dye 5b to dye 7b (5ae7a), the
one-photon absorption maxima in DMF show regular red shifts as
the branch number increases and the spectral intensities also vary
regularly, the absorption coefficients (
consistently and significantly as the branch number increases. Note
that the
max of dyes 5ae7a and 5be7b are of the order of 104 and
they increase in the ratios 1.0:2.5:2.7, 1.0:2.0:3.3 with the number
of chromophores. These can be assigned to the extended -delo-
3
max) of the maxima increase
molecules with D-p-A structures has a high photoluminescence
(PL) emission ability; hence, the single peak in the PL emission
spectra manifests that the state responsible for the PL emission is
not the localized excited state but the ICT state. Thus, the different
dipole moments of the ICT state and the ground state lead to the
shift in PL spectra [24c]. Note that the fluorescence quantum yields
3
p
calization and a certain coupling between the branches in dyes 6a,
7a and 6b, 7b. The enhancement can be more clearly seen by
(F) of dyes 5ae7a and 5be7b reduce with the ratios of 5.3:1.3:1.0
revealing the reduced molar absorbance
3
max/MW which is defined
and 2.6:1.4:1.0, respectively, as the branch number increases. This
indicates that these dyes employed the nitrogen atom as the linkage
allowing internal rotation in the monomer form in organic solvents,
whereby the fluorescence was reduced. This can be ascribed to the
partial conjugation structure resulting from the delocalization of
the lone pair electrons on nitrogen.
as divided by the molecular weight. The
3
3 max/MW values
max
increase with a ratio of 1.0:1.5:1.2 for dyes 5ae7a and 1.0:1.2:1.4 for
dyes 5be7b. This means that the unit molecular weight leads to
larger
ring.
3 max by incorporating more branches into the central triazine
Fig. 2. OPA spectra (left) and OPEF spectra (right) of dyes 5ae7a and 5be7b in DMF with a concentration of 1 ꢁ 10ꢀ5 mol Lꢀ1
.

H. Zhou et al. / Dyes and Pigments 94 (2012) 570e582
577
3.2.3. The linear optical properties of different electron-donating
group
As can be seen in Fig. 3, the OPA maxima and the OPEF maxima
of dyes 5ae5b, 6ae6b and 7ae7b in DMF all show obvious bath-
ochromic shifts from a to b, respectively. They are all in agreement
Two-photon fluorescence spectra of dye 7b in DMF pumped by
femtosecond laser pulses at 800 mw at different excitation wave-
lengths are presented in Fig. 4a (others are found in Fig. S2). Fig. 4b
shows 2PF spectra of dye 7b under different pump intensities
(others are found in Fig. S3), and the insets show logarithmic plots
of the fluorescence integral versus pumped powers with a slope of
1.93 when the input laser power is increasing, suggesting a two-
photon excitation mechanism. As no linear absorption was
observed in the range from 500 nm to 2000 nm, so the emission
excited by 790 nm laser wavelength can be attributed to the TPEF
mechanism. As shown in Fig. 4c, all the six dyes display 2PA activity
in the range of 720e880 nm in DMF.
with the order of the extension of the
ethoxyphenyl)-N-phenylaniline > triphenylamine. Note that the
maximum molar absorbance ( max) and the fluorescence quantum
yield ( ) of the dyes have to follow some rules. The maximum
molar absorbance ( max) of dyes 5ae5b and 6ae6b show the same
sequence of
p-systems: 4-ethoxy-N-(4-
3
F
3
3
>
3
and
3
>
3 max(6b), while dyes
max(5a)
max(5b)
max(6a)
7ae7b show a different sequence of
3
>
3
While the
max(7b)
max(7a).
fluorescence quantum yield (
all show the same sequence of F(5a)
F(7a) F(7b). Interestingly, the value of dyes 5be7b is depressed
in comparison with dyes 5ae7a, suggesting that although the
flexible 4-ethoxy-N-(4-ethoxyphenyl)-N-phenylaniline chains
function as solubilizing groups, it also enhances the nonradiative
decay pathways. Therefore, we draw a conclusion that the dyes
with triphenylamine as the donating end-groups appear to have
F
) of dyes 5ae5b, 6ae6b and 7ae7b
It can be seen from Table 1 that the fluorescence peak wave-
lengths are evidently red-shifted by 23e42 nm by comparing with
those of OPEF in DMF, which can be explained by the reabsorption
effect. For one-photon induced emission measurements, we used
dilute solutions (1 ꢁ 10ꢀ5 mol Lꢀ1), thus the reabsorption of the
fluorescence within the samples is negligible. In the case of two-
photon excitation, concentrated solutions (1 ꢁ 10ꢀ3 mol Lꢀ1) were
used. Since the 790 nm laser beam can pass through the whole
solution without linear depletion, the fluorescence emission is not
only from the surface layer, but also from inside the solution sample.
