Solvent-dependent ratiometric fluorescent merocyanine dyes: Spectral properties, interaction with BSA as well as biological applications

Article abstract of DOI:10.1016/j.dyepig.2016.02.020

A series of merocyanine dyes with excellent spectral properties were synthesized through changing the heterocyclic base structure for protein labeling and imaging in biological science, such as living cells and living mice. Dyes 7c-7h showed solvent-dependent changes on emission wavelength and fluorescence intensity in water and hydrophobic solvents, which were further analyzed by theoretical calculations and absorption spectra. The interaction of dye 7d with bovine serum albumin (BSA) showed an obvious ratiometric fluorescence response to solvent/protein environment, with the potential to report protein conformational changes. Investigation of the cytotoxicity and bioimaging capability displayed that dyes 7b-7h showed low cytotoxicity, and could stain living cell cytoplasm and be used for imaging in living mice with bright fluorescence at the application dose, which was suggested as fluorescent reagents for imaging in biological science.

Full text of DOI:10.1016/j.dyepig.2016.02.020

Dyes and Pigments 129 (2016) 163e173
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Solvent-dependent ratiometric fluorescent merocyanine dyes:
Spectral properties, interaction with BSA as well as biological
applications
,
*
Dengfeng Gao a, Anyang Li a, Li Guan a, Xianghan Zhang b, L.Y. Wang a
a Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, Ministry of Education, College of Chemistry and Materials Science, Northwest
University, Xi'an 710127, PR China
b School of Life Sciences and Technology, Xidian University, Xi'an 710071, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of merocyanine dyes with excellent spectral properties were synthesized through changing the
heterocyclic base structure for protein labeling and imaging in biological science, such as living cells and
living mice. Dyes 7ce7h showed solvent-dependent changes on emission wavelength and fluorescence
intensity in water and hydrophobic solvents, which were further analyzed by theoretical calculations and
absorption spectra. The interaction of dye 7d with bovine serum albumin (BSA) showed an obvious
ratiometric fluorescence response to solvent/protein environment, with the potential to report protein
conformational changes. Investigation of the cytotoxicity and bioimaging capability displayed that dyes
7be7h showed low cytotoxicity, and could stain living cell cytoplasm and be used for imaging in living
mice with bright fluorescence at the application dose, which was suggested as fluorescent reagents for
imaging in biological science.
Received 30 December 2015
Received in revised form
20 February 2016
Accepted 23 February 2016
Available online 27 February 2016
Keywords:
Merocyanine dyes
Spectral properties
BSA
© 2016 Elsevier Ltd. All rights reserved.
Biological applications
1. Introduction
at much below 400 nm, which leads to fast cell damage and pro-
duces high background from cellular autofluorescence when irra-
Cyanine dyes have been the object of continuous interest in the
scientific community due to their easy synthetic methods, tunable
absorption/emission spectra, high molar absorptivity and
moderate-to-high fluorescence quantum yield [1e5]. Instead of
two nitrogens for cyanines, merocyanine dyes consist of a poly-
methine chain connecting a nitrogen and an oxygen atom, where a
nitrogen atom is part of a donor and a carbonyl group as acceptor
[6,7]. Their unique structure framework has attracted much
attention in physics, medicine and biology [8e13], such as used as
nonlinear optical materials, photosensitizers in the photodynamic
treatment of cancer, radiosensitizers in solid tumor treatment,
diagnostic agents in medicine and potentiometric sensors. In recent
years, it has been increasing attention for merocyanine as protein
labeling fluorescent dye to study protein conformational changes,
ligand binding, or posttranslational modifications [14]. Dyes typi-
cally used as protein activity studies, including Dansyl, Aedans,
Adman etc., have several disadvantages, for example, they fluoresce
diating cells with near-UV light [14,15]. Usually dyes used as protein
activity studies not only have appropriate photophysical properties,
but also must maintain sufficient hydrophobic character to interact
with proteins, are water soluble and do not aggregate at concen-
trations appropriate for protein labeling [14]. Based on our previous
work [16,17], in terms of designing the new dyes, we focused on
merocyanine dyes with a rhodanine nucleus because the rhodanine
scaffold is a central part of biologically active compounds and
synthetic building blocks, and has good water solubility [18,19]. In
this work, we adjusted the properties of merocyanine dyes in water
and in hydrophobic solvents with heterocyclic base structure, and
prepared a series of merocyanine dyes. As expected, prepared dyes
fluoresced brightly at long wavelengths and showed a strong
ratiometric fluorescence response to solvent/protein environment,
with the potential as fluorescent probes to report protein confor-
mational changes. And the investigation of cytotoxicity and bio-
imaging capability in cells and mice showed that the new dyes
were of low cell cytotoxicity, could stain the cell cytoplasm and
exhibited bright fluorescence at 10 mM dose, and could be applied
in mice imaging, suggesting as potential fluorescent probes to
detect cell movement and cell positioning.
* Corresponding author.
E-mail address: wanglany@nwu.edu.cn (L.Y. Wang).
http://dx.doi.org/10.1016/j.dyepig.2016.02.020
0143-7208/© 2016 Elsevier Ltd. All rights reserved.

164
D. Gao et al. / Dyes and Pigments 129 (2016) 163e173
2. Experimental section
Table 1
Representative hydrogen bonds for dyes 7c, 7d, 7f, 7g and 7h.
2.1. Apparatus reagents and chemicals
dyes DeH/A
d(DeH)/Å d(H/A)/Å d(D/A)/Å :(DHA)/ꢀ
7c
7d
7f
C(8)eH(8)/O(1)
0.960
C(10)eH(10)/C(8) 0.960
C(21)eH(21)/O(1) 0.970
2.493
2.398
2.475
2.762
2.638
3.356
2.061
2.633
2.622
2.698
3.117
3.160
3.423
3.614
3.309
3.452
3.236
3.225
3.417
3.622
122.57
136.00
165.52
152.67
129.52
90.13
120.68
122.14
140.33
172.21
NMR spectra were recorded on a Varian INOVA-400 MHz
spectrometer (at 400 MHz for 1H NMR and 100 MHz for 13C NMR)
with tetramethylsilane (TMS) as internal standard. HRMS was
recorded on a microTOFQ II ESI-Q-TOF LC/MS/spectrometer. Single
crystal X-ray diffraction data were collected on a Bruker SMART
APEX II CCD X-ray crystallography. The absorption spectra were
recorded on a Purkinje General UV-1900 UVevis spectrometer. The
fluorescence spectra were measured on a HITACHI F-4500 fluo-
rescence spectrophotometer. Melting points were measured using
a XT-4 micromelting apparatus and were used uncorrected. Bio-
imaging of the dyes was performed on an Olympus FV1000
confocal microscopy. Imaging of living mice was conducted
employing Xenogen IVIS spectrum imaging system.
C(19)eH(19)/S(2)
C(18)eH(18)/O(1) 0.931
C(22)eH(22)/S(1) 0.960
C(16)eH(16)/O(1) 0.930
C(17)eH(17)/O(1) 0.930
C(17)eH(17)/O(1) 0.960
0.930
7g
7h
C(2) eH(2)/O(1)
0.930
heated at 70 ꢀC for 1 h and then cooled to room temperature. The
resulting precipitates were filtered and washed with acetone to
give 3-methyl-5-phenylaminomethylene rhodanine (2a). And the
mixture of crude product 2a (2.50 g, 10.0 mmol) dissolved in acetic
anhydride (8 mL) and triethylamine (0.16 mL) was stirred at 110 ꢀC
for 0.5 h, and concentrated to about one-half volume under
reduced pressure, and then stirred in methanol at room tempera-
ture for 1 h. The resulting precipitates were filtered and washed
with methanol to give 3a (2.74 g, 94%) as yellow crystals, m.p.
207e208 ꢀC (lit, m.p. 207e209 ꢀC [22]). Compound 3b (2.76 g, 90%)
was prepared with the similar synthetic method of 3a,
m.p.179e180 ꢀC (lit, m.p. 177e178 ꢀC [24]).
The solvents and chemicals were commercially available and
were used without further purification unless otherwise noted.
2.2. Preparation of the intermediates
The heterocyclic compounds 4, 5 and 6aec were prepared ac-
cording to the literature [20,21]. Compounds 3a-b were synthe-
sized according to a modified literature procedure [22,23]. A
mixture of 3-methyl rhodanine (1a; 4.41 g, 30.0 mmol) and N,N0-
diphenylformamidine (5.88 g, 30.0 mmol) in acetonitrile was
Fig. 1. X-ray crystal structures of dyes 7c (1), 7d (2), 7f (3), 7g (4) and 7h (5).

D. Gao et al. / Dyes and Pigments 129 (2016) 163e173
165
H
11.52 (s, 1H, -NH), 8.22 (d, J ¼ 7.2 Hz, 1H, ArH); 8.11 (d, J ¼ 13.2 Hz,
1H, eCH ¼ ), 7.96 (d, J ¼ 8.0 Hz, 1H, ArH), 7.77e7.65 (m, 1H, ArH),
7.55e7.37 (m, 2H, ArH), 6.96 (d, 1H, J ¼ 5.2 Hz, ArH), 6.32 (d, 1H,
J ¼ 13.2 Hz, eCH ¼ ), 4.04 (q, 2H, J ¼ 6.4 Hz, eCH2-), 1.18 (t, 3H,
J ¼ 6.4 Hz, eCH3). 13C NMR (100 MHz, CDCl3) 193.97, 169.94, 169.19,
168.26, 140.35, 131.20, 130.72, 129.40, 128.54, 127.37, 121.81, 111.14,
109.66, 109.43, 106.39, 92.78, 39.54, 27.97. HRMS (TOF MS ESI)
calculated for C18H15N2OSþ2 : 339.0626; found: 339.0644.
Ac
N
H
S
Ph
N C N Ph
S
S
S
S
N
Ac2O
Et3N
H
Ph
Ph
N
S
O
N
N
CH3CN
R2
R2
R2
O
O
2a R2=Me
2b R2=Et
1a R2=Me
1b R2=Et
3a R2=Me
3b R2=Et
CH3
3b
O
N
N
EtOH, Ac2O, Et3N
N
I
S
S
CH3
S
S
2.3.3. 3-Methyl-5-[2-(1,3,3-trimethyl-4,5-benzoindoles-2-ylidene)-
ethylidene]-rhodanine (7c)
7a
7b
4
5
yield 85%. m.p. 193e195 ꢀC. UVevis (MeOH) lmax: 516.5 nm, ε:
3b
1.21 ꢁ 105 L molꢂ1 cmꢂ1. 1H NMR (400 MHz, CDCl3).
d (ppm): 8.05
ClO4
CH3
HN
HN
EtOH, Ac2O, Et3N
(d, J ¼ 13.2 Hz, 1H, eCH ¼ ), 8.04 (d, J ¼ 8.8 Hz, 1H, ArH), 7.9e7.82
(m, 2H, ArH), 7.6e7.5 (m, 1H, ArH), 7.41e7.32 (m, 1H, ArH), 7.20 (d,
1H, J ¼ 8.8 Hz, ArH), 5.20 (d, 1H, J ¼ 13.2 Hz, eCH ¼ ), 3.50 (s, 3H,
eCH3), 3.42 (s, 3H, eCH3), 1.96 (s, 6H, eCH3). 13C NMR (100 MHz,
N
O
O
R2
S
N
3a-b
CDCl3)
d (ppm):190.71, 168.86, 165.88, 139.64, 130.74, 129.79,
S
129.76, 129.01, 128.92, 127.47, 126.35, 122.72, 120.74, 110.50, 108.39,
91.76, 48.49, 30.16, 29.08, 26.90. HRMS (TOF MS ESI) calculated for
CH3
PF6
EtOH, Ac2O, Et3N
N
R1
N
R1
C
21H21N2OSþ2 : 381.1095; found: 381.1098.
6a R1=Me
7c R1=Me, R2=Me
6b R1=n-propyl
6c R1=n-butyl
7d R1=n-propyl, R2=Me
7e R1=n-butyl, R2=Me
2.3.4. 3-Methyl-5-[2-(1-n-propyl-3,3-dimethyl-4,5-benzoindoles-
2-ylidene) ethylidene]-rhodanine (7d)
yield 90%. m.p. 170e171 ꢀC. UVevis (MeOH) lmax: 520.5 nm, ε:
7f R1=Me, R2=Et
1.29 ꢁ 105 L molꢂ1 cmꢂ1. 1H NMR (400 MHz, CDCl3).
d (ppm): 8.05
7g R1=n-propyl, R2=Et
7h R1=n-butyl, R2=Et
(d, J ¼ 13.2 Hz,1H, eCH ¼ ), 8.05e8.00 (m,1H, ArH), 7.87 (d, J ¼ 8 Hz,
1H, ArH), 7.83 (d, J ¼ 8.8 Hz, 1H, ArH), 7.58e7.50 (m, 1H, ArH),
7.42e7.33 (m, 1H, ArH), 7.18 (d, 1H, J ¼ 8.4 Hz, ArH), 5.25 (d, 1H,
J ¼ 13.2 Hz, eCH ¼ ), 3.83 (t, 2H, J ¼ 7.2 Hz, eCH2-), 3.50 (s, 3H,
eCH3), 1.96 (s, 6H, eCH3), 1.90e1.78 (m, 2H, eCH2-), 1.05 (t,
Scheme 1. Synthesis of dyes 7ae7h.
J ¼ 7.6 Hz, 3H, eCH3). 13C NMR (100 MHz, CDCl3)
d (ppm): 191.66,
2.3. Synthesis of dyes 7ae7h
169.46,166.85,140.42,131.99,130.94,130.82,130.02,129.93,128.54,
127.38, 123.80, 121.80, 111.06, 109.72, 92.80, 49.67, 44.71, 31.19,
27.99, 20.28, 11.62. HRMS (TOF MS ESI) calculated for C23H25N2OSþ2 :
409.1408; found: 409.1409.
A mixture of 4 (0.29 g, 1.00 mmol), 3b (0.31 g, 1.00 mmol) and
acetic anhydride (0.14 g, 1.37 mmol) in 15 mL ethanol was heated at
50 ꢀC for 1 h with vigorous stirring, then triethylamine (0.38 g,
3.76 mmol) was added and was stirred at 60 ꢀC for 3 h. The formed
precipitates were filtered and washed with ethanol to give 7a
(0.30 g) as dark green solid.
The same procedure described above but using compound 5
(0.27 g, 1.00 mmol) and 3b (0.31 g, 1.00 mmol) stirring at 70 ꢀC for
5 h gave crude product, which was recrystallised from ethanol to
give 7b (0.27 g) as violet black solid. And using compound 6a, 6b or
6c (1.00 mmol) and 3a or 3b (1.00 mmol) stirring at 60 ꢀC for 4 h
gave pure red dyes 7c (0.32 g), 7d (0.37 g), 7e (0.38 g), 7f (0.36 g), 7g
(0.35 g) and 7h (0.38 g), respectively.
2.3.5. 3-Methyl-5-[2-(1-n-butyl-3,3-dimethyl-4,5-benzoindoles-2-
ylidene)-ethylidene]-rhodanine (7e)
yield 89%. m.p.177e178 ꢀC. UVevis (MeOH) lmax: 521.0 nm, ε:
1.19 ꢁ 105 L molꢂ1 cmꢂ1. 1H NMR (400 MHz, CDCl3).
d (ppm): 8.05
(d, J ¼ 13.2 Hz, 1H, eCH ¼ ) 8.05e8.02 (m, 1H, ArH), 7.87 (d, J ¼ 8 Hz,
1H, ArH), 7.84 (d, J ¼ 8.8 Hz, 1H, ArH), 7.58e7.51 (m, 1H, ArH),
7.40e7.34 (m, 1H, ArH), 7.18 (d, J ¼ 8.8 Hz, 1H, ArH), 5.26 (d,
J ¼ 13.2 Hz, 1H, eCH ¼ ), 3.85 (t, J ¼ 7.2 Hz, 2H, eCH2-), 3.50 (s, 3H,
eCH3), 1.95 (s, 6H, eCH3), 1.72e1.81 (m, 2H, eCH2-), 1.51e1.43 (m,
2H, eCH2-), 1.03 (t, J ¼ 7.2 Hz, 3H, eCH3). 13C NMR (100 MHz, CDCl3)
d
(ppm): 191.67, 169.34, 166.84, 140.32, 131.98, 130.99, 130.81,
2.3.1. 3-Ethyl-5-[2-(1-methyl-quinoline-4-ylidene)-ethylidene]-
rhodanine (7a)
130.03, 129.93, 128.55, 127.38, 123.81, 121.80, 111.06, 109.67, 92.75,
49.67, 43.00, 31.21, 28.87, 27.96, 20.36, 13.89. HRMS (TOF MS ESI)
calculated for C24H27N2OSþ2 : 423.1565; found: 423.1546.
yield 91%. m.p. 259e260 ꢀC. UVevis (MeOH) lmax: 610.0 nm, ε:
1.28 ꢁ 105 L molꢂ1 cmꢂ1. 1H NMR (400 MHz, CDCl3).
d (ppm): 8.02
(d, J ¼ 6.8 Hz, 1H, ArH); 7.93 (d, J ¼ 12.8 Hz, 1H, eCH ¼ ), 7.58 (d,
J ¼ 8.0 Hz, 1H, ArH), 7.43e7.32 (m, 2H, ArH), 7.02 (d, J ¼ 6.8 Hz, 1H,
ArH), 6.74 (d, 1H, J ¼ 6.8 Hz, ArH), 6.06 (d, 1H, J ¼ 12.8 Hz, eCH ¼ ),
4.17 (q, 2H, J ¼ 6.0 Hz, eCH2-), 3.67 (s, 3H, eCH3), 1.28 (t, 3H,
2.3.6. 3-Ethyl-5-[2-(1,3,3-trimethyl-4,5-benzoindoles-2-ylidene)-
ethylidene]-rhodanine (7f)
yield 92%. m.p. 175e176 ꢀC. UVevis (MeOH) lmax: 517.5 nm, ε:
1.18 ꢁ 105 L molꢂ1 cmꢂ1. 1H NMR (400 MHz, CDCl3).
d (ppm): 8.05
J ¼ 6.0 Hz, eCH3). 13C NMR (100 MHz, CDCl3)
d 190.81, 166.68,
(d, J ¼ 7.6 Hz, 1H, ArH), 8.02 (d, J ¼ 13.2 Hz, 1H, eCH ¼ ); 7.87 (d,
J ¼ 9.2 Hz, 1H,ArH), 7.84 (d, J ¼ 8.8 Hz, 1H, ArH), 7.58e7.49 (m, 1H,
ArH), 7.41e7.34 (m, 1H, ArH), 7.20 (d, J ¼ 8.8 Hz, 1H, ArH), 5.19 (d,
J ¼ 13.2 Hz, 1H, eCH ¼ ), 4.19 (q, J ¼ 7.2 Hz, 2H, eCH2-), 3.42 (s, 3H,
eCH3), 1.96 (s, 6H, eCH3), 1.29 (t, J ¼ 7.2 Hz, 3H, eCH3). 13C NMR
150.43, 140.56, 132.90, 131.86, 131.44, 128.71, 123.65, 123.34, 119.23,
114.28, 109.65, 99.99, 39.58, 35.18, 12.37. HRMS (TOF MS ESI)
calculated for C17H17N2OSþ2 : 329.0782; found: 329.0784.
2.3.2. 3-Ethyl-5-[2-benzo[c,d]indole-2-ylidene)-ethylidene]-
rhodanine (7b)
(100 MHz, CDCl3) d (ppm): 191.37, 169.72, 166.72, 140.72, 131.48,
130.81, 130.75, 130.04, 129.97, 128.52, 127.38, 123.74, 121.79, 111.69,
109.43, 92.82, 49.50, 39.60, 30.12, 27.96, 12.38. HRMS (TOF MS ESI)
calculated for C22H23N2OSþ2 : 395.1252; found: 395.1247.
yield 79%. m.p. 239e240 ꢀC. UVevis (MeOH) lmax: 568.5 nm, ε:
4.50 ꢁ 104 L molꢂ1 cmꢂ1. 1H NMR (400 MHz, DMSO-d6).
d (ppm):

166
D. Gao et al. / Dyes and Pigments 129 (2016) 163e173
Table 2
The absorption and fluorescence spectral characteristics of dyes 7ae7h in different solvents.
Solvents
DMSO
Dyes
lmax/nm
lem/nm
Stockes shift/nm
ε ꢁ 10ꢂ4/L molꢂ1 cmꢂ1
Fluorescence intensity
7a
7b
7c
7d
7e
7f
7g
7h
7a
7b
7c
7d
7e
7f
7g
7h
7a
7b
7c
7d
7e
7f
7g
7h
7a
7b
7c
7d
7e
7f
620.5
570.0
528.5
530.0
530.0
529.5
531.0
531.0
610.0
568.5
516.5
520.5
521.0
517.5
521.5
522.0
617.5
571.0
517.0
521.5
522.5
519.0
521.0
522.5
461.0
538.0
558.0
539.0
559.0
569.0
535.5
532.5
e
e
16.98
3.16
e
583.0
567.0
568.0
568.6
568.8
568.2
568.4
e
53.0
67.0
68.0
68.6
68.8
68.2
68.4
e
685.0
2184.3
2046.1
2234.8
2257.0
1924.2
2009.7
e
13.02
10.99
12.73
12.15
10.10
10.79
12.81
4.50
12.10
12.90
11.90
11.75
9.62
10.74
15.43
4.75
12.29
10.56
12.55
11.41
9.60
MeOH
EtOH
H2O
593.2
563.4
565.2
565.6
563.8
566.2
565.2
e
63.2
63.4
65.2
65.6
63.8
66.2
65.2
e
230.3
945.5
891.6
829.2
873.6
846.9
993.7
e
594.4
561.8
563.4
562.6
562.2
563.4
564.8
e
64.0
61.8
63.4
62.6
62.2
63.4
64.8
e
401.6
975.8
1206.7
1082.0
1032.3
1075.8
1223.6
e
9.38
3.90
2.15
2.50
4.71
5.20
1.18
4.15
619.0
573.2
625.4
583.4
586.8
576.4
575.2
89.0
73.2
125.4
83.4
86.8
76.4
75.2
139.3
223.5
309.2
289.8
278.0
229.2
256.8
7g
7h
4.77
The lex for 7b was 530 nm and 7ce7h was 500 nm.
2.3.7. 3-Ethyl-5-[2-(1-n-propyl-3,3-dimethyl-4,5-benzoindoles-2-
ylidene)-ethylidene]-rhodanine (7g)
2.4. Sample preparation
yield 83%. m.p. 166e167 ꢀC. UVevis (MeOH) lmax: 521.5 nm, ε:
2.4.1. Sample preparation of dyes in different solvents
The stock solutions of dyes (1 ꢁ 10ꢂ3 mol Lꢂ1) in DMSO were
diluted to the corresponding concentration (1 ꢁ 10ꢂ5 mol Lꢂ1) with
different solvents (DMSO, methanol, ethanol, and water). The
UVevis and fluorescence spectra of these solutions were recorded
at room temperature.
9.62 ꢁ 104 L molꢂ1 cmꢂ1. 1H NMR (400 MHz, CDCl3).
d (ppm): 8.05
(d, J ¼ 7.2 Hz, 1H, ArH), 8.02 (d, J ¼ 13.6 Hz, 1H, eCH ¼ ); 7.87 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.83 (d, J ¼ 8.8 Hz, 1H, ArH), 7.58e7.50 (m, 1H,
ArH), 7.42e7.33 (m, 1H, ArH), 7.18 (d, J ¼ 8.8 Hz, 1H, ArH), 5.24 (d,
J ¼ 13.6 Hz, 1H, eCH ¼ ), 4.19 (q, J ¼ 7.2 Hz, 2H, eCH2-), 3.83 (t,
J ¼ 7.2 Hz, 2H, eCH2-), 1.96 (s, 6H, eCH3), 1.83 (m, 2H, eCH2-), 1.29
(t, J ¼ 6.8 Hz, 3H, eCH3), 1.04 (t, J ¼ 7.6 Hz, 3H, eCH3). 13C NMR
2.4.2. Sample preparation of dyes in the presence of BSA
The stock solution (1 ꢁ 10ꢂ3 mol Lꢂ1) of dye was obtained by
dissolving the dye in DMSO and further diluted with TE buffer
containing 10 mmol Lꢂ1 TriseHCl, 1 mmol Lꢂ1 EDTA at pH 7.5. The
(100 MHz, CDCl3)
d (ppm): 191.27, 169.27, 166.64, 140.46, 131.66,
130.88, 130.78, 130.00, 129.92, 128.56, 127.36, 123.76, 121.80, 111.23,
109.70, 92.82, 49.62, 44.68, 39.58, 28.01, 20.27, 12.37, 11.62.HRMS
C
24H27N2OSþ2 : 423.1565; found:
stock solution (200 m
g mLꢂ1) of BSA (bovine serum albumin) was
(TOF MS ESI) calculated for
423.1575.
prepared by dissolving BSA in TE buffer. Before spectroscopic
measurements, the working solutions of mixed BSA-dye were
freshly prepared by diluting the high concentration stock solution
with TE buffer to corresponding concentrations (BSA: 0, 10, 20, 30,
40, 50, 60, 70, 80 and 90
working solutions were prepared immediately before the experi-
ment. Measurements method was mentioned as above.
2.3.8. 3-Ethyl-5-[2-(1-n-butyl-3,3-dimethyl-4,5-benzoindoles-2-
ylidene)-ethylidene]-rhodanine (7h)
m
g mLꢂ1, dye: 1 ꢁ 10ꢂ5 mol Lꢂ1). All
yield 88%. m.p. 181e182 ꢀC. UVevis (MeOH) lmax: 522.0 nm, ε:
1.07 ꢁ 105 L molꢂ1 cmꢂ1. 1H NMR (400 MHz, CDCl3).
d (ppm): 8.05
(d, J ¼ 6.4 Hz, 1H, ArH), 8.03 (d, J ¼ 13.2 Hz, 1H, eCH ¼ ), 7.87 (d,
J ¼ 8.0 Hz, 1H, ArH), 7.84 (d, J ¼ 8.8 Hz, 1H, ArH), 7.59e7.49 (m, 1H,
ArH), 7.42e7.33 (m, 1H, ArH), 7.17 (d, J ¼ 8.8 Hz, 1H, ArH), 5.24 (d,
J ¼ 13.2 Hz, 1H, eCH ¼ ), 4.19 (q, J ¼ 6.8 Hz, 2H, eCH2-), 3.85 (t,
J ¼ 7.6 Hz, 2H, eCH2-), 1.96 (s, 6H, eCH3), 1.83e1.71 (m, 2H, eCH2-),
1.54e1.40 (m, 2H, eCH2-), 1.29 (t, J ¼ 6.8 Hz, 3H, eCH3), 1.03 (t,
2.5. Determination of cytotoxicity and cell staining
2.5.1. Cytotoxicity
MTT assay was performed to evaluate the toxicity of dyes 7ae7h
to mouse fibroblasts cells (L929) and human lung adenocarcinoma
cells (A549). The cells were seeded into 96-well plates at cell
J ¼ 7.6 Hz, 3H, eCH3). 13C NMR (100 MHz, CDCl3)
d (ppm): 191.26,
169.17, 166.62, 140.36, 131.65, 130.93, 130.79, 130.01, 129.93, 128.56,
127.36, 123.77, 121.80, 111.22, 109.66, 92.77, 49.62, 42.97, 39.57,
28.87, 27.98, 20.36, 13.90, 12.37.HRMS (TOF MS ESI) calculated for
density of 1.0 ꢁ 104 cells/well in 180
mL of culture medium and
maintained for 24 h. Then solutions of dyes 7ae7h with different
concentrations were added to 96-well plates, respectively, and
incubated for another 48 h. And three wells containing only cells
C
25H29N2OSþ2 : 437.1721 found: 437.1737.

D. Gao et al. / Dyes and Pigments 129 (2016) 163e173
167
Fig. 2. Absorption spectra of dyes 7a (a), 7b (b) in water (AH ¼ absorbance of aggre-
gation; AM ¼ absorbance of monomer). Inset: fluorescence spectra of dyes 7a (a), 7b (b)
in water, lex ¼ 530 nm.
Fig. 3. Absorption (a) and fluorescence (b) spectra of dye 7d in different solvents.
Inset: fluorescence spectra of dyes 7d in water, lex ¼ 500 nm.
10 min. Prior to imaging, the cells were washed three times with
PBS, and the fluorescence images were acquired through a confocal
laser scanning microscopy.
were suspended in a mixture solution of 198
mL complete medium
and 2 L DMSO. They were used as the control for investigating cell
m
viability. Three wells containing only the complete medium were
used as the blank control. Following that, the medium was removed
and washed three times with PBS. Finally, 180
medium and 20 L MTT solution (5 mg/mL) were added to each
well. After incubation for 4 h, the supernatant was removed and
200 L DMSO was added to each well. The trays were then vigor-
ously shaken to solubilize the formazan product and the absor-
bance at a wavelength of 490 nm was read on Microplate Reader
and analyzed. All experiments were conducted in triplicate. Data
were expressed as mean SD.
mL of fresh culture
2.6. Fluorescence imaging in living mice
m
Kunming mice (20e25 g) were given an intraperitoneal (i.p.)
m
injection of dye (400
m
L ꢁ 400
mM in saline, containing 1% DMSO).
The mice were anesthetized by inhalation of isoflurane. As a con-
trol, untreated mice were also prepared. The mice were then
imaged (20 min after the injection of the dye) by using a Xenogen
IVIS Spectrum Imaging System.
2.5.2. Fluorescence imaging in cells
3. Results and discussion
L929 cells and A549 cells were seeded into culture plates at a
density of 1 ꢁ 104 cells per plate in DMEM complete medium for
12 h. Then we chose dye 7d to study the effect of different con-
centrations on the L929 cell staining. The cells were incubated with
required concentrations of dye 7d (0.625, 1.25, 2.5, 5.0, 10.0,
3.1. Synthesis and characterization
The dyes 7aeh were synthesized by the reaction of the qua-
ternary ammonium salts having an active methyl 4, 5 or 6aec with
intermediates 3a-b, which were easily obtained by the reaction of
1a or 1b with N,N0-diphenylformamidine and then with acetic
anhydride. The formation reaction of dyes 7aeb proceeded
20.0 m
M) for 1 h at 37 ꢀC in humidified air and 5% CO2. For other
dyes, the concentration in working solutions was 10
cells were further incubated with Hoechst 33342 (5
m
m
M. Then the
g mLꢂ1) for

168
D. Gao et al. / Dyes and Pigments 129 (2016) 163e173
Table 3
Main orbital transitions and lem of dye 7d calculated by TD-B3LYP/6-31G*.
Solvent
DMSO
Transition charactera
D
E/eV
fb
lem (nm) theoretical
lem (nm)c experimental
LUMO/HOMO (0.698)
LUMO/HOMO-1(0.119)
LUMO/HOMO (0.696)
LUMO/HOMO-1 (0.133)
2.35
3.21
2.31
3.15
0.551
567
568
Water
0.533
579
618
a
The proportion of the main transition is given in parentheses.
Oscillator strength.
lex ¼ 500 nm.
b
c
Fig. 4. Molecular orbitals of dye 7d in DMSO (left) and water (right). The unit of energy is eV.
efficiently at 60e70ꢀC for 3e5 h in ethanol and afforded high yields
of 79e91% after recrystallization from ethanol, and the formation
reaction at 60 ꢀC for 4 h in ethanol gave pure 7ceh in 83e92%
yields.
their structures were confirmed by 1H NMR, 13C NMR and HRMS. 1H
NMR spectra of dyes 7ae7h showed the chemical shift of H
in ¼ CHeCH ¼ (5.19e6.32 ppm and 7.93e8.11 ppm) and the
coupling constants (3JHH) 12.8e13.6 Hz. And the chemical shift of H
on NH (11.52 ppm) in benzo[c,d]indole, C(CH3)2 (1.96 ppm) in
benzoindoline, NCH3 (3.42e3.50 ppm) and NCH2CH3 (4.04e4.19
and 1.03e1.29 ppm) in rhodanine ring was all displayed. 13C NMR
spectra of dyes 7ae7h showed the chemical shift of C in C]O
The purity of prepared dyes 7ae7h was established by TLC, and
(190.71e193.97
(150.43e168.26
ppm),
C]S
and
ppm),
¼CHeCH ¼ (130.74e132.90
91.76e96.44 ppm), C (26.90e27.99 ppm) of geminal dimethyl and C
(108.39e109.72 ppm) in aromatic heterocycle. The HRMS data of
dyes showed that the relative molecular mass of dyes 7ae7h was
consistent with the theoretical calculation results, and the error
was less than 5 ppm.
3.2. Crystal structures
Crystals suitable for X-ray analysis were obtained by slow
evaporation of a solution of dyes 7c, 7d, 7f, 7g or 7h in methanol-
dichloromethane. The crystal structures were given in Fig. 1 and
the representative hydrogen bonds were shown in Table 1. Other
data were exhibited in supplementary information. As shown in
Fig. 1, the study revealed that the rhodanine ring, methine chain
and benzoindoline ring were almost coplanar, and the N-alkyl
Fig. 5. Fluorescence spectra of dye 7d in different concentrations of BSA.

D. Gao et al. / Dyes and Pigments 129 (2016) 163e173
169
Table 4
Spectral characteristics of dye 7d with BSA.
Dyes
BSA concentrations
lem1/nm
lem2/nm
Fluorescence intensity1
Fluorescence intensity2
Stokes shifts1/nm
Stokes shifts2/nm
m
g/mL
7d
0
582.8
581.2
580.8
578.6
576.0
574.4
572.4
569.0
568.2
565.8
624.2
624.2
624.2
623.0
622.2
622.4
620.0
624.2
622.4
622.8
276.6
283.7
310.3
330.5
341.4
336.5
356.5
387.0
397.0
399.9
319.0
318.4
299.6
296.5
285.6
258.5
248.3
204.9
180.6
162.2
82.8
81.8
80.8
78.6
76.0
74.4
72.4
69.0
68.2
65.8
124.2
124.2
124.2
123.0
122.2
122.2
120.0
124.2
122.4
122.8
10
20
30
40
50
60
70
80
90
The lex ¼ 500 nm
Fig. 6. Fluorescence spectra of dye 7d in water upon addition of ethanol.
groups on benzoindoline ring and rhodanine were situated on the
opposite side. Further, the dye molecules were E-Z configuration.
The adjacent molecules were stacked through the p-p interaction,
with face-to-face distances of 8.87 Å (7c), 8.94 Å (7d), 7.28 Å (7f),
7.66 (7g) and 10.79 Å (7h) along a axis and the conjugated mole-
cules were arranged in a head-to-head fashion. Parameters in CIF
format were available as Electronic Supplementary Information
from Cambridge Crystallographic Data Center (CCDC:
1439727e1439731).
3.3. Spectral properties of dyes
3.3.1. Absorption and fluorescence spectra of dyes
We initially introduced the quinoline or benzo[c,d]indole ring to
merocyanine to obtained dyes 7a and 7b (Scheme 1), and investi-
gated their absorption and fluorescence spectra in four common
solvents (Table 2). The absorption and fluorescence spectra of dyes
7a and 7b in water were displayed in Fig. 2. It could be found that
both 7a and 7b mainly existed in H-aggregates states in water,
therefore, 7a showed little fluorescence and 7b displayed weak
fluorescence. To decrease aggregation, we introduced benzoindo-
line ring with bulky and nonplanar geminal dimethyl to mer-
ocyanine to make stacking unfavorable, and synthesized a series of
dyes 7ce7h and investigated their spectral properties (Table 2). Just
as we expected, the introduction of benzoindoline ring to the
merocyanine structures decreased the aggregation in water and the
dyes mainly existed in monomer form. Fig. 3 gave the absorption
and fluorescence spectra of dye 7d in four common solvents. It
Fig. 7. The viability of cells incubated with dyes at various concentrations for 48 h.
Error bars were based on triplicate samples. (a) L929 cells and (b) A549 cells.
could be seen that the dye existed in monomer form and emitted
stronger fluorescence at short wavelengths in hydrophobic solvents
such as methanol, ethanol or dimethyl sulfoxide, and in water it
mainly existed in monomer form and produced fluorescence at
long wavelengths. The difference of fluorescence spectra between
hydrophobic solvents and water could be attributed to effects of H-

170
D. Gao et al. / Dyes and Pigments 129 (2016) 163e173
Fig. 8. Confocal fluorescence images of dye 7d with different concentrations in L929 cells. (a. 0.625, b. 1.25, c. 2.5, d. 5.0, e. 10.0, f. 20.0 mM).
aggregates and solvation. Dye 7d formed some non-fluorescent H-
aggregates in water, which made its fluorescence intensity lower,
and it was readily solvated in polarity large water, which made its
transition energy lower, leading to emission maxima larger. To give
a deep insight of the solvent polarity effect at the molecular level,
the emission maxima (lem) of dye 7d were calculated by time-
dependent density functional theory (TD-DFT) [25] adopted the
polarizable continuum model (PCM) [26], using the Gaussian 09
program [27]. The geometries of excited state S1 of dye 7d molecule
were fully optimized at TD-B3LYP/6-31G* level in both water and
hydrophobic solvent dimethyl sulfoxide. The transition character,
orbital compositions, and energy gap of the molecular orbitals
(MOs) of dye 7d were showed in Table 3 and Fig. 4. It could be seen
that the calculated lem was in good agreement with the experi-
mental values. As shown in Fig. 4, LUMO energies in DMSO and
water were ꢂ2.41 eV and ꢂ2.51 eV, respectively, and HOMO en-
ergies in DMSO and water were ꢂ4.76 eV and ꢂ4.82 eV, respec-
tively. In the large polarity solvent water, the HOMO-LUMO energy
gap was smaller than that in DMSO, which showed that dye 7d was
different concentrations of BSA were shown in Fig. 5 and Table 4. It
could be seen that dye 7d exhibited two emission peaks at about
580 and 620 nm in TE buffer solution, and the fluorescence in-
tensity at long wavelength was decreased and that at short wave-
length was increased and accompanied by emission maxima blue
shift with the increasing concentrations of BSA. It could be attrib-
uted to the fact that after adding protein the dye must interact with
the protein surface, and moved into the hydrophobic regions of
protein, which was equivalent to hydrophobic solvents (Fig. 6), so
that the fluorescence intensity of dye was increased and emission
maximum was blue-shifted with the increase of protein. To inves-
tigate this point, the fluorescence spectra of dye 7d in different
amounts of ethanol aqueous solutions were determined. As shown
in Fig.6, the fluorescence intensity was increased and the emission
maxima were blue-shifted as the amounts of ethanol increased,
which were consistent with adding BSA to buffer solution. From the
above investigation, the important interaction of dye 7d with BSA
occurred, which was suggesting as a potential ratiometric fluores-
cent probe to report protein conformational changes.
readily solvated in water, leading a longer lem
.
From Table 2, it could also be found that dyes 7ae7h all absor-
bed light at long wavelengths advantageous in live cell imaging,
and dyes 7ce7h showed an obvious solvent-dependent change in
emission maxima and fluorescence intensity, for example, the
emission maxima and fluorescence intensity of dye 7d changed
more than 62 nm and 3.9-fold, respectively, from 563 nm to 1207 in
ethanol to 625 nm and 309 in water, which belonged to solvent-
dependent ratiometric fluorescent dye.
3.4. Cytotoxicity and fluorescence imaging in cells
For biomedical applications, the toxicity of dye was a major
concern. Dyes 7ae7h were taken to perform an MTT assay on L929
and A549 cells with different working concentrations (0.313, 0.625,
1.25, 2.5, 5, 10
m
M) to evaluate cytotoxicity. As shown in Fig. 7, the
M. The
cell viability was about 80% at the concentration of 10
m
results indicated that the dyes had low cytotoxicity to cells and
suitable for bioimaging.
3.3.2. The interaction of dye 7d with BSA
In order to examine the feasibility of the novel dyes for cellular
imaging applications, staining of the living cells with the dyes was
studied using confocal fluorescence microscopy. The dye 7d was
chosen to study the effect of dye concentrations on the fluorescence
Based on the above study results, we investigated the interac-
tion of dye 7d with BSA to explore the possibility as protein fluo-
rescent probe. The fluorescence spectra of dye 7d in the presence of

D. Gao et al. / Dyes and Pigments 129 (2016) 163e173
171
Fig. 9. Fluorescence images of L929 and A549 cells incubated with dyes (10 mM) and Hoechst 33342. (a) Fluorescence image (red channel). (b) Emission of Hoechst 33342 from the
blue channel (nuclear staining). (c) The overlay image of (a) and (b). (d) Bright field image of cells shown in panel. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
imaging. As shown in Fig. 8, the fluorescence signals of the cells
became stronger gradually when increasing the concentrations of
dye, and the signal was stabilized after the amount of dye 7d
cytoplasm was sustained for 24 h at least. Taken together, the
fluorescence imaging in living cells showed the dyes would be able
to act as potential cytoplasmic staining and fluorescent reagents to
clearly observe cells, and analyze the characteristics of cells and
determine the developmental stages of cells.
reached 10
mM. Therefore, the working concentration of dye was
determined to be 10
mM for staining L929 and A549 cells.
We used dyes 7ae7h for cell staining, and investigated fluo-
rescence imaging of stained cells. The results showed that dye 7a
exhibited no fluorescence in cells, while dye 7b displayed weaker
fluorescence signal, and dyes 7ce7h displayed brighter light, and
dyes 7be7h could all stain cells and mainly distributed in the
cytoplasm. Fig. 9 showed the fluorescence images of L929 and
A549 cells incubated with dyes 7b, 7d and 7f as well as commer-
cially available Hoechst 33342. As expected, the fluorescence sig-
nals from cellular cytoplasmic and nuclear regions could be easily
detected in cells. And dyes 7b, 7d and 7f could be accumulated in
cells, and mainly distributed in the cytoplasm (Fig. 9a, colored in
red), and commercial dye Hoechst 33342 stained nuclei (Fig. 9b,
colored in blue). In addition, the fluorescence signal of cellular
3.5. Imaging of dyes in living mice
Encouraged by the results of fluorescence imaging in living cells,
we wondered whether the new dyes may be potentially useful for
imaging applications in living animals. Toward this end, we eval-
uated the suitability of the dyes for imaging in mice. Kunming mice
were divided into two groups; one group was untreated as the
control group. And dye 7d or 7h (400
mL ꢁ 400
mM in saline, con-
taining 1% DMSO) was injected into the intraperitoneal cavity of the
mice in the second group. Twenty minutes after injecting dye so-
lution, the mice were imaged. As shown in Fig. 10b1 and b2, a larger
fluorescence signal was noted than in the untreated group

172
D. Gao et al. / Dyes and Pigments 129 (2016) 163e173
Fig. 10. Representative fluorescent images of dyes 7d and 7h in mice: (a) control group; (b) The fluorescent images of mice after intraperitoneal (i.p.) injected with dyes.
and BSA association study. J Org Chem 2014;79:5511e20.
(Fig. 10a1 and a2). Thereby, these results established that the new
dyes may be effective for living animal bio-imaging applications.
[3] Volkova KD, Kovalska VB, Balanda AO, Losytskyy MY, Golub AG, Vermeij RG,
et al. Specific fluorescent detection of fibrillar a-synuclein using mono- and
trimethine cyanine dyes. Bioorg Med Chem 2008;16:1452e9.
[4] Miletto I, Gilardino A, Zamburlin P, Dalmazzo S, Lovisolo D, Caputo G, et al.
Highly bright and photostable cyanine dye-doped silica nanoparticles for
optical imaging: photophysical characterization and cell tests. Dyes Pigment
2010;84:121e7.
[5] Volkova KD, Kovalska VB, Balanda AO, Vermeij RJ, Subramaniam V,
Slominskii YL, et al. Cyanine dye-protein interactions: looking for fluorescent
probes for amyloid structures. J Biochem Biophys Methods 2007;70:727e33.
[6] Nuesch F, Moser GE, Shklover V, Gratzel M. Merocyanine aggregation in
mesoporous networks. J Am Chem Soc 1996;118:5420e31.
4. Conclusion
A series of novel merocyanine dyes 7ae7h were prepared and
the crystal structures of dyes 7c, 7d, 7f, 7g and 7h were analyzed by
X-ray diffraction. Dyes 7ce7h fluoresced brightly at long wave-
lengths in different solvents and displayed solvent-dependent
properties, which was reasonably explained by theoretical calcu-
lations and absorption spectra. And dye 7d exhibited strong inter-
action with BSA and showed an obvious ratiometric fluorescence
response to solvent/protein environment. And dyes 7be7h showed
excellent fluorescence imaging capability in living cells and mice.
These characteristics of new dyes demonstrated their potential
applications in protein labeling and imaging in biological science.
[7] Park H, Chang SK. Signaling of water content in organic solvents by sol-
vatochromism of
a hydroxynaphthalimide-based merocyanine dye. Dyes
Pigment 2015;122:324e30.
[8] Krause S, Stolte M, Wurthner F, Koch N. Influence of merocyanine molecular
dipole moments on the valence levels in thin films and the interface energy
level alignment with Au(111). J Phys Chem C 2013;117:19031e7.
[9] Nahain AA, Lee JE, Jeong JH, Park SY. Photoresponsive fluorescent reduced
graphene oxide by spiropyran conjugated hyaluronic acid for in vivo imaging
and target delivery. Biomacromolecules 2013;14:4082e90.
[10] Kreß KC, Bader K, Stumpe J, Eichhorn SH, Laschat S, Fischer T. Rigidified
merocyanine dyes with different aspect ratios: dichroism and photostability.
Dyes Pigment 2015;121:46e56.
Acknowledgment
[11] Wang S, Kim SH. New solvatochromic merocyanine dyes based on barbituric
acid and meldrums acid. Dyes Pigment 2009;80:314e20.
[12] Aiken S, Clayton D, Gabbutt CD, Heron BM, Kolla SB. Cationic merocyanine
dyes by photomodulation of [4-(naphthopyran-3-yl) phenyl]methines. Dyes
Pigment 2013;97:118e23.
[13] Henary M, Pannu V, Owens EA, Aneja R. Near infrared active heptacyanine
dyes with unique cancer-imaging and cytotoxic properties. Bioorg Med Chem
Lett 2012;22:1242e6.
We appreciate the financial support for this research by a grant
from Xi'an the Scientific and Technological Plan Projects (CXY1511
(5)); National Natural Science Foundation of China under Grant
Nos. 21575111, 81201137; Natural Science Foundation of Shaanxi
Province (2012JC2-10).
[14] Toutchkine A, Kraynov V, Hahn K. Solvent-sensitive dyes to report protein
conformational changes in living cells. J Am Chem Soc 2003;125:4132e45.
[15] He TC, Hu WB, Shi HF, Pan QF, Ma GH, Huang W, et al. Strong nonlinear optical
phosphorescence from water-soluble polymer dots: towards the application
of two-photon bioimaging. Dyes Pigment 2015;123:218e21.
[16] Zhang XH, Liu Q, Shi HJ, Wang LY, Fu YL, Wei XC, et al. Synthesis, spectral
properties of cell-permeant dimethine cyanine dyes and their application as
fluorescent probes in living cell imaging and flow cytometry. Dyes Pigment
2014;100:232e40.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2016.02.020.
References
[17] Xiang JF, Liu YX, Sun D, Zhang SJ, Fu YL, Zhang XH, et al. Synthesis, spectral
properties of rhodanine complex merocyanine dyes as well as their effect on
K562 leukemia cells. Dyes Pigment 2012;93:1481e7.
[1] Escobedo JO, Rusin O, Lim S, Strongin RM. NIR dyes for bioimaging applica-
tions. Curr Opin Chem Biol 2010;14:64e70.
[2] Pisoni DS, Todeschini LA, Borges AC, Petzhold CL, Rodembusch FS, et al.
Symmetrical and asymmetrical cyanine dyes. Synthesis, spectral properties,
[18] Renault S, Bertrand S, Carreaux F, Bazureau JP. Parallel solution-phase

D. Gao et al. / Dyes and Pigments 129 (2016) 163e173
173
synthesis of 2-Alkylthio-5-arylidene-3,5-dihydro-4H-imidazol-4-one by one-
pot three-component domino reaction. J Comb Chem 2007;9:935e42.
[19] Nitsche C, Klein CD. Aqueous microwave-assisted one-pot synthesis of N-
substituted rhodanines. Tetrahedron Lett 2012;53:5197e201.
[20] Liu Q, Su XH, Wang LY, Sun W, Lei YB, Wen ZY. An OFF-ON-OFF type of pH
fluorescentsensor: benzo[c,d]indole-based dimethine cyaninedye-synthesis,
of 2-alkyl-3,5-diaryl-l,3,4-thiadiazolium cations. Can J Chem 1987;65:2713e6.
[25] Scalmani G, Frisch MJ, Mennucci B, Tomasi J, Cammi R, Barone V. Geometries
and properties of excited states in the gas phase and in solution: theory and
application of a time-dependent density functional theory polarizable con-
tinuum model. J Chem Phys 2006;124:094107.
[26] Tomasi J, Mennucci B, Cammi R. Chem Rev 2005;105:2999e3093.
[27] Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR,
Scalmani G, Barone V, Mennucci B, Petersson GA, