Styrylcyanine-based fluorescent probes with red-emission and large Stokes shift for the detection of viscosity

Article abstract of DOI:10.1016/j.jphotochem.2017.05.035

Based on the mechanism of intramolecular charge transfer (ICT), two new styrylcyanine dyes, DPA-1 and DPA-2, composed of an electron-rich N-phenylaniline and a cationic benzothiazene connected with ethylene(s) bridge, were designed and synthesized. These two dyes exhibited red-emission (653/607?nm) and simultaneously impressive large Stokes shift (111/92?nm) due to intramolecular charge transfer effect, twisted geometry and extended conjugation system. When their solution viscosity increased from 1.01?cP to 234?cP in the water-glycerol system, the fluorescence intensity of the synthetic dyes was enhanced by 81-fold and 64-fold, respectively. Additionally, a favorable linear relationship between the fluorescence intensity and the environmental viscosity was observed within the above viscosity range for the obtained samples (R²?>?0.99), which led to the establishment of a method for the quantitative determination of the solution viscosity. Such dyes with improved photophysical properties, including emission wavelength and Stokes shift, could be used as promising candidates for intracellular viscosity detection. Moreover, the mechanism of fluorescence emission of the resultant products toward viscosity was further investigated. Of the two probes studies, DPA-1 is better than DPA-2 in terms of emission wavelength, Stokes shift and the sensitivity of the fluorescence intensity to viscosity.

Full text of DOI:10.1016/j.jphotochem.2017.05.035

Accepted Manuscript
Title: Styrylcyanine-based fluorescent probes with
red-emission and large Stokes shift for the detection of
viscosity
Authors: Xiangbin Cao, Jianhui Liu, Pan Hong, Guanglan Li,
Ce Hao
PII:
S1010-6030(17)30259-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2017.05.035
JPC 10662
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
25-2-2017
19-4-2017
22-5-2017
Please cite this article as: Xiangbin Cao, Jianhui Liu, Pan Hong, Guanglan Li,
Ce Hao, Styrylcyanine-based fluorescent probes with red-emission and large Stokes
shift for the detection of viscosity, Journal of Photochemistry and Photobiology A:
Chemistryhttp://dx.doi.org/10.1016/j.jphotochem.2017.05.035
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

Styrylcyanine-based fluorescent probes with red-emission
and large Stokes shift for the detection of viscosity
Xiangbin Cao, Jianhui Liu*, Pan Hong, Guanglan Li and Ce Hao*
School of Petroleum and Chemical Engineering, Dalian University of Technology,
Panjin, Liaoning 124221, P. R. China
* Corresponding author: liujh@dlut.edu.cn (J. Liu)
haoce@dlut.edu.cn (C. Hao)
Graphical Abstract
Highlights




●Two novel styrylcyanine dyes were developed based on ICT.
●The probes exhibited red-emission and large Stokes shift.
●The probes displayed high sensitivity to viscosity.
●The fluorescence intensity was affected by intramolecular rotation.
Abstract: Based on the mechanism of intramolecular charge transfer (ICT), two new
styrylcyanine dyes, DPA-1 and DPA-2, composed of an electron-rich N-phenylaniline
and a cationic benzothiazene connected with ethylene(s) bridge, were designed and
synthesized. These two dyes exhibited red-emission (653/607 nm) and simultaneously
impressive large Stokes shift (111/92 nm) due to intramolecular charge transfer effect,
twisted geometry and extended conjugation system. When their solution viscosity
increased from 1.01 cP to 234 cP in the water-glycerol system, the fluorescence
1

intensity of the synthetic dyes was enhanced by 81-fold and 64-fold, respectively.
Additionally, a favorable linear relationship between the fluorescence intensity and
the environmental viscosity was observed within the above viscosity range for the
obtained samples (R2 > 0.99), which led to the establishment of a method for the
quantitative determination of the solution viscosity. Such dyes with improved
photophysical properties, including emission wavelength and Stokes shift, could be
used as promising candidates for intracellular viscosity detection. Moreover, the
mechanism of fluorescence emission of the resultant products toward viscosity was
further investigated. Of the two probes studies, DPA-1 is better than DPA-2 in terms
of emission wavelength, Stokes shift and the sensitivity of the fluorescence intensity
to viscosity.
Keywords: red-emission; large Stokes shift; styrylcyanine; viscosity; intramolecular
charge transfer
1. Introduction
Diffusion-mediated cellular processes, including transportation of mass and
signal, interactions between biomolecules and diffusion of reactive metabolites, can
be influenced by intracellular viscosity [1,2]. Indeed, abnormal changes in viscosity
are connected to a wide range of diseases and dysfunctions [3], such as atherosclerosis
and diabetes [4,5]. Therefore, the detection of intracellular viscosity is important for
biological analysis and disease diagnosis [6].
Fluorescent molecular rotors are good candidates to be used for determining the
viscosity of a microenvironment [2]. These fluorescent molecules can freely rotate in
low-viscosity media, resulting in the weak intrinsic fluorescence. Once the
intramolecular rotation is restricted in viscous media, the fluorescence intensity or
lifetime of the dyes will increase [7-10]. To date, various kinds of molecular rotors
have been used to make great achievements in intracellular viscosity research.
However, traditional molecular rotors are limited to 9-(dicyanovinyl)-julolidine
(DCVJ) and their derivatives [7,8,11]. Porphyrin-based rotors suffer from
complicated synthesis and low sensitivity [1,12]. The applications of BODIPY-based
rotors [10,13-16] for in vivo analysis may be limited by their short wavelength, due to
the significant auto-fluorescence of biomolecules by excitation with visible light
[17,18]. On the other hand, the cyanine dyes have been used as chemosensors for
intracellular viscosity detection due to their perfect photophysical properties and
outstanding biocompatibility [2,3,19-23]. For instance, Peng et al. reported a
2

