Water-soluble two-photon absorption benzoxazole-based pyridinium salts with the planar cationic parts: crystal structures and bio-imaging

Article abstract of DOI:10.1016/j.dyepig.2017.08.033

Six new benzoxazole-based compounds 1–6 of the donor-bridge-accepter (D-π-A) type with different anions were obtained by methylation and anion exchange reaction. The single crystals of 1, 3 and 6 were obtained by slow evaporation of acetonitrile and determined by X-ray crystallography. Ellipsoidal structures of the three compounds showed that the cationic parts all maintained good planarity, which was beneficial for two-photon absorption (2PA) properties and accorded with the results of two-photon excited fluorescence tests. Biological imaging experiments showed water-soluble six compounds had excellent penetrability in living cells and could located in cytoplasm in one- and two-photon cell imaging. Biological imaging result of compound 6 was more excellent than other compounds, which indicated synergy of anions and cations played a very important role in cell imaging.

Full text of DOI:10.1016/j.dyepig.2017.08.033

Accepted Manuscript
Water-soluble two-photon absorption benzoxazole-based pyridinium salts with the
planar cationic parts: Crystal structures and bio-imaging
Dan Liu, Haiyan Wang, Hong Li, Huihui Zhang, Qingsong Liu, Zepeng Wang,
Xiaoping Gan, Jieying Wu, Yupeng Tian, Hongping Zhou
PII:
S0143-7208(17)31531-0
10.1016/j.dyepig.2017.08.033
DYPI 6195
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 13 July 2017
Revised Date: 14 August 2017
Accepted Date: 18 August 2017
Please cite this article as: Liu D, Wang H, Li H, Zhang H, Liu Q, Wang Z, Gan X, Wu J, Tian Y, Zhou H,
Water-soluble two-photon absorption benzoxazole-based pyridinium salts with the planar cationic parts:
Crystal structures and bio-imaging, Dyes and Pigments (2017), doi: 10.1016/j.dyepig.2017.08.033.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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ACCEPTED MANUSCRIPT
Excellent solubility in water media of six new benzoxazole-based compounds 1-6 with different
anions could apply in biological imaging. Ellipsoidal structures of 1, 3 and 6 showed that the
cationic parts all maintained good planarity, which was beneficial for two-photon absorption (2PA)
properties. The two-photon cell imaging results showed that 1-6 had excellent penetrability in
living cells and could located in cytoplasm.

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Water-soluble two-photon absorption benzoxazole-based pyridinium salts
with the planar cationic parts: crystal structures and bio-imaging
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Dan Liu ‡ a, Haiyan Wang ‡ a, Hong Li a, Huihui Zhang a, Qingsong Liu a, Zepeng Wang a, Xiaoping
Ganbc, *, Jieying Wu a, Yupeng Tian a, Hongping Zhou a, *
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a
College of Chemistry and Chemical Engineering, Anhui University and Key Labotatory of
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Functional Inorganic Materials Chemistry of Anhui Province, Anhui Province Key Laboratory of
Chemistry for Inorganic/Organic Hybrid Functionalized Materials, 230601, Hefei, P.R. China.
b School of Science, Anhui Agricultural University, 230036 Hefei, P. R. China.
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Co-operative Innovation Research Center for Weak Signal-Detecting Materials and Devices
Integration, Anhui University, Hefei 230601, P. R. China.
‡ These authors contributed equally to this work and should be considered as co-first authors.
Corresponding author. Fax: +86-551-63861279
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E-mail: zhpzhp@263.net
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Abstract
Six new benzoxazole-based compounds 1-6
of the donor-bridge-accepter (D-π-A) type with different anions were obtained by
methylation and anion exchange reaction. The single crystals of 1, 3 and 6 were obtained
by slow evaporation of acetonitrile and determined by X-ray crystallography. Ellipsoidal
structures of the three compounds showed that the cationic parts all maintained good
planarity, which was beneficial for two-photon absorption (2PA) properties and accorded
with the results of two-photon excited fluorescence tests. Biological imaging experiments
showed water-soluble six compounds had excellent penetrability in living cells and could
located in cytoplasm in one- and two-photon cell imaging. Biological imaging result of
compound 6 was more excellent than other compounds, which indicated synergy of anions
and cations played a very important role in cell imaging.
Keywords: pyridinium, 2PA properties, bio-imaging
30 1. Introduction
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It is necessary to design and synthesize organic materials with larger 2PA cross-sections
(δ2PA) because of their potential application, such as photodynamic therapy [1-5],
photochemical delivery of biological messengers [6-10], confocal microscopy [11-13],
three-dimensional date storage [14-25] and optical power limiting [26]. Among these,
two-photon microscopy was a powerful technique for noninvasive live cell imaging, due to
two-photon excitation could achieve deeper penetration, less photobleaching and higher
spatial resolution compared with one-photon excitation [27]. In two-photon bio-imaging
applications, an excellent stain not only requires a high signal-to-noise ratio which depends
on large δ2PA values, but also demands good biological compatibility which depends on the
water solubility. Therefore, design and synthesis of 2PA compounds with appropriate 2PA
cross-sections in water solution is an important way to realize the application in bioimaging.