As a result, the reabsorption of the shorter wavelength fluorescence
by the concentrated sample can no longer be neglected. The short
wavelength side of the two-photon fluorescence was reabsorbed by
the solution and red shifts of the fluorescence spectra were readily
observed [17]. As shown in Fig. 5, the intensities of TPEF of dyes
5be7b exhibit the sequence of 5b < 6b << 7b. These similarities
between TPEF and OPEF indicate that both the emissions for a given
dye are from the same excited state, though their initial
FrankeCondon states may be different. The difference between
TPEF and OPEF is mainly during the excitation process: two-photon
absorption vs single-photon absorption.
>
F(5b) F(6a) F(6b) and
,
>
>
F
high
F
and
3
values compared with the dyes with 4-ethoxy-N-
max
(4-ethoxyphenyl)-N-phenylaniline as the donating end-groups.
3.3. Two-photon properties
The two-photon excited fluorescence spectra (TPEF) of six dyes
in DMF were recorded at their maximum excitation wavelength
with a pulse duration of 140 fs under 800 mw (milliwatt). The 2PA
excited fluorescence spectra (TPEF) data of dyes 5ae7a and 5be7b
are described in Table 1, which were measured in DMF
(c ¼ 1 ꢁ 10ꢀ3 mol Lꢀ1). Representative TPEF spectra of dye 7b are
shown in Fig. 4a and b, while the others are included in the Sup-
porting Information (Figs. S2 and S3).
Fig. 3. OPA spectra (left) and OPEF spectra (right) of dyes 5ae5b, 6ae6b and 7ae7b in DMF with a concentration of 1 ꢁ 10ꢀ5 mol Lꢀ1
.

578
H. Zhou et al. / Dyes and Pigments 94 (2012) 570e582
Fig. 4. (a) Two-photon fluorescence spectra of dye 7b in DMF pumped by femtosecond laser pulses at 800 mw at different excitation wavelength. (b) 2PF spectra of dye 7b in DMF
under different pumped powers at 790 nm, insert contains the logarithmic plots of the fluorescence integral of chromophores versus different excitation intensities. (c) Two-photon
absorption cross sections of dyes 5ae7a and 5be7b in 720e880 nm regions.
As shown in Table 1 and Fig. 5, the 2PA cross section values (
increases obviously as the branch number increases in the series of
dyes 5ae7a and 5be7b. The reduced 2PA cross section /MW, which
is defined as divided by the molecular weight, varies in a propor-
tion of 1.0:2.6:2.4 for dyes 5ae7a and 1.0:1.6:4.2 for dyes 5be7b,
respectively. This means that the unit molecular weight enables
enhanced 2PA cross section values as the branch number increases.
s
)
This leads to the indication of some interactions between branches
in the molecule, thus resulting in charge redistribution and
extended delocalization. Consequently, the enhancement effect
works in both linear and non-linear 2PA processes for dyes 5ae7a
and 5be7b. Note that the non-linear spectroscopic behavior of dye
6b resembles that of dye 5b, but does not resemble that of dye 7b. It
is different from dye 5ae7a that dye 6a resembles that of dye 7a.
s
s
Fig. 5. The two-photon fluorescence spectra of dyes 5ae7a and 5be7b in DMF (c ¼ 1 ꢁ 10ꢀ3 mol Lꢀ1).

H. Zhou et al. / Dyes and Pigments 94 (2012) 570e582
579
Fig. 6. The two-photon fluorescence spectra of dyes 5ae5b, 6ae6b and 7ae7b in DMF (c ¼ 1 ꢁ 10ꢀ3 mol Lꢀ1).