pentamethine cyanine dye RY3 with a formyl group as the rotor to determine the
intracellular viscosity [2]. Moreover, Belfield et al. [24] reported two near-infrared
viscosity probes based on squaraine, where biomolecules exhibit the least absorption
and auto-fluorescence background [8,25]. However, the small Stokes shift of cyanine
dyes can cause the self-quenching and influence the accuracy of the measurement,
due to the significant overlap of the absorption and emission spectra from the probes
[8,25,26]. Thus, it is urgent to design a red-shifted emission fluorescent probe with
large Stokes shift for the determination of intracellular viscosity.
Generally, the emission wavelengths of the dyes can be increased by introducing a
conjugated vinyl group as reported by Telore and Peng [27,28]. Also, some studies
have shown that the large Stokes shift can be usually achieved by the intramolecular
charge transfer (ICT) effect [25,29,30]. Benzothiazene is often chosen as the acceptor
(A) moiety due to its excellent ICT structures and outstanding photophysical
performances [27,31-34]. In this study, N-methyldiphenylamine was used as the
donor (D) moiety, as they have been employed as hole-transporting materials
inorganic light-emitting diodes (OLED) [35]. The acceptor and donor moieties were
connected by ethylene, which enables to extend the conjugation system, hence
releasing the longer wavelength fluorescence [36,37]. Based on this consideration, we
anticipated that the resulting dyes would simultaneously achieve large Stokes shift
and red-emission. We also postulate that the obtained samples could be used for
microviscosity detection owing to the presence of several rotatable C-C bonds
according to previous reports [21,23]. In addition, the rotation of chemical bonds
increases the flexibility of the molecular skeleton, and thus increases the Stokes shift,
which has been reported by some researchers [8,28]. In this study, two new
styrylcyanine dyes (DPA-1 and DPA-2) based on ICT principle were constructed by
the ethylene bridged N-methyldiphenylamine and benzothiazene (Scheme 1). The
relationship between the fluorescence and the viscosity was also further investigated.
2. Experimental section
2.1. Materials and instruments
All the solvents and reagents were obtained from commercial suppliers and used
directly without any further purification. All reactions were carried out with agitation
using a magnetic stirrer. The NMR spectra were recorded with a Bruker Avance 500
MHz instrument, using tetramethysilane (TMS) as an internal standard. Mass
spectrometry (MS) was performed on Q-TOF-MS instruments. UV–Vis absorption
3

spectra were recorded with an Agilent Carry 60 spectrophotometer. The target dye
molecules were dissolved in dimethyl sulfoxide (DMSO) to produce 1 mM of stock
solutions. Fluorescence spectroscopy was conducted with a F97Pro
spectrophotometer (Shanghai Lengguang Technology Co., Ltd., Shanghai, China).
The fluorescence intensity was adjusted to a suitable range by modifying the
excitation and emission slit widths. DPA-1 and DPA-2 were excited at 470 nm and
490 nm, respectively.
2.2. Synthesis of DPA-1
The thiazole-based quaternary ammonium salt 4 and other intermediates (3a and
3b) were synthesized according to published procedures [35,38,39]. Benzothiazolium
bromide salt 4 (0.64 g, 2.0 mmol) and the diformylated derivative 3a (0.06 g, 0.25
mmol) were mixed in 10 mL of anhydrous ethanol and a catalytic amount of pyridine
(0.1 ml) was added. The reaction mixture was refluxed under nitrogen for 48 h, and
then filtered in vacuo. The crude solid product was further purified by
recrystallization in ethanol. The yield was 94%.
1H NMR (500 MHz, DMSO-d6), δ (ppm): 8.44 (dd, J = 8.0, 1.4 Hz, 2H), 8.30 (d,
J = 15.5 Hz, 2H), 8.17-8.12 (m, 2H), 8.09-8.04 (m, 4H), 8.03 (d, J = 2.0 Hz, 2H), 7.80
(ddd, J = 8.5, 7.3, 1.5 Hz, 2H), 7.78-7.74 (m, 2H), 7.43-7.37 (m, 4H), 7.37-7.33 (m,
6H), 7.33 -7.28 (m, 4H), 6.25 (s, 4H), 3.52 (s, 3H). 13C NMR (126 MHz, DMSO-d6),
δ (ppm): 173.2, 151.0, 150.1, 141.7, 134.5, 132.5, 130.0, 129.6, 128.9, 128.8, 128.4,
128.2, 127.4, 125.0, 121.1, 117.2, 111.1, 51.7, 39.6. MS (ESI): m/z calcd: for
C45H37Br2N3S2 341.6212 [M -2Br]2+; found: 341.6209.
2.3. Synthesis of DPA-2
DPA-2 was prepared in a similar manner as described for DPA-1. The yield was
88%.
1H NMR (500 MHz, DMSO-d6), δ (ppm): 8.38 (dd, J = 8.1, 1.3 Hz, 1H), 8.20 (d,
J = 15.2 Hz, 1H), 8.11-8.02 (m, 1H), 7.92-7.87 (m, 2H), 7.84 (d, J = 15.3 Hz, 1H), 7.75
(ddd, J = 8.5, 7.2, 1.3 Hz, 1H), 7.70 (ddd, J = 8.2, 7.2, 1.1 Hz, 1H), 7.53-7.46 (m, 2H),
7.41- 7.36 (m, 2H), 7.36-7.29 (m, 6H), 6.87-6.82 (m, 2H), 6.17 (s, 2H), 3.41 (s, 3H).
13C NMR (126 MHz, DMSO-d6), δ (ppm): 172.8, 153.1, 151.2, 146.6, 141.7, 134.6,
133.1, 130.5, 129.7, 129.6, 128.9, 128.3, 127.8, 127.3, 126.8, 126.6, 124.7, 123.8,
116.8, 114.6, 107.9, 51.3, 40.8. MS (ESI): m/z calcd: for C29H25BrN2S 433.1733 [M -
Br]+; found: 433.1747.
4