For instance, Wong’s group [28] designed and synthesized a 2,7-carbazole dicationic salt
(2,7-9E-BHVC) which acted as a two-photon turn-on fluorescent probe for nucleic acid
(NA)，the effective 2PA cross-sections (δeff) were 1.67 GM and 30 GM in the absence and
presence of NA in phosphate buffer saline solution (PBS), respectively. Zhang’s group [29]
synthesized four tetraphenylethylene (TPE) salt with different anion group based
nanoparticles to apply on 2PA bio-imaging in water solution. The results showed that the
sizes, the surface charge and hydrophobic TPE fragment could all facilitate the uptake and
interaction with lipid bilayers of cancer cells, thus the nanoparticles showed good
permeability to cancer cells. Wang and Zhang [30] synthesized a positive charged
water-soluble polythiophene (PTO) that the 2PA cross-section was more than 300 GM in
water solution, which could be successfully implied in Hela cells imaging.
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Researchers committed to the application of two-photon materials in biological research
because of their intrinsic properties, especially water solubility to avoid
organic solvents, which could effectively reduce the toxicity to cells or tissues.
However, the larger 2PA cross-sections usually required a large conjugated system by the
longer synthetic route, which lowered the solubility in water solution and enhanced cost,
subsequently. So it was still a challenge to design water-soluble 2PA materials to be applied
to bio-imaging. Based on the above, we designed and synthesized six new D–π–A type
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compounds 1-6 based on benzoxazole groups, investigated the influence of different anions
on photophysical property, in which the benzoxazole group acted as electron-donor, the
pyridine cationic as electron-acceptor. The small molecules 1-6 exhibited excellent
solubility and linear, nonlinear optical properties in water, which were successfully carried
out the 2PA bio-imaging applications.
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2. Experimental section
2.1 Materials and instruments
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All commercially available chemicals were of analytical grade or chromatographic grade.
Every solvent was purified using conventional methods beforehand. IR spectra were
recorded with a Nicolet FT-IR NEXUS 870 spectrometer (KBr discs) in the 4000–400
cm-1 region. 1H and 13C NMR spectra were recorded on a Bruker AV 400 Ultrashield
spectrometer using DMSO-d6 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). The mass
spectra were obtained on a Bruker Autoflex III smart beam mass spectrometer The X-ray
diffraction measurements were performed on a CCD area detector using graphite
monochromated MoKα radiation (λ = 0.71069 Å) at 298(2) K. Intensity data were collected
in the variable ω/2θ-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 the
SHELXTL-97 program package. CCDC: 1544353 (for 1), 1535136 (for 3), 1535139 (for
6).
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Scheme 1 Synthetic route to 1-6.
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2.2 Synthesis and characterization
Six compounds were prepared according to the synthetic routes showed in Scheme 1.
The experiment details and structure characterization data were described in the
experimental section and electronic supporting information (ESI). The intermediate L1 was
synthesized efficiently according to our previous work [31].
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2.2.1 (E)-4-(4-(benzo[d]oxazolyl-2-yl) styryl) pyridine (L2)
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L2: L1 (8.00 g, 14.50 mmol), 4-Pyridinecarboxaldehyde (1.28 g, 12.00 mmol), K2CO3
(4.97 g, 36.00 mmol), 5 drops methyltrioctylammonium chloride were added in
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three-necked flask with 10 mL N, N-Dimethylacetamide and refluxed at 140 C for 7 h.
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The reaction mixture was cooled to room temperature and poured into dichloromethane and
stirred vigorously, the solid was removed by filtration, the filtrate was washed with
saturated brine, then purified with chromatography (Petroleum ether: Ethyl acetate = 5:1) to
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give 2.50 g of white powder. Yield: 70%. H NMR: (400 MHz, DMSO-d6), δ (ppm): 8.59
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(d, J = 8.0 Hz, 2H), 8.25 (d, J = 8.0 Hz, 2H), 7.90 (d, J = 8.0 Hz, 2H), 7.84-7.80 (t, J = 8.0
Hz, 2H), 7.66 (d, J = 16.0 Hz, 1H), 7.62 (d, J = 4.0 Hz, 2H), 7.47-7.41 (m, 3H). 13C NMR
(100 MHz, DMSO-d6), δ (ppm):161.96, 150.11, 143.82, 141.55, 139.59, 131.85, 128.30,
127.81, 127.71, 126.00, 125.61, 124.94, 121.04, 119.81, 110.91. MS (ESI): m/z, found:
299.1105; molecular formula C20H14N2O, requires [M+H]+, 299.1106.