As shown in Fig. 6, by comparing dye 5a and dye 5b, dye 6a and
dye 6b, dye 7a and dye 7b, respectively, introducing an ethoxy
terminal groups results in an obvious red-shift in two-photon
fluorescence maxima and showing increased TPEF intensity from
b to c. The possible reason is that the ethoxy group increases the
electron-donating strength of the end group and also extends the
conjugation length of the system. In addition, it was found that dye
side effect of dyes that must be controlled when dealing with living
cells or tissues. Fig. 7 shows the cell viability for MCF-7 cells treated
with dyes 6ae7a, 6be7b at different concentrations for 24 h. The
results clearly indicated that MCF-7 cells incubated with concen-
tration of 1 mm of dye 7a remained almost 100% viable after 24 h of
feeding time, demonstrating the superior biocompatibility of the
compound dye 7a. This is also supported by live/dead co-staining
studies. Besides, it was found that high concentration only leads
to a gradual decrease of viable cells as shown in Fig. 7. As a result,
cytotoxicity tests definitely indicate that the low-micromolar
concentrations of dye 7a would not give rise to obvious toxic
effects on living cells over a period of 24 h, and dye 7a is indeed has
great potentials for further biological studies.
7b has a larger
s value of 1018 GM than the other five dyes because
of the introduction of the ethoxy group.
According to Beljonne’s exciton model [18], the extra enhance-
ment in 2PA response for octupolar dyes can be achieved when the
core allows significant electronic coupling between the individual
branches. With respect to our system, a nitrogen atom as the
linkage was adopted to combine 1, 3, 5-triazine core with substit-
uents to accomplish a large 2PA cross section (1018 GM) which
indicates that the lone pair electrons on the amino nitrogen atom of
dye 7b (also in 5b and 6b) may have delocalized onto the conju-
gated system of the molecule and this may increase intramolecular
charge-transfer. Moreover, these well-conjugated structures allow
sufficient electronic coupling between the branches, which has also
been experimentally confirmed by the above-mentioned linear and
non-linear photophysical data.
3.4. Cytotoxicity tests (MTT assay)
Considering their application in 2PF imaging, the MTT assay was
performed to ascertain the cytotoxic effect of dyes 6ae7a, 6be7b
against MCF-7 cells over a 24 h period. Cytotoxicity is a potential
Fig. 7. MTT assay of MCF-7 cells treated with dyes 6ae7a, 6be7b at different
concentrations for 24 h.

580
H. Zhou et al. / Dyes and Pigments 94 (2012) 570e582
Fig. 8. (A) Bright-field image of the MCF-7 cells stained with dye 7a (1
at 720e750 nm. (C) Merged image.
m
m) for 2 h at 37 ꢂC. (B) Two-photon fluorescence microscopy (2PFM) image of the same cells with excitation
Fig. 9. (A) 2PFM image of the MCF-7 cells stained with dye 7a (excitation at 720e750 nm). (B) Labeling of MCF-7 cells with SYTO-9Ò
lex ¼ 543 nm, lem ¼ 560e580 nm) for 10 min after stained with dye 7a. (D) Merged image.
(
lex ¼ 488 nm, lem ¼ 500e530 nm) and (C) PI
(
3.5. Two-photon microscopy bio-imaging application of dye 7a
live cells (Fig. 9D) than dead cells. These results demonstrate the
bio-imaging application of dye 7a by labeling MCF-7 cells and also
its low toxicity for living cells (also supported by MTT).
To evaluate the performance of dye 7a in living cells, a two-
photon fluorescence microscopy (2PFM) imaging, live/dead cos-
taning was performed using 2
m
M SYTO9Ò (live) and 10
m
M PI
4. Conclusions
(dead) and a cytotoxicity analysis were conducted. MCF-7 cells
(human breast cancer cell line) were the testing candidates and
were cultured and stained with dye 7a. A bright-field image
(Fig. 8A) of each cell was taken immediately prior to the 2PFM
imaging. The 2PFM image and the merged image show that after
2 h incubation with MCF-7 cells (Fig. 8B,C), the complex went
through the membrane and just localized uniformly in the cyto-
plasm. The intense fluorescence is mainly from dye 7a internalized
in the MCF-7 cell cytoplasm and the distribution in the nucleolus is
significantly lower, suggesting that the cell cytoplasm can only be
labeled by dye 7a.