2.4. Fluorescence quantum yield measurements
The relative fluorescence quantum yield was determined according to the
following equation [40]:
(
⁄
)(
⁄
)( ⁄ )( ⁄ )
훷x = 훷푠 퐹x 퐹s 퐴s 퐴x 휆s 휆x 푛x 푛s
Where, Φ is the quantum yield, F is the integrated area under the corrected
emission spectrum, A is absorbance at the excitation wavelength, λ is the excitation
wavelength, n denotes the refractive index of the solution (since the concentrations of
the solutions is very low, ranging from 10-7-10-8 mol/L, the refractive indices of the
solutions were replaced with those of the solvents), and the subscripts x and s refer to
the unknown and the standard, respectively. Here, rhodamine B (ΦF = 0.97 in
ethanol) was used as the reference standard.
2.5. Viscosity and fluorescence spectral measurement
The solvents were prepared by mixing deionized water-glycerol systems in
different proportions. The viscosity was recorded using a rotational viscometer (NDJ-
5S, Shanghai Hengping Scientific Instrument Co., Ltd., Shanghai, China). A 100 μL
volume of stock solution (1.0 mM in DMSO) was added to the water-glycerol mixture
(10 mL) to give a final concentration of 10.0 μM. The air bubbles in these solutions
were removed by sonicating for 30 min. The solution was kept for one hour at a
constant temperature (20℃), then the absorption and emission spectra were measured
in a UV-Vis spectrophotometer and a fluorescence spectrophotometer, respectively.
The quantitative relationship between fluorescence intensity and the viscosity of
solvent was fitted by the Fӧrster-Hoffmann equation as follows [41]:
log 푰풇 = 퐂 + 풙 log 휼
Where, If stands for the fluorescence intensity, C represents the constant related to
probe concentration and temperature, x is the probe-dependent constant and η denotes
the viscosity of the solvent.
2.6. Low temperature fluorescence spectra
The fluorescence intensity of the probes was measured in a glycerol/water (7:3,
v/v) mixture (10.0 μM) at 0, 5, 15, 20, 25, 35 and 40℃at the corresponding excitation
wavelength.
2.7. Fabrication of PMMA films
5

An 80 μL volume of stock solution was added to 400 μL of CH2Cl2 containing
14.0 mg of polymethylmethacrylate (PMMA). Then, the resulting mixture was coated
onto the surface of a thin quartz plate (2.5 cm diameter). The solvent was then
allowed to evaporate.
2.8. Computational study
All the calculations of geometry optimizations of the dyes were carried out using
density functional theory (DFT) and time-dependent density functional theory
(TDDFT) in the Gaussian 09 software package [42]. The B3LYP functional [43] and
6-31G* basis set [44] were used to calculate the geometry optimization and scan the
potential energy curves in both S0 state and S1 state. Based on the optimized
geometry, we calculated the absorption spectrum in the level of PBE0/6-311G*
[45,46] with the method of IEFPCM model in water. Additionally, the potential-
energy curves of the ground state (S0) were qualitatively scanned by constrained
optimizations, keeping the dihedral angles fixed at a series of values. The potential
energy curves of S1 were obtained in a similar manner as described for S0 using the
TDDFT method.
3. Results and Discussion
3.1. Design and synthesis of the DPA-1 and DPA-2
As mentioned above, N-methyldiphenylamine has been employed as a hole-
transporting material in OLED [35], and therefore it was chosen as the donor part in
our studies. As for acceptor unit, benzothiazene was chosen due to its electron-
deficient property, enabling to have a better effect to accept the incoming electrons. In
addition, benzothiazene tends to construct ICT structures and achieve excellent
photophysical performances [27,31,32]. Overall, this kind of donor-π-bridge-acceptor
(D-π-A) provides them with large conjugated system and strong ICT effect, and this
will be beneficial for the attainment of a large Stokes shift and a long wavelength
emission [25,33]. Moreover, the quaternized aromatic amino groups endow the probes
with good water solubility, while the benzyl group introduced further regulates lipid
solubility [3] and helps it to accumulate in mitochondria [33]. In addition, we
anticipated that DPA-1 and DPA-2 would be used for microviscosity detection due to
the presence of several rotatable C-C bonds on these two molecules [21,23].
The synthetic route was designed for our two DPA analogues in four steps，
starting from commercially available diphenylamine (1), which can be methylated to
6