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2.2.2 (E)-4-(4-(benzo[d]oxazolyl-2-yl) styryl)-1-methylpyridinium-1-ium iodide (1)
1: L2 (1.16 g, 3.80 mmol) and idomethane (1.70 g, 14.00 mmol) were dissolved in 40
mL THF and refluxed at 65oC for 4 h. The mixture was filtered and the solid was washed
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with THF for 3 times, 1.22 g of yellow product was obtained. Yield: 72%. H NMR: (400
MHz, DMSO-d6), δ (ppm): 8.92 (d, J = 4.0 Hz, 2H), 8.33-8.27 (m, 4H), 8.10 (d, J = 16.0
Hz, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.86-7.82 (t, J = 8.0 Hz, 2H), 7.70 (d, J = 16.0 Hz, 1H),
7.50-7.43 (m, 2H), 4.28 (s, 3H). 13C NMR: (100 MHz, DMSO-d6), δ (ppm): 161.66, 151.91,
150.24, 145.25, 141.49, 139.07, 138.30, 128.80, 127.88, 127.40, 125.86, 125.31, 125.05,
123.84, 119.94, 110.99, 47.05. MS (APCI): m/z, found: 313.1337; molecular formula
C21H17IN2O, requires [M-I-]+, 313.1335.
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2.2.3
(E)-4-(4-(benzo[d]oxazolyl-2-yl)
styryl)-1-methylpyridinidium-1-
ium
4-methylbenzene sulfonate
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2: An acetonitrile solution of sliver p-toluenesulfonate (0.15 g, 0.68 mmol) was added to
acetonitrile solution of 1 (0.30 g, 0.68 mmol), the mixture was refluxed at 80oC for 4 h,
then the precipitate was removed by filtration. 0.18 g of yellow product was obtained by
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concentrating filtrate. Yield: 54%. H NMR: (400 MHz, DMSO-d6), δ (ppm): 8.91 (d, J =
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8.0 Hz, 2H), 8.32 (d, J = 8.0 Hz, 2H), 8.27 (d, J = 8.0 Hz, 2H), 8.09 (d, J =16.0 Hz, 1H),
7.98 (d, J = 8.0 Hz, 2H), 7.86-7.82 (t, J = 8.0 Hz, 2H), 7.69 (d, J =16.0 Hz, 1H), 7.49-7.43
(m, 4H), 7.11 (d, J = 8.0 Hz, 1H), 4.27 (s, 3H), 2.28 (s, 3H). 13C NMR (100 MHz,
DMSO-d6), δ (ppm): 161.67, 151.90, 150.25, 145.77, 145.28, 141.49, 139.07, 138.31,
137.48, 128.80, 127.99, 127.89, 127.40, 125.87, 125.45, 125.35, 125.07, 123.84, 119.95,
111.00, 47.01, 20.74. MS (APCI): m/z, found: 313.1331; molecular formula C28H24N2O4S,
requires [M-(CH3-Ph-SO3)-]+, 313.1335.
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2.2.4
(E)-4-(4-(benzo[d]oxazolyl-2-yl)
styryl)-1-methylpyridinium-1-
ium
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hexafluoroPhosphate (3), (E)-4-(4-(benzo[d]oxazolyl-2-yl) styryl)-1-methylpyridinium-1-
ium trifluorooacetate (4), (E)-4-(4-(benzo[d]oxazolyl-2-yl) styryl)-1-methylpyridinium-1-
ium nitrate (5)
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3-5: Yellow powders 3-5 were prepared according to the similar procedure of 2 by using
silver hexafluoorophosphate (0.15 g, 0.68 mmol), silver trifluoroacetate (0.15 g, 0.68 mmol)
or silver nitrate (0.12 g, 0.68 mmol) instead of sliver p-toluenesulfonate. Yield: 13% (0.04
g), 34% (0.10 g) and 44% (0.10 g) for 3, 4, 5, respectively. The characterization of these
compounds was as follows.
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3: H NMR: (400 MHz, DMSO-d6), δ (ppm): 8.92 (d, J = 4.0 Hz, 2H), 8.32 (d, J = 8.0
Hz, 2H), 8.28 (d, J = 4.0 Hz, 2H), 8.10 (d, J = 16.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 2H),
7.86-7.82 (t, J = 8.0 Hz, 2H), 7.70 (d, J = 16.0 Hz, 1H), 7.49-7.43 (m, 2H), 4.28 (s, 3H).