We have reported one-step syntheses of a series of multi-
branched two-photon absorbing triazine dyes by changing the
ratio of RNH2 and 2, 4, 6-trichloro-1, 3, 5-triazine. The studies further
demonstrated that having a
way to transfer charge as well as a
cross section, and their 2PA cross section values (
s
-electron pair as bridge is an efficient
bridge to induce a large 2PA
) increase with
p
d
increasingelectron-donating strength of the end-groups and branch
number. Among the nine dyes, dye 7b was found to exhibit a large
2PA across section values (1018 GM) as measured by the two-photon
induced fluorescence methods, and low-micromolar concentrations
of dye 7a were tested showing no obvious toxic effect in imaging
MCF-7 cells. Moreover, we also demonstrated that dye 7a offers
a potential application in two-photon fluorescence imaging due to
its excellent biocompatibility and low toxicity. We believe that the
design method and fabrication strategy are potentially applicable to
the derivatization of analogous two-photon absorption dyes.
In order to observe the cell viability of the MCF-7 cells after
stained with dye 7a, costaining was performed using 2
lex ¼ 488 nm, lem ¼ 500e530 nm) and 10 M PI (lex ¼ 543 nm,
m
M SYTO-9Ò
(
m
lem ¼ 560e580 nm) for 10 min after staining with dye 7a in PBS
(Fig. 9A). When SYTO-9Ò dye and propidium iodide (PI) are used in
combination, intact cells are labeled green and cells with damaged
membranes are labeled red. CLSM (Confocal Laser Scanning
Microscope) images showed the appearance of both green (Fig. 9B)
and red (Fig. 9C) cells, representing live and dead cells, respectively.
Because SYTO-9Ò dye is able to penetrate all the cells and colour
them green, the red cells represented dead cells which are
permeable to PI as well, whose bound SYTO-9Ò stain was displaced
by PI due to its high affinity for DNA [32], leaving the cells red. Most
of the MCF-7 cells were mainly green, indicating a larger number of
Acknowledgments
This work was supported by Program for New Century Excellent
Talents in University (China), Doctoral Program Foundation of
Ministry of Education of China (20113401110004), Science and
Technological Fund of Anhui Province for Outstanding Youth
(10040606Y22), National Natural Science Foundation of China

H. Zhou et al. / Dyes and Pigments 94 (2012) 570e582
581
[12] (a) McDonagh AM, Humphrey MG, Samoc M, Luther-Davies B. Organometallic
complexes for nonlinear optics. 17.1 synthesis, third-order optical nonline-
arities, and two-photon absorption cross section of an alkynylruthenium
dendrimer. Organometallics 1999;18:5195e7;
(21071001, 51142011 and 21101001), Natural Science Foundation
of Education Committee of Anhui Province (KJ2012A024,
KJ2010A030), the 211 Project of Anhui University, the Team for
(b) Spangler CW. Recent development in the design of organic materials for
optical power limiting. J Mater Chem 1999;9:2013e20.
[13] Cho BR, Son KH, Lee SH, Song YS, Lee YK, Jeon SJ, et al. Two photon absorption
properties of 1,3,5-tricyano-2,4,6-tris(styryl)benzene derivatives. J Am Chem
Soc 2001;123:10039e45.
[14] Kim HM, Seo MS, Jeon SJ, Cho BR. Two-photon absorption properties of hexa-
substituted benzene derivatives. Comparison between dipolar and octupolar
molecules. Chem Commun 2009;47:7422e4.
Scientific
Innovation
Foundation
of
Anhui
Province
(2006KJ007TD), Ministry of Education Funded Projects Focus on
Returned Overseas Scholar, and Anhui University Student Innova-
tive Experiment Plan (XJ103575023, KYXL201100337). Thank
Lingxia Zheng of Department of Physcics and Materials Science of
City University of Hong Kong for her help in writing the paper.
[15] Porrès L, Mongin O, Katan C, Charlot M, Pons T, Mertz J, et al. Enhanced two-
photon absorption with novel octupolar propeller-shaped fluorophores
derived from triphenylamine. Org Lett 2004;6:47e50.