obtain N-methyldiphenylamine (2) via nucleophilic substitution with iodomethane.
Then, it was formylated by the Vilsmeier-Haack reaction to obtain the required
intermediates, 3a and 3b, according to the previously reported literature. The thiazole-
based quaternary ammonium salt (4) was synthesized by mixing 2-methyl
benzothiazole and benzyl bromide in the absence of solvent at 110℃for 3 h with
excellent yield (96%). The ammonium salt 4 (1 eq.) was heated with 3a (0.125 eq.) in
the presence of a catalyzed amount of pyridine to give one of the final products, DPA-
1, by Knoevenagel condensation. The analogue DPA-2 was obtained in a similar way
by the reaction of 4 with excess of 3b (0.25 eq.). The crude products were purified by
recrystallization and characterized by 1H NMR, 13C NMR and MS.
3.2. Spectral properties of DPA-1
To characterize the spectral properties of the sensors, we first studied the solvent
dependency of DPA-1. It can be observed from data shown in Table S1 that the
maximum emission wavelength varied substantially with the polarity of the solvents
(dichloromethane, THF, acetonitrile, DMSO and water), while little change was
observed in the maximum absorption (around 542 nm), which further indicated the
occurrence of the ICT effect on DPA-1 as reported by Wang and Fan [33,47]. As
anticipated, DPA-1 exhibits an absorption maximum (λabs) at 542 nm and an emission
maximum (λem) at 653 nm (Fig. 1) in water, which are greatly red-shifted compared
with the reported rotor DCVG (λem = 502 nm). This result may be attributed to the
extended conjugation system of DPA-1. Moreover, it exhibits a notably larger Stokes
shift (111 nm) than that of DCVG (42 nm), which may be ascribed to the ICT effect
and the twisted geometry. These intriguing photophysical properties make them
suitable candidates for biological analysis. These intriguing photophysical properties
make them suitable candidates for biological analysis. It should be noted that DPA-1
shows quite low fluorescence quantum yield in these solvents possibly because the
excited dye releases substantial energy by non-radiative deactivation, and therefore
results in weak fluorescence [27,48]. In addition, the fluorescence response of DPA-1
to a narrow polarity range with different proportions of water and 1, 4-dioxane was
evaluated. The evaluation revealed that when the solvent polarity decreases by adding
1, 4-dioxane to water, DPA-1 shows small responses by a 6.7-fold fluorescence
enhancement, as shown in Fig. 2. This result indicates that polarity changes have little
interference in the fluorescence intensity of DPA-1, which is consistent with
previous report [3].
7

3.3. Fluorescence response to solvent viscosity
Unlike its lower response to polarity, DPA-1 showed significant fluorescence
enhancement upon changes of solution viscosity. The viscous media with different
viscosity could be obtained by mixing glycerol and water at different volume ratio. As
the viscosity of the solution increased from 1.01 cP in water to 234 cP in 90% of
glycerol, the fluorescence intensity of DPA-1 at 650 nm was substantially increased
by 81-fold (Fig. 3a). We consider this result to be promising because this is attributed
to viscosity-dependent rotation between N-methyldiphenylamine and benzothiazene
(Scheme 2). Such rotation is restricted in the presence of viscous solution, i.e., this
rotation is negatively correlated with viscosity. In this case, the energy of the excited
state is reserved for emission without non-radiative energy dissipation throughout the
rotation [15,23]. Meanwhile, no apparent changes were observed in absorption of
DPA-1 with the changes of solution viscosity, which indicated that the molecular
structure of the probe remained unaltered [21].
As illustrated in Fig. 3b, DPA-1 fits the Fӧrster–Hoffmann linear relationship
between the fluorescence intensity (log I650) and the viscosity (log η) (R2 = 0.998, x =
0.814). This linear correlation is crucial and provided a reliable option for DPA-1 to
be employed to detect the viscosity in various media. Also, the presence of some
additives, like cysteine (Cys), glutathione (GSH), Fe3+ and Cu2+, had no obvious
influence on the fluorescence at 650 nm for DPA-1 (Fig. 4). This better selectivity of
DPA-1 to viscosity over some other substances suggested that they had experienced
no interference in the fluorescence intensity at 650 nm as reported by Wang [23].
3.4. Low temperature fluorescence spectra
The relation between fluorescence intensity and the temperature was also
examined. As the temperature decreased from 40℃to 0℃, the fluorescence intensity
of DPA-1 at 650 nm was enhanced by 3-fold, although no wave-shift were observed
in the fluorescence spectra (Fig. 5a). This indicated that the decrease of temperature
can effectively restrain the intramolecular rotation between N-methyldiphenylamine
and benzothiazene in the DPA-1, and thus resulted in the enhancement of the
fluorescence, which is consistent with the literature [2,20]. Moreover, a good linear
relationship was observed between log (I650) and temperature (Fig. 5b), which is
expected since the viscosity is inversely proportional to the temperature, according to
“Andrade’s equation”.
8