13C NMR (100 MHz, DMSO-d6), δ (ppm): 161.66, 151.91, 150.25, 145.27, 141.49, 139.08,
138.31, 128.80, 127.90, 127.41, 125.87, 125.34, 125.07, 123.84, 119.95, 111.00, 47.04. MS
(APCI): m/z, found: 313.1348; molecular formula C21H17F6N2OP, requires [M-(PF6)-]+,
313.1335.
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4: H NMR: (400 MHz, DMSO-d6), δ (ppm): 8.92 (d, J = 4.0 Hz, 2H), 8.31 (d, J = 8.0
Hz, 2H), 8.27 (d, J = 8.0 Hz, 2H), 8.09 (d, J =16.0 Hz, 1H), 7.97 (d, J = 8.0 Hz, 2H),
7.85-7.81 (t, J = 8.0 Hz, 2H), 7.69 (d, J =16.0 Hz, 1H), 7.49-7.42 (m, 2H), 4.28 (s, 3H). 13C
NMR (100 MHz, DMSO-d6), δ (ppm): 161.66, 151.90, 150.24, 145.26, 141.48, 139.06,
138.29, 128.78, 127.86, 127.38, 125.84, 125.31, 125.04, 123.83, 119.93, 110.98, 46.99. MS
(APCI): m/z, found: 313.1335; molecular formula C23H17F3N2O3, requires [M-(F3CCOO)-]+,
313.1335.
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5: H NMR: (400 MHz, DMSO-d6), δ (ppm): 8.91 (d, J = 8.0 Hz, 2H), 8.32 (d, J = 8.0
Hz, 2H), 8.27 (d, J = 8.0 Hz, 2H), 8.10 (d, J =16.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 2H),
7.86-7.82 (t, J = 8.0 Hz, 2H), 7.70 (d, J =16.0 Hz, 1H), 7.50-7.43 (m, 2H), 4.28 (s, 3H). 13C
NMR (100 MHz, DMSO-d6), δ (ppm): 161.66, 151.91, 150.24, 145.27, 141.49, 139.07,
138.30, 128.79, 127.88, 127.40, 125.86, 125.86, 125.32, 125.05, 123.83, 119.94, 110.99,
47.01. MS (APCI): m/z, found: 313.1333; molecular formula C21H17N3O4, requires
[M-(NO3)-]+, 313.1335.
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2.2.5
(E)-4-(4-(benzo[d]oxazolyl-2-yl)
styryl)-1-methylpyridinium-1-
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tetraphenylborate (6)
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6: Sodium tetraphenylboron (0.23 g, 0.68 mmol) and 1 were dissolved in ethanol. Then
the mixture was refluxed at 80oC for 6 h. 0.27 g of yellow product was obtained by
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filtration and washed the solid with ethanol for 3 times. Yield: 63%. H NMR: (400 MHz,
DMSO-d6), δ (ppm): 8.89 (d, J = 8.0 Hz, 2H), 8.32 (d, J = 8.0 Hz, 2H), 8.26 (d, J = 8.0 Hz,
2H), 8.09 (d, J =16.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.86-7.81 (t, J = 10.0 Hz, 2H), 7.69
(d, J =16.0 Hz, 1H), 7.50-7.43 (m, 2H), 7.18 (s, 8H), 6.94-6.90 (t, J = 8.0 Hz, 8H),
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6.80-6.76 (t, J = 8.0 Hz, 4H), 4.26 (s, 3H). C NMR (100 MHz, DMSO-d6), δ (ppm):
164.02, 163.56, 162.56, 161.57, 151.91, 150.26, 145.27, 141.49, 139.09, 138.30, 135.49,
128.80, 127.91, 127.43, 125.88, 125.28, 123.84, 121.47, 119.95, 111.00, 47.02. MS (APCI):
m/z, found: 313.1348; molecular formula C45H37N2O4, requires [M-(B(Ph)4)-]+, 313.1335.
20 3. Results and discussion
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3.1. Crystal structures
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To investigate the structure - property relationship of the synthesized compounds, we
elucidated the molecule structure in solid state. Fortunately, the single crystals of
compounds 1, 3 and 6 were obtained by slow evaporation from acetonitrile solution. Their
molecular structure diagrams with atom numbering scheme and stacking structures
involved some weak interactions among the adjacent molecules were depicted in Fig. S2.
The one-dimensional structures of 1, 3 and 6 were depicted in Fig.S2. The crystallographic
data were summarized in Table S1. From the Fig. 1, we could see that the dihedral angle
(θ1) between the phenlybenzoxazole ring and the plane P1 are 6.363o, 5.997o, 6.610o for 1,
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3 and 6, respectively. The dihedral angle (θ2) between the plane P1 and the plane P2 are
17.165o, 10.502o, 6.589o for 1, 3 and 6, respectively. The bond lengths of the -C=C- bond in
1 is 1.327Å. The bond lengths of 3 and 6 in the same position are similar to those of 1,
which means there exist a good delocalization system in the section of these compounds.
