Aza-crown ether derivatives based on stilbene: Two-photon absorption and bioimaging

Article abstract of DOI:10.1016/j.dyepig.2014.03.034

Two novel aza-crown ether derivatives based on stilbene were synthesized and characterized. Their linear photophysical properties were systematically investigated in various solvents. The nonlinear photophysical properties were investigated by two-photon induced fluorescence method. The two-photon absorption cross sections values measured by two-photon excited fluorescence were determined to be 328.2 GM and 246.55 GM for the dyes derived from monoaza 15-crown-5 and 18-crown-6, respectively, which accorded with dipole moment changes under photoexcitation. In addition, a single-photon fluorescence cell imaging experiment proved that dye derived from monoaza 15-crown-5 was suitable for biomedical imaging.

Full text of DOI:10.1016/j.dyepig.2014.03.034

Dyes and Pigments 107 (2014) 133e139
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Aza-crown ether derivatives based on stilbene: Two-photon
absorption and bioimaging
,
b
,
c
a
a
a
Wei Li a, Mingdi Yang a , Linpan Wang , Weiju Zhu , Lina Ye , Jieying Wu ,
*
Yupeng Tian a, Hongping Zhou a
,
*
a College of Chemistry and Chemical Engineering, Anhui University and Key Laboratory of Functional Inorganic Materials Chemistry of Anhui Province,
230601 Hefei, PR China
b School of Materials and Chemical Engineering, Anhui Jianzhu University, Hefei 230601, PR China
c Department of Civil Engineering, Anhui Vocational and Technical College, 230051 Hefei, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two novel aza-crown ether derivatives based on stilbene were synthesized and characterized. Their
linear photophysical properties were systematically investigated in various solvents. The nonlinear
photophysical properties were investigated by two-photon induced fluorescence method. The two-
photon absorption cross sections values measured by two-photon excited fluorescence were deter-
mined to be 328.2 GM and 246.55 GM for the dyes derived from monoaza 15-crown-5 and 18-crown-6,
respectively, which accorded with dipole moment changes under photoexcitation. In addition, a single-
photon fluorescence cell imaging experiment proved that dye derived from monoaza 15-crown-5 was
suitable for biomedical imaging.
Received 30 January 2014
Received in revised form
23 March 2014
Accepted 25 March 2014
Available online 3 April 2014
Keywords:
Aza-crown ether
Stilbene
SPEF
Ó 2014 Published by Elsevier Ltd.
d
Bioimaging
TPFM
1. Introduction
macrocycles, dendrimers, polymers, and multi-branched mole-
cules. Reinhardt synthesized a variety of donore
eA) and donore edonor (De eD) derivatives, in which fluo-
rene, biphenyl, or naphthyl groups are employed as the mobile
electron bridge, and showed that the TPA cross sections become
larger if a planar fluorene was used as the -center [4]. The TPA
peacceptor (De
The development of materials displaying two-photon absorp-
tion (2PA) [1] has attracted great interest in past decades due to a
variety of potential applications in photonics and optoelectronics,
such as three-dimensional optical data storage, fluorescence im-
aging, optical limiting, up-converted lasing and microfabrication
[2].
p
p
p
p
-
p
properties of fluorene derivatives were subsequently optimized by
Belfield by introducing a variety of donors and acceptors [5]. The
The applications of 2PA materials stimulated substantial
research on structure-property relationship [3]. The significant
issue is how to synthesize the materials with large 2PA cross sec-
tions. Some efficient molecular design strategies were put forward
to provide guidelines for the development of materials with large
important role of the
demonstrated by Prasad, who showed that the TPA cross sections of
the De eD chromophores based on dithienothiophene as the p-
center were larger by an order of magnitude than those of the
fluorene derivatives [6]. By combining synthesis, characterization,
and theory, Marder, Perry, and Webb found that bis(styryl)-
benzene derivatives with donoreacceptoredonor (DeAeD) and
acceptoredonoreacceptor (AeDeA) structural motifs, which were
linear quadrupolar molecules, showed exceptionally large TPA
cross sections [7].
p-center for the design of large TPA dyes was
p
two-photon absorption cross sections
acceptoredonor (DeAeD)-type molecules, donore
eA)-type molecules, donore edonor (De eD)-type molecules,
(s
), including donore
peacceptor (De
p
p
p
* Corresponding authors. College of Chemistry and Chemical Engineering, Anhui
University and Key Laboratory of Functional Inorganic Materials Chemistry of Anhui
Province, 230601 Hefei, PR China. Tel.: þ86 551 63861279; fax: þ86 551 63861259.
E-mail addresses: zhpzhp@263.net, zhouhongping@ahu.edu.cn (H. Zhou).
Most organic dyes with large conjugated structures have a major
defect that solubility is poor for bioimaging, as a result, it is
necessary to enhance their biocompatibility by optimizing the
http://dx.doi.org/10.1016/j.dyepig.2014.03.034
0143-7208/Ó 2014 Published by Elsevier Ltd.

134
W. Li et al. / Dyes and Pigments 107 (2014) 133e139
Table 1
structure [8]. In the design of the target molecules, the aza-crown
ether often used as phase transfer catalyst was introduced. The
aza-crown ether serves as the electron donor, meanwhile, it was
also expected to endow the molecules hydrophilia. And that crown
ether compounds having good solubility and environmental sta-
bility are also multidentate ligands that exhibit selectivity for
specific metal ions in solutions containing other chemically similar
ions, which can be applied to identify ions. In this paper, we
designed and synthesized two novel aza-crown ether derivatives-
Photophysical properties of dye 1 and dye 2 in four different polar solvents.
Fc
Dnd
a
b
Ligand
Dye 1
Solvents
lmax
lmax
Benzene
DCM
THF
DMF
Benzene
DCM
395
395
395
398
391
392
393
393
455,482
494
485
511
458, 479
489
0.373
0.361
0.367
0.333
0.372
0.368
0.370
0.330
3338.43
5073.54
4697.90
5306.15
3741.39
5018.40
4698.40
5875.82
Dye 2
THF
DMF
482
511
based TPA dyes with dumbbell-shape (DepeD), aza-crown ether
as electron donor and stilbene as the conjugated chain. Compared
with simple dimethyl amino substituents, the two dyes showed the
similar two-photon absorption property and better solubility.
Furthermore, a single-photon fluorescence cell imaging experiment
proved the suitability of dye 1 for biomedical imaging.
a
Peak position of the longest absorption band.
Peak position of SPEF, excited at the absorption maximum.
b
c
Quantum yields determined by using quinine sulfate as standard.
d
Stokes’ shift in cmꢀ1
.
ray lamp, in the process, 4, 4-Bis(diethylphosphonomethyl)
biphenyl(1.36 g, 3.0 mmol) was added in batches. After completion
of the reaction (monitored by Thin Layer chromatography (TLC)),
the mixture was dissolved in Dichloromethane (150 mL). The re-
sidual solid was filtered and filtrate was concentrated. Yellow-
green needle product (1.24 g, 1.565 mmol) was obtained by
recrystallized from dichloromethane. Yield: 52.3%. mp 233 ꢁC; 1H
2. Experimental section
2.1. Apparatus and materials
Chemicals were purchased and used as received. Every solvent
was purified by conventional methods beforehand. IR spectra were
recorded with a Nicolet FT-IR NEXUS 870 spectrometer (KBr discs)
in the 4000e400 cmꢀ1 region. 1H NMR and 13C NMR were recorded
on 400 MHz and 100 MHz NMR instruments using CDCl3 as solvent
respectively. The mass spectra were obtained on a Bruker Autoflex
III smartbeam mass spectrometer and a Finnigan LCQ Spectrometer.
Elemental analyses data were measured by a Perkin Elmer 240B
elemental analyzer. The one-photon absorption (OPA) spectra were
recorded on a SPECORD S600 spectrophotometer. The one-photon
excited fluorescence (OPEF) spectra measurements were per-
formed using a Hitachi F-7000 fluorescence spectrophotometer. For
time-resolved fluorescence measurements, the fluorescence sig-
nals were collimated and focused onto the entrance slit of a
monochromator with the output plane equipped with a photo-
multiplier tube (HORIBA HuoroMax-4P). Two-photon absorption
NMR (400 MHz, CDCl3, TMS) (Fig. S1):
d
7.59 (J ¼ 8, d, 4H), 7.53(J ¼ 8,
d, 4H), 7.40(J ¼ 8, d, 4H), 7.07(J ¼ 16, d, 2H), 6.92(J ¼ 16, d, 2H),
6.66(J ¼ 8, d, 4H), 3.78e3.76(m, 8H), 3.67e3.64(m, 32H). 13C NMR
(100 MHz, CDCl3, TMS) (Fig. S2):
d 146.25, 137.87, 136.16, 127.68,
126.80, 125.87, 125.37, 124.24, 122.60, 110.54, 70.32, 69.21, 69.15,
67.54, 51.55. IR (KBr): 3054 (m),1610 (m),1486 (s), 1208 (s), 1142 (s).
MS (ESI): 397.22 [(Mþ2)/2]þ. Anal. Calcd for C48H60N2O8: C, 72.70;
H, 7.63; N, 3.53%. Found: C, 72.35; H, 7.28; N, 3.26%.
2.2.2. Synthesis of dye 2 (Scheme 1)
Dye 2 was obtained as orange powders in 40.6% yield by
following a similar procedure of Dye 1. mp 207 ꢁC;1H NMR
(400 MHz, CDCl3, TMS) (Fig. S3):
d
7.59(J ¼ 8, d, 4H), 7.53(J ¼ 8, d,
4H), 7.39(J ¼ 8, d, 4H), 7.06(J ¼ 16, d, 2H), 6.92(J ¼ 16, d, 2H),
(TPA) cross sections (d) of the samples were obtained by two-
6.68(J ¼ 8, d, 4H), 3.72-3.70(m, 8H), 3.67e3.64(m, 40H). 13C NMR
photon excited fluorescence (TPEF) method [9] at femtosecond
laser pulse and Ti: sapphire system (680e1080 nm, 80 MHz, 140 fs)
as the light source.
(100 MHz, CDCl3, TMS)(Fig. S4):
d 147.58, 138.90, 137.17, 130.21,
127.84, 127.10, 126.90, 126.40, 123.62, 111.71, 70.81, 70.74, 68.72,
62.26, 62.19, 51.24. IR (KBr): 3038 (m), 1610 (m), 1491 (s), 1197 (s),
1122 (s). MS(ESI): 441.25 [(Mþ2)/2]þ. Anal. Calcd for C52H68N2O10
:
2.2. Synthesis
C, 70.88; H, 7.78; N, 3.18%. Found: C, 71.12; H, 7.53; N, 3.31%.
2.2.1. Synthesis of dye 1 (Scheme 1)
t-BuOK (1.2 g, 10 mmol) and 4-(1, 4, 7, 10-tetraoxa-13-aza
cyclopentadecyl) benzaldehyde (1 g, 3 mmol) were placed into a
dry mortar and milled vigorously for about 90 min under infrared
2.2.2.1. Linear absorption and single-photon excited fluorescence
(SPEF). The photophysical properties of dyes 1 and 2 are summa-
rized in Table 1. The linear absorption spectra (one-photon
Scheme 1. Preparation of the dyes 1 and 2.

W. Li et al. / Dyes and Pigments 107 (2014) 133e139
135
Fig. 1. Linear absorption and fluorescence of dye 1 (left) and dye 2 (right) in four organic solvents of different polarities.
absorption (OPA)) were measured in solvents of different polarities
at a concentration of 1 ꢂ 10ꢀ5 mol Lꢀ1, in which influence of the
solvent has been excluded. The one-photon-excited fluorescence
(OPEF) spectra were measured with the same concentrations as
those of the linear absorption spectra.