3.5. Emission spectrum of DPA-1 in solid state
To further verify the hypothesis of free intramolecular rotation, the solid-state
emission of DPA-1 was investigated. As previously stated, the free intramolecular
rotation is suppressed with the increase of the viscosity of the solution, thus favoring
fluorescence. In contrast to the situation in 90% glycerol, DPA-1 in the PMMA film
displayed stronger fluorescence (Fig. 6). This result indicated that intramolecular
rotation cannot be completely restricted by glycerol, and on the contrary, rigid
environment of the solid state is able to restrict non-radiative deactivation induced by
free rotation of DPA-1 to a greater degree, hence releasing more intense fluorescence
[49]. Meanwhile, it is noteworthy that the maximal emission wavelength of DPA-1 in
the PMMA film is slightly blue-shifted compared with the situation in 90% glycerol,
which may be ascribed to the aggregation of the dyes molecules [49,50].
3.6. Photophysical and sensing properties of DPA-2
Similarly, the spectral properties of DPA-2 were also investigated (Fig. S2-S8
and Table S2). Compared with its DPA-1 analogue, in water, DPA-2 displayed a
blue-shifted absorption at 515 nm and an emission wavelength at 607 nm (Fig. S2).
This may be ascribed to its larger conjugate structure due to an additional
benzothiazene, which would provide a strategy for the design of new red-shifted
emission probes. A larger quantum yield (ΦF = 0.009) was observed from DPA-2
compared to that of DPA-1 (ΦF = 0.005), probably due to the less deactivation of its
pathway (Scheme 2). Furthermore, DPA-2 exhibited a lower viscosity sensitivity (64-
fold) compared to that of DPA-1 (81-fold). This phenomenon may apparently be
attributed to its larger quantum yield. The reduction of the intrinsic fluorescence
quantum yields is beneficial to improve the environmental sensitivity of the viscosity
probes, which is consistent with the literature [48].
To further investigate the corresponding photophysical and sensing properties,
we compared our probes with those of other previously reported studies as list in
Table S3. Compared with these viscosity probes, such as RY3 and dA-SQ, the
obvious advantage of our products is that they display a larger Stokes shift (> 90 nm),
which can be attributed to the ICT effect and the twisted geometry. In addition to this,
it is noteworthy that the dyes developed by us exhibited red emission and large Stokes
shift simultaneously, which may effectively avoid the influence of auto-fluorescence
and self-quenching during in vivo analysis [25]. Moreover, as can be seen from the
Table S3, the fluorescence intensity of the synthesized dyes exhibit comparable or
9

outperform the sensitivity to solution viscosity (81-fold and 64-fold), which benefits
measurement accuracy. Therefore, these intriguing properties make them suitable
candidates for measurement of intracellular viscosity.
3.7. Theoretical calculations
To gain a better insight into the molecular geometries and absorption spectra, we
performed theoretical calculations in the framework of the DFT and TDDFT methods.
The highest occupied molecular orbital (HOMO) of DPA-1 is mainly located on the
N-methyldiphenylamino group and the lowest unoccupied molecular orbital (LUMO)
on the benzothiazenyl moiety, indicating the electron density transfer from the
electron-rich N-methyldiphenylamino group to the electron-poor benzothiazenyl
moiety, as shown in Fig. 7. This is characteristic of the ICT [48]. The transition
(HOMO → LUMO) of the DPA-2 also involves an ICT process, however the charge
transfer is smaller than that of DPA-1. Moreover, according to our calculations, the
energy difference from DPA-1 (2.26 eV) is smaller than that of DPA-2 (2.60 eV),
leading to a blue-shift in the electronic spectra of DPA-2 compared to DPA-1. The
result is in line with the above experimental results of the study on absorption spectra,
which indicates that our theoretical calculations are reliable for this study.
Moreover, the mainly non-radiative deactivation of the synthesized probes was
further investigated based on the DFT/TDDFT. According to the literature
[23,48,51,52], the low quantum yield is mainly caused by the non-radiative
deactivation in the excited state, which may be ascribed to rotation of the methine
chain. The main non-radiative deactivation occurs in the single excited state with a
remarkably low energy gap to the ground state, which is formed during the rotation of
the methine chain [48,53]. As illustrated in Scheme 3, several C-C bonds are involved
in the rotation (vinyl double bond is negligible because of significant structure overlap
during the rotation and only one side of DPA-1 is considered to simplify the
computational processes). The quantum chemical calculations based on the
DFT/TDDFT could explain which chemical bond dominates the non-radiative
deactivation [23,48]. The potential energy curves of the resultant products in the S0
and S1 states with different torsion dihedral angles are qualitatively scanned (Fig. 8,
S10). The activation energy and energy gap for rotations around different C-C bonds
of the resulting dyes in the S0 and S1 states are calculated. As listed in Table 1, the
result indicated that rotation about φ1 is more difficult than that about φ2 for the DPA-
1 because of the higher rotational energy barrier in the excited state and the larger
10

energy gap to the ground state. Accordingly, the rotation of the chemical bond close
to benzothiazene (φ2) should be responsible for the mainly non-radiative deactivation
of DPA-1. However, the results of the calculation of the activation energy and energy
gap for DPA-2 suggested that the non-radiative deactivation was dominated by the
rotation of the chemical bond close to N-methyldiphenylamine (φ1). The rotation of
different chemical bonds causes mainly non-radiative deactivation for the two
analogues, which may be associated with molecular symmetry [48]. In addition,
DPA-2 showed higher intrinsic fluorescence quantum yield than DPA-1, due to a
higher rotating energy barrier. The result is consistent with the experimental results.
Of the two synthetic probes, DPA-1 demonstrated higher sensitivity (81-fold) toward
viscosity than DPA-2 (64-fold), due to its lower quantum yield.
4. Conclusions
In conclusion, we have developed two novel styrylcyanine dyes based on ICT
and confirmed their usefulness as fluorescent probes. Our synthetic dyes exhibited
larger Stokes shift derived from, the strong ICT effect and twisted geometry, and red-
emission from the extended conjugation system. The obtained samples also showed
remarkable fluorescence enhancement on increasing the environmental viscosity due
to the restrain of intramolecular rotation. Their larger Stokes shift and red-emission
give them the potential for biological analysis. Meanwhile, a linear relationship
between the fluorescence intensity and the environmental viscosity could be used for
quantitative determination of microviscosity.
Acknowledgements
This work has been supported by the National Natural Science Foundation of
China (Grant Nos. 21373042 and 21677029).
References
[1] M. K. Kuimova, S. W. Botchway, A. W. Parker, M. Balaz, H. A. Collins, H. L. Anderson, K.
Suhling, P. R. Ogilby, Imaging intracellular viscosity of a single cell during photoinduced cell
death, Nat. Chem. 1 (2009) 69-73.
[2] X. Peng, Z. Yang, J. Wang, J. Fan, Y. He, F. Song, B. Wang, S. Sun, J. Qu, J. Qi, M. Yan,
Fluorescence ratiometry and fluorescence lifetime imaging: using a single molecular sensor
for dual mode imaging of cellular viscosity, J. Am. Chem. Soc. 133 (2011) 6626-6635.
[3] N. Jiang, J. Fan, S. Zhang, T. Wu, J. Wang, P. Gao, J. Qu, F. Zhou, X. Peng, Dual mode
monitoring probe for mitochondrial viscosity in single cell, Sensors Actuators B: Chem. 190
(2014) 685-693.
[4] G. Deliconstantinos, V. Villiotou, J. C. Stavrides, Modulation of particulate nitric-oxide synthase
11