The ellipsoidal structures of three compounds show that the benzoxazole group, the π -
bridge and the electron acceptor maintain the good planarity, which is beneficial to the
intramolecular charges delocalization and increases the electron mobility in the conjugate
system and further enlarges the 2PA properties.
Fig. 1 ORTEP diagram (with 50% thermal ellipsoid probability) structures of 1, 3 and 6.
3.2. Optical measurements
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3.2.1 One-photon absorption and emission properties
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One-photon absorption and fluorescence emission spectra were measured in six solvents
with different polarities at the concentration of 1×10-5 mol·L-1. As shown in Fig.2 and Fig.
S3, considering all the six compounds possessed the similar linear optical properties, we
just used 1 as example to discuss in detail. It could be seen from Fig.2a that compound 1
exhibited two peaks located at ̴ 280 and ̴ 370 nm, of which the high-energy band was
mainly attributed to the π-π* transitions of the benzoxazole part, while the low-energy band
was likely assigned to whole molecule π-π* transitions [32]. Fig.2b illustrated the
fluorescence emission spectra of compound 1 in different polarities solvents. We could see
that there was one emission peak located at from 504 nm to 511 nm and compounds 2-6
had similar absorption and emission properties as compound 1, which may be the same
cationic group.
Fig. 2 Linear absorption and emission spectra of compound 1 in six solvents.
3.2.2 Theoretical calculations
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To further investigate the influence of the structure on the characteristic of charge
transition of the six compounds, time-dependent density functional theory (TDDFT) was
carried out to investigate the energy level of the ground and excited states. The Gaussian 09
program and B3LYP/6-31G basis sets were used for calculations. The isosurface of the
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highest occupied and the lowest unoccupied molecular orbital (HOMO and LUMO) were
shown in Fig. 3. Take compounds 1 and 3 as examples. For 1, the electron-cloud was
delocalized throughout the entire backbones, the HOMO and LUMO diagrams clearly
showed the π-π* transitions characteristics upon photoexcitation. This result suggested that,
on the one hand, the optical transition from LUMO to HOMO was a direct one, which
offers a high fluorescent efficiency, on the other hand, the molecules had no intramolecular
charge transfer process. In sharp contrast, the HOMO and LUMO of 3 exhibited the
obvious intramolecular charge transfer characteristics, which may be favorable for the 2PA
of the compounds. In this process, most of the electron clouds of the HOMO were evenly
distributed in the electrondonating groups (phenylbenzoxazole), while the majority of the
electron clouds of the LUMO were transferred and evenly distributed electron-accepting
motifs (pyridine cationic moieties). The theoretical calculations showed that the
intramolecular charge transfer trend of compound 6 was consistent with that of compound
3.
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Fig. 3 Energy levels of the HOMO and LUMO, energy gaps and electron cloud distributions of 1, 3 and
6 molecules calculated by the B3LYP/6-31G program.
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3.2.3 Fluorescence lifetime
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 (χ2).
The fluorescence lifetimes of 1-6 in water were 0.786 ns, 0.779 ns, 0.789 ns, 0.789 ns,
0.784 ns and 0.460 ns, respectively. The experimental results showed that the fluorescence
lifetimes of benzoxazole-based pyridinium with different anions had no obvious distinction
except 6, perhaps due to the hydrophobicity of the tetraphenylborate in aqueous which was
unfavorable to fluorescence emission.
Fig. 4 Time-resolved fluorescence curves of 1-6 in H2O.
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3.2.4 Two-photon excited fluorescence spectra and 2PA cross-sections.
Two-photon fluorescence spectra of 1-6 in H2O by femtosecond laser pulses at 300 mW
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as different excitation wavelengths were presented in Fig. 5a and Fig. S4a. Fig. 5b and Fig.
S4b showed TPEF spectra under different pump intensities and logaritnmic plots of the
fluorescence intergral versus pumped powers with a slope of 1.91 (1), 1.88 (2), 1.72 (3),
1.81 (4), 1.77 (5) and 1.79 (6) when the input laser power was increasing, suggesting a
two-photon excitation mechanism. 2PA cross-sections of the samples were obtained by
two-photon excited fluorescence method at femtosecond laser pulse and Ti: sapphire
system (680-1080 nm, 80 MHz, 140 fs) as the light source in H2O at a concentration of
1.0×10-4 mol·L-1. The 2PA cross-sections were determined by comparing their TPEF with
that of fluorescein, according to following eqn:
Φref Cref nref
F
δ
= δref
Φ
C
n F
ref
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Where the ref subscripts stand for the reference molecule (here fluorescein dissolved in the
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aqueous NaOH solution (1 mol·L-1) at concentration of 1× 10-3 mol·L-1 was used as
reference). δ was the 2PA cross-sections value,
c was the concentration of the solution, n is
the refractive index of the solution, was the TPEF integral intensities of the solution
F
emitted at the exciting wavelength, and Ф was the fluorescence quantum yield. The δ value
of reference was taken from the literature [33].