As shown in Fig.1, we can see that strong absorption peaks fall in
390 nm nearby region, and weak absorption peaks fall in 300 nm
nearby region. According to the density functional theory (DFT)
The OPA spectra of dyes 1 and 2 exhibit almost no red-shift with
the increasing of solvent polarity, which show negligible sol-
vatochromic behavior. Upon increasing polarity of the solvent, as
shown in Fig.1 and Table 1, the fluorescent emission peaks of dyes 1
and 2 show remarkable bathochromic shifts and the Stokes shifts
also show a tendency to monotonically increase. This can be
explained by the fact that the excited state may possess a higher
polarity than the ground state. An increased dipoleedipole inter-
action between the solute and solvent leads to a lowering of the
energy level of the excited state [12,13]. Normalized UV absorption
spectra in ethanol solvent are special (Fig. S5), there is a marked
hypochromatic shift. It may be that the ethanol is protic solvent
calculations (Fig. 2), these peaks can be belong to pep* transition of
aromatic ring system [10]. DFT calculations were carried out using
Gaussian 03 program. The nonlocal density function of B3LYP with
6-31G basis sets was used for the calculations [11].
Fig. 2. Energy level and electron density distribution of frontier molecular orbitals of dye 1 (Left) and dye 2 (Right).

136
W. Li et al. / Dyes and Pigments 107 (2014) 133e139
Fig. 3. LipperteMataga plots for dyes 1-2.
that interacts with the crown ether ring, which reduces the degree
of intramolecular electron delocalization [14].
corresponding with their linear and nonlinear optical properties
[17].
As described above, the fluorescence spectra show large Stokes
shifts which depend upon the solvent polarity. Further significant
solvatochromism is observed, suggesting a large change in dipole
moment between the ground and emissive excited states. The
LipperteMataga equation is the most widely used equation to
evaluate the dipole moment changes of the dyes with photoexci-
tation [15,16] and LipperteMataga plots for dyes 1 and 2 are given
in Fig. 3. The LipperteMataga equation is as follow:
Photographs of solutions containing dye 1 are displayed in Fig. 4,
from which one can clearly see dye 1 presents different colors in
different solvents under the irradiation of ultraviolet light with
365 nm. Chromaticity coordinates [18] figure shows the distribu-
tion of the different color areas in parallel. The great sensitivity to
every solvents exhibits potential and useful application for the
development of efficient sensors for the detection of volatile
organic compounds (VOCs) [19].
The fluorescence quantum yields (F) were determined by using
quinine sulfate as the reference according to the literature method
[20]. Quantum yields is corrected as follows.
ꢀ
ꢁ
2 f
D
3
Dn
¼
me
ꢀ
mg 2 þ b
4
p
0Zca3
3
ꢀ 1
n2 ꢀ 1
D
f ¼
ꢀ
A
A
rh2s Ds
sh2r Dr
2n2 þ 1
2
3 þ 1
Fs
¼
Fr
in which Dn
¼
nabs
ꢀ
nem stands for Stokes shift, nabs and nem are
absorption and emission frequency (cmꢀ1), Z is the Planc’s con-
stant, c is the velocity of light in vacuum, a is the Onsager radius and
where the s and r indices designate the sample and reference
samples, respectively, A is the absorbance at lexc is the average
,
h
b is a constant.
index, n is the dielectric constant, me and mg are the dipole moments
of the emissive and ground states, respectively and is the
D
f is the orientation polarizability,
3
is the refractive
refractive index of the appropriate solution, and D is the integrated
area under the corrected emission spectrum [21].
3
0
Time-resolved fluorescence measurements were performed in
Fig. 5 and the detailed data of the fluorescence decay curves of dyes
1 and 2 are listed in Table 2. The decays were analyzed by the ‘least-
squares’ method. The quality of the exponential fits was evaluated
permittivity of the vacuum. (me
the LipperteMataga plot.
ꢀ
mg)
2 is proportional to the slope of
The large slopes of dyes 1 and 2 shown in the LipperteMataga
plot infer large dipole moment changes for dyes 1 and 2 with
photoexcitation [16]. The slope of the best-fit line is related to the
dipole moment change between the ground and excited states
by the goodness of fit (
to be ꢃ15% from sample concentrations and instruments. The mean
excited-state lifetime ( ) of dyes 1 and 2 became longer with the
c
2). The experimental errors were estimated
s
(
me
ꢀ
mg). The values of me
ꢀ
mg were calculated as 18.72 D for dye 1
increasing of the polarity of the solvents in general trend except in
ethanol (Fig. S6 and Table S1), which could be explained by the
hydrogen bonds between solute and ethanol molecules consume
and 16.76 D for dye 2. The large values of dyes 1 and 2 indicate that
the molecule in the excited state has an extremely polar structure,
Fig. 4. The photographs of dye 1 in four solvents (benzene, DCM, THF and DMF) under the irradiation of 365 nm wavelength and chromaticity coordinates figures of dye 1.

W. Li et al. / Dyes and Pigments 107 (2014) 133e139
137
Table 2
Fluorescence lifetimes of dyes 1 and 2 in different solvents.
c2
a
b
c
b
c
b
c
d
Sample
ldet
s1
A1
s 2
(ns)
A2
s 3
(ns)
A3
<
s
>
(nm) (ns)
(ns)
Dye1-benzene 395
0.75 0.94 1.56 0.06
0.92 0.37 1.24 0.63
0.98 0.26 1.64 0.55 0.29 0.19 1.2119 1.07
0.63 0.15 1.50 0.85
0.79 0.96 1.78 0.04
0.70 0.32 1.30 0.68
0.55 0.33 1.61 0.67
0.68 0.29 1.61 0.71
0.7986 1.41
1.1216 0.868
Dye1-DCM
Dye1-THF
Dye1-DMF
395
395
398
1.3695 1.17
0.8296 1.54
Dye2-benzene 391
Dye2-DCM
Dye2-THF
Dye2-DMF
392
393
393
1.108
1.25
1.2536 1.09
1.3403 1.03
a
Detection wavelength.
Fluorescence lifetime.
Fractional contribution.
Weighted mean lifetime.
b
c
d
Fig. 8. Two-photon absorption cross sections of dyes 1 and 2 (chloroform solution
10ꢀ3 M) in 680e920 nm regions.
Fig. 5. The normalized fluorescence lifetimes at lex ¼ 395 nm for dye 1 (Left) and dye 2 (Right) in four solvents.
Fig. 6. The TPEF spectra of dye 1 (Left) and dye 2 (Right) in CHCl3 pumped by femtosecond laser pulses at 300 mw under different excitation wavelengths (1 ꢂ 10ꢀ3 mol Lꢀ1).
Fig. 7. Output fluorescence intensity (Iout) vs. the square of input laser power (Iin) for dye 1 (Left) and dye 2 (Right), excitation carried out at 740 nm, with c ¼ 1 ꢂ 10ꢀ3 mol/L in
CHCl3.

138
W. Li et al. / Dyes and Pigments 107 (2014) 133e139
Fig. 9. (A) single-photon fluorescence microscopy (SPFM) image of HepG2 cells with excitation at 511 nm. (B) Bright-field image of HepG2 cells stained with dye1. (C) Merged image.
some energy from the excited state molecules [22]. The lifetimes (
of dyes 1 and 2 decay as a tripple exponential in ethanol, while in
other four solvents the mean lifetime ( ) are mainly the result of
s
)
is mainly a result of dye 1 internalized in the HepG2 cell cytoplasm
and the distribution in the nucleolus is significantly lower, sug-
gesting that only the cell cytoplasm can be labeled by dye 1. These
results demonstrate the potential bio-imaging applications of dye 1
by labeling HepG2 cells. However, two-photon fluorescence mi-
croscopy (TPFM) imaging was not performed successfully in DMSO,
the TPEF spectra of dyes 1 and 2 were tested in CHCl3 rather than
DMSO because of the poor solubility of the dyes in DMSO.
s
combined action of single-exponential and double-exponential.
In the wavelength range 450e900 nm, there is no linear ab-
sorption for the dyes, which indicates that there are no molecular
energy levels corresponding to an electron transition in the spectral
range. In test process, laser induced frequency up-converted fluo-
rescence in this spectral range appeared, which can be ascribed to
two-photon-excited fluorescence (TPEF). Upon excitation from 450
to 920 nm, it is impossible to produce single-photon-excited up-
converted fluorescence. From Fig. 6, we can conclude that the op-
timum excitation wavelength of dyes 1 and 2 are not associated
with the laser energy, but the two-photon fluorescence intensity is
associated with excitation wavelength, the optimal excitation
wavelength of two dyes is 740 nm. Dyes 1 and 2 in CHCl3 were
pumped by femtosecond laser pulses at different pump intensities
with 740 nm excitation wavelength. Fig. 7 shows a logelog plot of
the excited fluorescence signal vs. excited light power [23]. The
slope value is 1.849 for dye 1 and 2.141 for dye 2, respectively. It
provides direct evidence for the squared dependence of excited
fluorescence intensity and input laser power, suggesting a two-
photon excitation mechanism.
3. Conclusions
In this contribution, two novel aza-crown ether derivatives
based on stilbene compounds with moderate two-photon absorp-
tion cross sections were synthesized. Photophysical properties and
the two-photon fluorescence microscopy (TPFM) were systemati-
cally investigated. The two dyes exhibited positive solvato-
fluorochromic properties in various solvents mainly due to the
pe
p* transition of aromatic ring system, which are in line with DFT
calculations. The solvatochromic behavior of dye 1 offers potential
application in the detection of volatile organic compounds (VOCs).
Moreover, we also demonstrated that dye 1 had the potential for
application in single-photon fluorescence imaging.
TPA cross sections (
d
) of samples is determined by Eq.:
Acknowledgments
Fref cref nref
F
This work was supported by the Program for New Century
Excellent Talents in University (China), the Doctoral Program
Foundation of the Ministry of Education of the People’s Republic of
China (20113401110004), the National Natural Science Foundation
of China (21271003, 21271004 and 51372003), the Ministry of Ed-
ucation of the People’s Republic of China, the Natural Science
Foundation of Education Committee of Anhui Province
(KJ2012A024), the Natural Science Foundation of Anhui Province
(1208085MB22), the 211 Project of Anhui University, the Ministry
of Education Funded Projects Focus on Returned Overseas Scholar.
d
¼
dref
F
c
n Fref
where the ‘ref’ subscript represents the reference molecule (here
fluorescein in aqueous solution of sodium hydroxide at a concen-
tration of 1.0 ꢂ 10ꢀ3 mol Lꢀ1 was used as the reference).
d 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 of
the solution emitted at the exciting wavelength, and
F is the
fluorescence quantum yield. The dref value of reference was taken
from the literature [24]. Two-photon absorption cross sections of
dyes 1 and 2 in 680e920 nm region are displayed in Fig. 8. The two-
photon absorption cross sections of dyes 1 and 2 are 328.2 GM and
246.55 GM in 740 nm, respectively, which are in good agreement
with the calculated values of dipole moment.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2014.03.034.
2.2.2.2. Application of dye 1 for single-photon microscopy bio-
imaging. To evaluate the performance of dye 1 in living cells, sin-
gle- and two-photon fluorescence microscopy (SPFM and TPFM)
imaging is performed in Fig. 9. HepG2 cells were the testing can-
didates and were cultured and stained with dye 1. A bright-field
image of each cell was taken immediately prior to the SPFM im-
aging. The SPFM image and the merged image show that after 2 h
incubation with HepG2 cells, dye 1 went through the membrane
and localized uniformly in the cytoplasm. The intense fluorescence
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