activity and peroxynitrite synthesis in cholseterol-enriched endothelial-cell membranes
Biochem. Pharmacol. 49 (1995) 1589-1600.
[5] O. Nadiv, M. Shinitzky, H. Manu, D. Hecht, C. T. Roberts, D. Leroith, Y. Zick, Elevated protein
tyrosine phosphatase activity and increased membrane viscosity are associated with impaired
activation of the insulin receptor kinase in old rats, Biochem. J. 298 (1994) 443-450.
[6] M. A. Haidekker, T. P. Brady, D. Lichlyter, E.A. Theodorakis, A ratiometric fluorescent viscosity
sensor, J. Am. Chem. Soc. 128 (2006) 398-399.
[7] M. A. Haidekker, E. A. Theodorakis, Molecular rotors--fluorescent biosensors for viscosity and
flow, Org. Biomol. Chem. 5 (2007) 1669-1678.
[8] J. Shao, S. Ji, X. Li, J. Zhao, F. Zhou, H. Guo, Thiophene-Inserted Aryl-Dicyanovinyl
Compounds: The Second Generation of Fluorescent Molecular Rotors with Significantly
Redshifted Emission and Large Stokes Shift, Eur. J. Org. Chem. 2011 (2011) 6100-6109.
[9] F. Zhou, J. Shao, Y. Yang, J. Zhao, H. Guo, X. Li, S. Ji, Z. Zhang, Molecular Rotors as
Fluorescent Viscosity Sensors: Molecular Design, Polarity Sensitivity, Dipole Moments
Changes, Screening Solvents, and Deactivation Channel of the Excited States, Eur. J. Org.
Chem. (2011) 4773-4787.
[10] G. Y. Marina K. Kuimova, James A. Levitt, and Klaus Suhling., Molecular Rotor Measures
Viscosity of Live Cells via Fluorescence Lifetime Imaging, J. Am. Chem. Soc. 130 (2008)
6672-6673.
[11] M. A. Haidekker, T. P. Brady, S.H. Chalian, W. Akers, D. Lichlyter, E. A. Theodorakis,
Hydrophilic molecular rotor derivatives - synthesis and characterization, Bioorg. Chem. 32
(2004) 274-289.
[12] K. P. Ghiggino, J. A. Hutchison, S. J. Langford, M. J. Latter, M. A. P. Lee, P.R. Lowenstern, C.
Scholes, M. Takezaki, B. E. Wilman, Porphyrin-Based Molecular Rotors as Fluorescent Probes
of Nanoscale Environments, Adv. Funct. Mater. 17 (2007) 805-813.
[13] J. Li, Y. Zhang, H. Zhang, X. Xuan, M. Xie, S. Xia, G. Qu, H. Guo, Nucleoside-Based
Ultrasensitive Fluorescent Probe for the Dual-Mode Imaging of Microviscosity in Living Cells,
Anal. Chem. 88 (2016) 5554-5560.
[14] H. Zhu, J. Fan, M. Li, J. Cao, J. Wang, X. Peng, A "distorted-BODIPY"-based fluorescent probe
for imaging of cellular viscosity in live cells, Chem. Eur. J. 20 (2014) 4691-4696.
[15] Z. Yang, Y. He, J. H. Lee, N. Park, M. Suh, W. S. Chae, J. Cao, X. Peng, H. Jung, C. Kang, J.
S. Kim, A self-calibrating bipartite viscosity sensor for mitochondria, J. Am. Chem. Soc. 135
(2013) 9181-9185.
[16] L. Wang, Y. Xiao, W. Tian, L. Deng, Activatable rotor for quantifying lysosomal viscosity in
living cells, J. Am. Chem. Soc. 135 (2013) 2903-2906.
[17] R. Weissleder, A clearer vision for in vivo imaging, Nat. Biotechnol. 19 (2001) 316-317.
[18] R. Weissleder, V. Ntziachristos, Shedding light onto live molecular targets, Nat. Med. 9 (2003)
123-128.
[19] W. Sun, S. Guo, C. Hu, J. Fan, X. Peng, Recent Development of Chemosensors Based on
Cyanine Platforms, Chem. Rev. 116 (2016) 7768-7817.
[20] M. Wang, Y. Zhang, X. Yue, S. Yao, M.V. Bondar, K.D. Belfield, A Deoxyuridine-Based Far-
Red Emitting Viscosity Sensor, Molecules 21 (2016).
[21] X. Wang, F. Song, X. Peng, Aversatile fluorescent probe for imaging viscosity and hypochlorite
in living cells, Dyes Pigm 125 (2016) 89-94.
12