Fig. 5 (a) The TPEF spectra of compound 1 in H2O by femtosecond laser pulses at 300 mV under the
different excitation wavelengths. (b) Output fluorescence (Iout) versus the square of input laser power
(Iin) under the optimal excitation wavelengths that at 760 nm for 1 with c =1.0×10-4 mol·L-1 in H2O.
Fig. 6 showed the 2PA cross-sections of compounds 1-6 in the 700-880 nm range, the
maximum 2PA cross-sections were 134 GM, 75 GM, 176 GM, 191 GM, 266 GM and 103
GM for 1, 2, 3, 4, 5 and 6 (1 GM=10-50 cm-4 s per photon), respectively. The values of the
2PA cross-sections of compounds 1-6 were somewhat different and may be attributed to the
different hydrophilicity or hydrophobicity of different anion in H2O. All the results showed
that compounds 1-6 possessed good 2PA properties, which indicated compounds 1-6 had
potential applications in two-photon bio-imaging.
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Fig. 6 2PA cross-sections of 1-6 in H2O versus excitation wavelength of identical energy of 300 mW
(from a 140 fs, 80 MHz, Ti: sapphire laser).
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3.3. Cell image
To further exploit the potential application of the six compounds that emitted
two-photon excited fluorescence in water solution, we carried out one- and two-photon
bio-imaging experiment through the stained 1–6 live Hela cells. As shown in Fig. S5, the
time-dependent two-photon excited fluorescence of compound 6 was carried out as an
example. The result indicated that compound 6 had good light stability and was very
suitable for being used in biological applications. The tested compounds were dissolved in
H2O and then serially diluted in complete culture medium. In two-photon microscopy
imaging, the optimal excitation wavelength of each compound was used. As shown in Fig.
7a, green fluorescence from the cells indicated that the compounds 1-6 could be
effectively internalized by Hela cells and was very suitable for use in biological
applications. In Fig. 7b, the TPEF image of Hela cells showed red fluorescence upon
excitation. In Fig. 7c, the fluorescent images and the merged images conformed that
compounds 1-6 all went through the membrane and localized in the cytoplasm exclusively.
Due to the good lipophilicity of tetraphenylboron, compound 6 was more easily to
penetrate the cell membrane and the phenomenon of dyeing the cytoplasm was more
pronounced, which manifested that the practical application of 2PA materials in biology is
not only related to δ and quantum yield, but also to the nature of itself.
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Fig. 7 Fluorescence images of Hela cells with compounds 1-6: (a) One-photon fluorescence
microscopy image; (b) Two-photon fluorescence microscopy image; (c) Merged image.
19 4. Conclusion
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In summary, we synthesized six water-soluble benzoxazolyl pyridinium derivatives 1-6.
One- and two-photon optical properties had been researched systematically and their
structure–property relationships were summarized clearly through the combination of
experimental data and theoretical calculations. Based on their excellent one- and
two-photon optical properties, good solubility in water medium, as well as suitable cell
permeability, we utilized them successfully to carry out one- and two-photon fluorescence
bio-imaging.
27 Acknowledgements
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This wok was supported by General Program of National Natural Science Foundation of
China (51472002, 51432001), Science and Technology Plan of Anhui Province
(1604b0602016), the Ministry of Education of China, Higher Education Revitalization Plan
Talent Project of Anhui Province (2013), the Natural Science Foundation of Education
Committee of Anhui Province (KJ2016A218) and Open Foundation of Co-operative
Innovation Research Center for Weak Signal-Detecting Materials and Device Integration of
Anhui University (Y01008411).
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Reference
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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[1] Garcia G, Hammerer F, Poyer F, et al. Carbohydrate-conjugated porphyrin dimers:
Synthesis and photobiological evaluation for a potential application in one-photon
and two-photon photodynamic therapy. Bioorganic & medicinal chemistry
2013;21(1):153-165.
[2] Li L, Zhang C W, Chen G Y J, et al. A sensitive two-photon probe to selectively
detect monoamine oxidase B activity in Parkinson’s disease models. Nature
communications;2014, 5:3276–3285.
[3] Geng J, Goh C C, Tomczak N, et al. Micelle/silica co-protected conjugated polymer
nanoparticles for two-photon excited brain vascular imaging. Chemistry of Materials
2014;26(5):1874-1880.
[4] Hu W, He T, Jiang R, et al. Inner salt-shaped small molecular photosensitizer with
extremely enhanced two-photon absorption for mitochondrial-targeted photodynamic
therapy. Chemical Communications 2017;53(10):1680-1683.