Appendix A. Supporting information
[16] Collings JC, Poon SY, Le DC, Charlot M, Katan C, Palsson LO, et al. The synthesis
and one- and two-photon optical properties of dipolar, quadrupolar and
octupolar donoreacceptor molecules containing dimesitylboryl groups. Chem
Eur J 2009;15:198e208.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.dyepig.2012.03.017.
[17] Xu F, Wang ZW, Gong QH. Synthesis, characterization, and optical properties
of two-photon-absorbing octupolar molecule with an s-triazine core. Opt
Mater 2007;29:723e7.
References
[18] Beljonne D, Wenseleers W, Zojer E, Shuai ZG, Vogel H, Pond SJK, et al. Role of
dimensionality on the two-photon absorption response of conjugated
molecules: the case of octupolar compounds. Adv Funct Mater 2002;12:
631e41.
[19] Jiang YH, Wang YC, Hua JL, Tang J, Li B, Qian SX, et al. Multibranched triar-
ylamine end-capped triazines with aggregation-induced emission and large
two-photon absorption cross-sections. Chem Commun 2010;46:4689e91.
[20] Jiang YH, Wang YC, Wang B, Yang JB, He NN, Qian ShX, et al. Synthesis, two-
photon absorption and optical limiting properties of multi-branched styryl
derivatives based on 1,3,5-triazine. Chem Asian J 2011;6:157e65.
[21] (a) Murase T, Fujita M. Highly blue luminescent triazineeamine conjugated
oligomers. J Org Chem 2005;70:9269e78;
[1] Pawlicki M, Collins HA, Denning RG, Anderson HL. Two-photon absorption
and the design of two-photon dyes. Angew Chem Int Ed 2009;48:3244e66.
[2] Terenziani F, Katan C, Badaeva E, Tretiak S, Blanchard-Desce M. Enhanced
two-photon absorption of organic chromophores: theoretical and experi-
mental assessments. Adv Mater 2008;20:4641e78.
[3] (a) Yao Sh, Ahn HY, Wang XH, Fu J, Stryland EWV, Hagan David, et al.
Donoreacceptoredonor fluorene derivatives for two-photon fluorescence
lysosomal imaging. J Org Chem 2010;75:3965e74;
(b) Andrade CD, Yanez CO, Rodriguez L, Belfield KD. A series of fluorene-based
two-photon absorbing molecules: synthesis, linear and nonlinear character-
ization, and bioimaging. J Org Chem 2010;75:3975e82;
(c) Dvornikov AS, Walker EP, Rentzepis PM. Two-photon three-dimensional
optical storage memory. J Phys Chem A 2009;113:13633e44;
(d) Tian H, Feng YL. Next step of photochromic switches? J Mater Chem 2008;
18:1617e22;
(e) Barsu C, Cheaib R, Chambert S, Queneau Y, Maury O, Cottet D, et al. Neutral
pushepull chromophores for nonlinear optical imaging of cell membranes.
Org Biomol Chem 2010;8:142e50;
(f) Lin TC, Haung YJ, Chen YF, Hu CL. Two-photon absorption and effective
broadband optical power limiting properties of a multi-branched chromo-
phore containing 2,3-diarylquinoxalinyl moieties as the electron-pulling
units. Tetrahedron 2010;66:1375e82;
ꢀ
ꢀ
ꢀ
(b) El-Sedik M, Almonasy N, Nepras M, Bures F, Dvorák M, Michl M. Synthesis,
absorption and fluorescence properties of N-triazinyl derivatives of 2-ami-
noanthracene. Dyes Pigm 2012;92:1126e31;
ꢀ
ꢀ
(c) Nepras M, Almonasy N, Michl M, Dvorák M, Fidler V. Electronic structure,
spectra and photophysical properties of N-triazinylderivatives of 1-amino-
pyrene. Semi-empirical theoretical study. Dyes Pigm 2012;92:1331e6.
[22] Yan YX, Fan HH, Guo YH, Lam CK, Huang H, Sun YH, et al. Synthesis and two-
photon absorption property of new
p-conjugated dendritic fluorophores
containing styrylpyridyl moieties. Mater Chem Phys 2007;101:329e35.
[23] (a) Fink R, Frenz C, Thelakkat M, Schmidt HW. Synthesis and characterization
of aromatic poly(1,3,5-triazineeether)s for electroluminescent devices.
Macromolecules 1997;30:8177e81;
(g) LaFratta CN, Fourkas JT, Baldacchini T, Farrer RA. Multiphoton fabrication.
Angew Chem Int Ed 2007;46:6238e58;
(h) Cumpston BH, Ananthavel SP, Barlow S, Dyer DL, Ehrlich JE, Erskine LL,
et al. Two-photon polymerization initiators for three-dimensional optical data
storage and microfabrication. Nature 1999;398:51e4.
(b) Inomata H, Goushi K, Masuko T, Konno T, Imai T, Sasabe H, et al. High-
efficiency organic electrophosphorescent diodes using 1,3,5-triazine electron
transport materials. Chem Mater 2004;16:1285e91;
(c) Zhong H, Xu E, Zeng D, Du J, Sun J, Ren S, et al. New optoelectronic
materials based on bitriazines: synthesis and properties. Org Lett 2008;10:
709e12;
[4] Zipfel WR, Williams RM, Webb WW. Nonlinear magic: multiphoton micros-
copy in the biosciences. Nat Biotechnol 2003;21:1369e77.
(d) Ren S, Fang Q, Yu F, Bu D. Synthesis and optical and electrochemical
[5] Denk W, Strickler JH, Webb WW. Two-photon laser scanning fluorescence
microscopy. Science 1990;248:73e6.
properties of new
p-conjugated 1,3,5-triazine-containing polymers. J Polym
Sci A Polym Chem 2005;43:6554e61.
ꢀ
[6] (a) Hrobáriková V, Hrobárik P, Gajdos P, Fitilis I, Fakis M, Persephonis P, et al.
[24] (a) Kannan R, He GS, Lin TC, Prasad PN, Vaia RA, Tan LS. Toward highly
active two-photon absorbing liquids. Synthesis and characterization of
1,3,5-Triazine-Based octupolar molecules. Chem Mater 2004;16:185e94;
(b) Meng F, Li B, Qian S, Chen K, Tian H. Enhanced two-photon properties
of tri-branched styryl derivatives based on 1,3,5-triazine. Chem Lett 2004;
33:470;
Benzothiazole-based fluorophores of donor-
p-acceptor-p-donor type dis-
playing high two-photon absorption. J Org Chem 2010;75:3053e68;
(b) Kim HM, Cho BR. Two-photon materials with large two-photon cross
sections. Structureeproperty relationship. Chem Commun; 2009:153e64;
(c) Kamada K, Iwase Y, Sakai K, Kondo K, Ohta KJ. Cationic two-photon
absorption chromophores with double- and triple-bond cores in symmetric/
asymmetric arrangements. Phys Chem C 2009;113:11469e74.
(c) Li B, Tong R, Zhu R, Meng F, Tian H, Qian S. The ultrafast dynamics and
nonlinear optical properties of tribranched styryl derivatives based on
1,3,5-triazine. J Phys Chem B 2005;109:10705e10;
(d) Cui YZ, Fang Q, Xue G, Xu GB, Yin L, Yu WT. Cooperative enhancement
of two-photon absorption of multibranched compounds with vinylenes
attaching to the s-triazine core. Chem Lett 2005;34:644.
[7] Fitilis I, Fakis M, Polyzos I, Giannetas V, Persephonis P, Vellis P, et al. A two-
photon absorption study of fluorene and carbazole derivatives. The role of the
central core and the solvent polarity. Chem Phys Lett 2007;447:300e4.
[8] (a) Chakrabarti S, Ruud K. Large two-photon absorption cross section:
molecular tweezer as a new promising class of compounds for nonlinear
optics. Phys Chem Chem Phys 2009;11:2592e6;
[25] (a) Ning ZJ, Tian H. Triarylamine: a promising core unit for efficient photo-
voltaic materials. Chem Commun; 2009:5483e95;
(b) Zein S, Delbecq F, Simon D.
A TD-DFT investigation of two-photon
(b) Chen CY, Pootrakulchote N, Wu SJ, Wang M, Li JY, Tsai JH, et al. New
ruthenium sensitizer with carbazole antennas for efficient and stable thin-
film dye-sensitized solar cells. J Phys Chem C 2009;113:20752e7;
(c) Zhang XH, Wang ZS, Cui Y, Koumura N, Furube A, Hara K. Organic sensi-
tizers based on hexylthiophene-functionalized indolo[3,2-b]carbazole for
efficient dye-sensitized solar cells. J Phys Chem C 2009;113:13409e15;
(d) Hrobárik P, Sigmundová I, Zahradník P, Kasák P, Arion V, Franz E, et al.
Molecular engineering of benzothiazolium salts with large quadratic hyper-
polarizabilities: can auxiliary electron-withdrawing groups enhance
nonlinear optical responses? J Phys Chem C 2010;114:22289e302;
(e) Moylan CR, Twieg RJ, Lee VY, Swanson SA, Betterton KM, Miller RD.
Nonlinear optical chromophores with large hyperpolarizabilities and
enhanced thermal stabilities. J Am Chem Soc 1993;115:12599e600;
(f) Whitacker CM, Patterson EV, Kott KL, McMahon RJ. Nitrogen and oxygen
donors in nonlinear optical materials: effects of alkyl vs phenyl substitution
on the molecular hyperpolarizability. J Am Chem Soc 1996;118:9966e73.
absorption of fluorene derivatives. Phys Chem Chem Phys 2009;11:694e702;
(c) Nguyen KA, Day PN, Pachter R. Effects of conjugation in length and
dimension on two-photon properties of fluorene-based chromophores. Theor
Chem Acc 2008;120:167e75;
(d) Wang CK, Macak P, Luo Y, Ågren H. Effects of
p centers and symmetry on
two-photon absorption cross sections of organic chromophores. J Chem Phys
2001;114:9813.
[9] Rudberg E, Salek P, Helgaker T, Agren H. Calculations of two-photon charge-
transfer excitations using Coulomb-attenuated density-functional theory. J
Chem Phys 2005;123:184108.
[10] Reinhardt BA, Brott LL, Clarson SJ, Dillard AG, Bhatt JC, Kannan R, et al. Highly
active two-photon dyes: design, synthesis, and characterization toward
application. Chem Mater 1998;10:1863e74.
[11] Albota M, Beljonne D, Bredas JL, Ehrlich JE, Fu JY, Heikal AA, et al. Design of
organic molecules with large two-photon absorption crosssections. Science
1998;281:1653e6.

582
H. Zhou et al. / Dyes and Pigments 94 (2012) 570e582
[26] Demas JN, Crosby GA. Measurement of photoluminescence quantum yields.
Review J Phys Chem 1971;75:991e1024.
[27] Gray TG, Rudzinski CM, Meyer EE, Holm RH, Nocera DG. Spectroscopic and
photophysical properties of hexanuclear rhenium(III) chalcogenide clusters. J
Am Chem Soc 2003;125:4755e70.
[28] Lee SK, Yang WJ, Choi JJ, Kim CH, Jeon SJ, Cho BR. 2,6-Bis[4-(p-dihex-
ylaminostyryl)styryl]anthracene derivatives with large two-photon cross
sections. Org Lett 2005;7:323e6.
[30] Whittall IR, Humphrey MG. Organometallic complexes for nonlinear optics.
3.1 molecular quadratic hyperpolarizabilities of ene-, imine-, and azo-linked
ruthenium
CHC6H4NO2)(PPh3)2(
s
-acetylides: X-ray Crystal structure of Ru((E)-4,40-C^CC6H4CH]
h
-C5H5). Organometallics 1996;15:1935e41.
[31] Wang XM, Zhou YF, Yu WT, Wang Ch, Fang Q, Jiang MH, et al. Two-photon
pumped lasing stilbene-type chromophores containing various terminal
donor groups: relationship between lasing efficiency and intramolecular
charge transfer. J Mater Chem 2000;10:2698e703.
[29] Xu C, Webb WW. Measurement of two-photon excitation cross sections of
molecular fluorophores with data from 690 to 1050 nm. J Opt Soc Am B 1996;
13:481e91.
[32] Bunthof CJ, Bloemen K, Breeuwer P, Rombouts FM, Abee T. Flow cytometric
assessment of viability of lactic acid bacteria. Appl Environ Microbiol 2001;67:
2326e35.