[22] Y. Baek, S. J. Park, X. Zhou, G. Kim, H. M. Kim, J. Yoon, A viscosity sensitive fluorescent dye
for real-time monitoring of mitochondria transport in neurons, Biosens. Bioelectron. 86 (2016)
885-891.
[23] F. Liu, T. Wu, J. Cao, S. Cui, Z. Yang, X. Qiang, S. Sun, F. Song, J. Fan, J. Wang, X. Peng,
Ratiometric detection of viscosity using a two-photon fluorescent sensor, Chem. Eur. J. 19
(2013) 1548-1553.
[24] Y. Zhang, X. Yue, B. Kim, S. Yao, K.D. Belfield, Deoxyribonucleoside-modified squaraines as
near-IR viscosity sensors, Chem. Eur. J. 20 (2014) 7249-7253.
[25] X. J. Peng, F. L. Song, E. Lu, Y. N. Wang, W. Zhou, J. L. Fan, Y. L. Gao, Heptamethine cyanine
dyes with a large stokes shift and strong fluorescence: A paradigm for excited-state
intramolecular charge transfer, J. Am. Chem. Soc. 127 (2005) 4170-4171.
[26] L. Fan, Y. J. Fu, Q. L. Liu, D. T. Lu, C. Dong, S. M. Shuang, Novel far-visible and near-infrared
pH probes based on styrylcyanine for imaging intracellular pH in live cells, Chem. Commun.
48 (2012) 11202-11204.
[27] H. Li, Q. Yao, J. Fan, J. Du, J. Wang, X. Peng, An NIR fluorescent probe of uric HSA for renal
diseases warning, Dyes Pigm 133 (2016) 79-85.
[28] R. D. Telore, N. Sekar, Carbazole-containing push–pull chromophore with viscosity and
polarity sensitive emissions: Synthesis and photophysical properties, Dyes Pigm 129 (2016) 1-
8.
[29] Z. Liu, C. Zhang, W. He, F. Qian, X. Yang, X. Gao, Z. Guo, A charge transfer type pH
responsive fluorescent probe and its intracellular application, New J. Chem. 34 (2010) 656.
[30] A. Kovalchuk, J.L. Bricks, G. Reck, K. Rurack, B. Schulz, A. Szumna, H. Weisshoff, A charge
transfer-type fluorescent molecular sensor that "lights up" in the visible upon hydrogen bond-
assisted complexation of anions, Chem. Commun. (2004) 1946-1947.
[31] B. Zhu, F. Yuan, R. Li, Y. Li, Q. Wei, Z. Ma, B. Du, X. Zhang, A highly selective colorimetric
and ratiometric fluorescent chemodosimeter for imaging fluoride ions in living cells, Chem.
Commun. 47 (2011) 7098-7100.
[32] X. Chen, H. Li, L. Jin, B. Yin, A ratiometric fluorescent probe for palladium detection based
on an allyl carbonate group functionalized hemicyanine dye, Tetrahedron Lett. 55 (2014) 2537-
2540.
[33] N. Jiang, J. Fan, F. Xu, X. Peng, H. Mu, J. Wang, X. Xiong, Ratiometric fluorescence imaging
of cellular polarity: decrease in mitochondrial polarity in cancer cells, Angew. Chem. Int. Ed.
Engl. 54 (2015) 2510-2514.
[34] S. Tatay, P. Gavina, E. Coronado, E. Palomares, Optical mercury sensing using a
benzothiazolium hemicyanine dye, Org. Lett. 8 (2006) 3857-3860.
[35] K. C. Majumdar, B. Chattopadhyay, S. Chakravorty, N. Pal, R.K. Sinha, A new type of
symmetrical banana-shaped material based on N-methyldiphenylamine as a core moiety
exhibiting an Ad→A2 transition, Tetrahedron Lett. 49 (2008) 7149-7152.
[36] X. Zhang, L.Chi, S. Ji, Y. Wu, P. Song, K. Han, H. Guo, T. D. James, and J. Zhao, Rational
design of d-PeT phenylethynylated-carbazole monoboronic acid fluorescent sensors for the
selective detection of r-hydroxyl carboxylic acids and monosaccharides, J. Am. Chem. Soc.
131 (2009) 17452–17463.
[37] J. Y. S. Ji, Q. Yang, S. Liu, M. Chen, and J. Zhao, Tuning the intramolecular charge transfer of
alkynylpyrenes: effect on photophysical properties and its application in design of OFF-ON
13

fluorescent thiol probes, J. Org. Chem. 74 (2009) 4855-4865.
[38] A. Latrofa, M. Franco, A. Lopedota, et al., Structural modifications and antimicrobial activity
of N-cycloalkenyl-2-acylalkylidene-2,3-dihydro-1,3-benzothiazoles, Farmaco 60 (2005) 291-
297.
[39] A. C. Hernandez-Perez, S. K. Collins, A visible-light-mediated synthesis of carbazoles, Angew.
Chem. Int. Ed. Engl. 52 (2013) 12696-12700.
[40] H. H. T. Rance A. Velapoldi Corrected Emission Spectra and Quantum Yields for a Series of
Fluorescent Compounds in the Visible Spectral Region, J. Fluoresc 14 (2004) 465-472.
[41] T. Forster, G. Hoffmann, Viscosity dependence of fluorescent quantum yields of some dye
systems, Zeitschrift Fur Physikalische Chemie-Frankfurt 75 (1971) 63-&.
[42] T. G. Frisch MJ, Schlegel HB, Scuseria GE, Robb MA,Cheeseman JR, et al., Gaussian 09,
revision C.01., Wallingford CT: Gaussian, Inc (2010).
[43] A.D. Becke, Density-functional thermochemistry. III. The role of exact exchange, J. Chem.
Phys. 98 (1993) 5648-5652.
[44] W. J. H. R. Ditchfield, and J. A. Pople, Self-Consistent Molecular Orbital Methods. 9. Extended
Gaussian-type basis for molecular-orbital studies of organic molecules, J. Chem. Phys. 54
(1971) 724.
[45] K. B. J. P. Perdew, and M. Ernzerhof, Generalized gradient approximation made simple, Phys.
Rev. Lett. 77 (1996) 3865-3868.
[46] A. D. M. a. G. S. Chandler, Contracted Gaussian-basis sets for molecular calculations. 1. 2nd
row atoms, Z=11-18, J. Chem. Phys. 72 (1980) 5639-5648.
[47] Y. Zhang, K. Wang, G. Zhuang, Z. Xie, C. Zhang, F. Cao, G. Pan, H. Chen, B. Zou, Y. Ma,
Multicolored-fluorescence switching of ICT-type organic solids with clear color difference:
mechanically controlled excited state, Chem. Eur. J. 21 (2015) 2474-2479.
[48] J. Cao, T. Wu, C. Hu, T. Liu, W. Sun, J. Fan, X. Peng, The nature of the different environmental
sensitivity of symmetrical and unsymmetrical cyanine dyes: an experimental and theoretical
study, Phys. Chem. Chem. Phys. 14 (2012) 13702-13708.
[49] T. A. Oelkrug D, Gierschner J, Egelhaaf H, Hanack M, Hohloch M, et al., Tuning of
fluorescence in films and nanoparticles of oligophenylenevinylenes, J. Phys. Chem. B 102
(1998) 1902-1907.
[50]A. Mishra, R.K. Behera, P. K. Behera, B.K. Mishra, G.B. Behera, Cyanines during the 1990s: A
Review, Chem. Rev. 100 (2000) 1973-2012.
[51] P. A. Hunt, M.A. Robb, Systematic control of photochemistry: The dynamics of
photoisomerization of a model cyanine dye, J. Am. Chem. Soc. 127 (2005) 5720-5726.
[52] G. L. Silva, V. Ediz, D. Yaron, B. A. Armitage, Experimental and computational investigation
of unsymmetrical cyanine dyes: Understanding torsionally responsive fluorogenic dyes, J. Am.
Chem. Soc. 129 (2007) 5710-5718.
[53] M. Dekhtyar, W. Rettig, M. Sczepan, Small S-0-S-1 energy gaps for certain twisted
conformations of unsymmetric polymethine dyes: quantum chemical treatment and
spectroscopic manifestations, Phys. Chem. Chem. Phys. 2 (2000) 1129-1136.
Figure Caption
14

Styrylcyanine-based fluorescent probes with red-emission
and large Stokes shift for the detection of viscosity
Scheme 1. Synthetic routes for DPA-1 and DPA-2.
15

Fig. 1. Normalized absorption and emission spectra of DPA-1 in water (containing 1%
DMSO).
Fig. 2. The emission spectra of DPA-1 (10 μM) in different solution. The solid lines represent
the probe in different proportional water and 1, 4-dioxane mixtures; the dash curve represents
DPA-1 in water-glycerol (10:90, v/v) under the same condition.
16

Scheme 2. Hypothetical rotation of the probes in low viscosity media.
Fig. 3. (a) Fluorescence emission spectra of DPA-1 (10 μM) in water and glycerol mixtures
with different viscosity. Inset: the photographs of DPA-1 in water (left) and 90% glycerol
(right) under a 365 nm UV lamp.(b) The linear relationship between log I650 and log η.
Fig. 4. The fluorescence spectra titration of DPA-1 (10 μM) in water by GSH (1 mM); Cys (1
mM); Fe3+(0.1 mM); Cu2+ (0.1 mM). Luminescence measurements were carried out three
minutes after the addition of analytes at room temperature.
17

Fig. 5. (a)The fluorescence spectra of DPA-1 (10 μM) at different temperatures in 70%
glycerol. (b) Dependence between log I650 and temperature.
Fig. 6. (a) Fluorescence spectra of DPA-1 in different states. The black color represents DPA-
1 in PMMA film; the red color in the figure is the dye in 90% glycerol (10 μM).
18

Fig. 7. Frontier molecular orbital profiles after geometry optimization of DPA-1 and DPA-2,
calculated at the DFT/B3-LYP/6-31G* level. Absorption spectra were studied at the
TDDFT/PBE0/6-311G* level in water with IEFPCM model.
Scheme 3. Definition of angles, φ1 and φ2, used to describe the rotation about different C-C
bonds. φ1 represents the dihedral angle of C1-C2-C3-C4 and φ2 represents the dihedral angle
of C3-C4-C5-S1.
19

Fig. 8. Potential-energy curves of DPA-1 at S0 (black) and S1 states (red) rotation along with
chemical bond φ1 (a) and chemical bond φ2 (b), calculated at the TDDFT/B3-LYP/6-31G*
level.
IC: internal conversion.
20

Styrylcyanine-based fluorescent probes with red-emission
and large Stokes shift for the detection of viscosity
Table 1 Activation energies of S1 and energy gaps between the S0 and S1 states rotation
around different chemical bands (φ1 and φ2) for DPA-1 and DPA-2.
DPA-1
Egap(kcal mol-1)
DPA-2
Egap(kcal mol-1)
Chemical bands
Ea (kcal mol-1)
7.26
Ea(kcal mol-1)
15.67
φ1
φ2
34.01
28.19
17.22
17.88
5.07
18.11
21