[5] Alfonso Hernandez L, Nelson T, Gelin M F, et al. Interference of interchromophoric
energy-transfer pathways in π-conjugated macrocycles. The journal of physical
chemistry letters 2016;7(23):4936-4944.
[6] Hisaki I, Nakagawa S, Ikenaka N, et al. A series of layered assemblies of
hydrogen-bonded, hexagonal networks of C 3-symmetric π-conjugated molecules: a
potential motif of porous organic materials[J]. Journal of the American Chemical
Society, 2016, 138(20): 6617-6628. Okada K, Yagi A, Segawa Y, et al. Synthesis and
properties of [8]-, [10]-,[12]-, and [16] cyclo-1, 4-naphthylenes. Chemical Science
2017;8(1):661-667.
[7] Iyoda M, Shimizu H. Multifunctional π-expanded oligothiophene macrocycles.
Chemical Society Reviews 2015;44(18):6411-6424.
[8] Cui M, Ono M, Watanabe H, et al. Smart near-infrared fluorescence probes with
donor–acceptor structure for in vivo detection of β-amyloid deposits. Journal of the
American Chemical Society 2014;136(9):3388-3394.
[9] Rathore K, Lim C S, Lee Y, et al. Dual-color imaging of cytosolic and mitochondrial
zinc ions in live tissues with two-photon fluorescent probes. Organic & biomolecular
chemistry 2014;12(21):3406-3412.
11

ACCEPTED MANUSCRIPT
1
2
[10] Hu Y, Heo C H, Kim G, et al. One-photon and two-photon sensing of biothiols using
a bis-pyrene-Cu (II) ensemble and its application to image GSH in the cells and
tissues. Analytical chemistry 2015;87(6):3308-3313.
3
4
[11] Yi M, Yang S, Peng Z, et al. Two-photon graphene oxide/aptamer nanosensing
conjugate for in vitro or in vivo molecular probing. Analytical chemistry
2014;86(7):3548-3554
5
6
7
[12]Xu W, Zuo J, Wang L, et al. Dinuclear ruthenium (II) polypyridyl complexes as single
and two-photon luminescence cellular imaging probes. Chemical Communications
2014;50(17):2123-2125.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
[13]Yuan L, Wang L, Agrawalla B K, et al. Development of targetable two-photon
fluorescent probes to image hypochlorous acid in mitochondria and lysosome in live
cell and inflamed mouse model. Journal of the American Chemical Society
2015;137(18):5930-5938.
[14]Dvornikov A S, Walker E P, Rentzepis P M. Two-photon three-dimensional optical
storage memory. The Journal of Physical Chemistry A 2009;113(49):13633-13644.
[15]Li Z, Pucher N, Cicha K, et al. A straightforward synthesis and structure–activity
relationship of highly efficient initiators for two-photon polymerization.
Macromolecules 2013;46(2):352-361.
[16]Morales A R, Frazer A, Woodward A W, et al. Design, synthesis, and structural and
spectroscopic studies of push–pull two-photon absorbing chromophores with
acceptor groups of varying strength. The Journal of organic chemistry
2013;78(3):1014-1025.
[17]Marder S R, Gorman C B, Tiemann B G, et al. Stronger acceptors can diminish
nonlinear optical response in simple donor-acceptor polyenes. Journal of the
American Chemical Society 1993;115(7):3006-3007.
[18]Pawlicki M, Collins H A, Denning R G, et al. Two Photon Absorption and the Design
of
Two Photon
Dyes.
Angewandte
Chemie
International
Edition
2009;48(18):3244-3266.
[19]Terenziani F, Katan C, Badaeva E, et al. Enhanced Two Photon Absorption of
Organic Chromophores: Theoretical and Experimental Assessments. Advanced
Materials 2008;20(24):4641-4678.
[20]Guo E Q, Ren P H, Zhang Y L, et al. Diphenylamine end-capped 1, 4-diketo-3,
6-diphenylpyrrolo [3, 4-c] pyrrole (DPP) derivatives with large two-photon
absorption cross-sections and strong two-photon excitation red fluorescence.
Chemical Communications 2009;(39):5859-5861.
[21]Gu M, Tannous T, Sheppard C J R. Three-dimensional confocal fluorescence imaging
under ultrashort pulse illumination[J]. Optics communications, 1995, 117(5):
406-412.
[22]Dvornikov A S, Walker E P, Rentzepis P M. Two-photon three-dimensional optical
storage memory. The Journal of Physical Chemistry A 2009;113(49):13633-13644.
[23]Tian H, Feng Y. Next step of photochromic switches. Journal of Materials Chemistry
2008;18(14):1617-1622.
[24]al push-pull chromophores for nonlinear optical imaging of cell membranes. Organic
& biomolecular chemistry 2010;8(1):142-150.
12

ACCEPTED MANUSCRIPT
1
2
[25]Denk W, Strickler J H, Webb W W. Two-photon laser scanning fluorescence
microscopy. Science 1990;248(4951):73-76.
3
[26]Wu H, Yu H, Yang Z, et al. Designing a deep-ultraviolet nonlinear optical material with
a large second harmonic generation response. Journal of the American Chemical
Society 2013;135(11):4215-4218.
4
5
6
[27]Liu X, Sun Y, Zhang Y, et al. A 2, 7-carbazole-based dicationic salt for fluorescence
detection of nucleic acids and two-photon fluorescence imaging of RNA in nucleoli
and cytoplasm. Organic & biomolecular chemistry 2011;9(10):3615-3618.
[28]Huang Y, Zhang G, Hu F, et al. Emissive nanoparticles from pyridinium-substituted
tetraphenylethylene salts: imaging and selective cytotoxicity towards cancer cells in
vitro and in vivo by varying counter anions. Chemical Science
2016;7(12):7013-7019.
[29]Lan M, Zhao S, Xie Y, et al. Water-Soluble Polythiophene for Two-Photon Excitation
Fluorescence Imaging and Photodynamic Therapy of Cancer. ACS Applied Materials
& Interfaces 2017-9(17):14590-14595.
[30]Ge X, Gan X, Yao S, et al. Rationally designed two-photon absorption compounds
based on benzoxazole derivatives: structure–property relationships and bio-imaging
applications. Journal of Materials Chemistry B 2016-4(16):2785-2793.
[31]Wang L, Zheng Z, Yu Z, et al. Schiff base particles with aggregation-induced enhanced
emission: random aggregation preventing π–π stacking. Journal of Materials
Chemistry C 2013;1(42):6952-6959.
[32]Wang L, Zheng Z, Yu Z, et al. Schiff base particles with aggregation-induced enhanced
emission: random aggregation preventing π–π stacking. Journal of Materials
Chemistry C 2013;1(42):6952-6959.
7
8
9
10
11
12
13
14
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16
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20
21
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Figure captions
Scheme 1 Synthetic route to 1-6.
Fig. 1 ORTEP diagram (with 50% thermal ellipsoid probability) structures of 1, 3 and
6.
Fig. 2 Linear absorption and emission spectra of compound 1 in six solvents.
Fig. 3 Energy levels of the HOMO and LUMO, energy gaps and electron cloud
distributions of 1, 3 and 6 molecules calculated by the B3LYP/6-31G program.
Fig. 4 Time-resolved fluorescence curves of 1-6 in H2O.
Fig. 5 (a) The TPEF spectra of compound 1 in H2O by femtosecond laser pulses at
300 mV under the different excitation wavelengths. (b) Output fluorescence (Iout)
versus the square of input laser power (Iin) under the optimal excitation wavelengths
that at 760 nm for 1 with c =1.0×10-4 mol·L-1 in H2O.
Fig. 6 2PA cross-sections of 1-6 in H2O versus excitation wavelength of identical
energy of 300 mW (from a 140 fs, 80 MHz, Ti: sapphire laser).
Fig. 7 Fluorescence images of Hela cells with compounds 1-6: (a) One-photon
fluorescence microscopy image; (b) Two-photon fluorescence microscopy image; (c)
Merged image.

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Scheme 1. Synthetic route to 1-6.
Fig. 1 ORTEP diagram (with 50% thermal ellipsoid probability) structures of 1, 3 and
6.

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Fig. 2 Linear absorption and emission spectra of compound 1 in six solvents.
Fig. 3 Energy levels of the HOMO and LUMO, energy gaps and electron cloud
distributions of 1, 3 and 6 molecules calculated by the B3LYP/6-31G program.
Fig 4 Time-resolved fluorescence curves of 1-6 in H2O.

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Fig. 5 (a) The TPEF spectra of compound 1 in H2O by femtosecond laser pulses at
300 mV under the different excitation wavelengths. (b) Output fluorescence (Iout)
versus the square of input laser power (Iin) under the optimal excitation wavelengths
that at 760 nm for 1 with c =1.0×10-4 mol·L-1 in H2O.
Fig. 6 2PA cross-sections of 1-6 in H2O versus excitation wavelength of identical
energy of 300 mW (from a 140 fs, 80 MHz, Ti: sapphire laser).

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Fig. 7 Fluorescence images of Hela cells with compounds 1-6: (a) One-photon
fluorescence microscopy image; (b) Two-photon fluorescence microscopy image;
(c) Merged image.

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Highlights
‹ Six benzoxazole-based compounds 1-6 with different anions were obtained by
methylation and anion exchange reaction.
‹ Compounds 1-6 showed good two-photon absorption properties in water solvent.
‹ Compounds 1-6 were applied in live cell imaging, which highlighted their
practical performance.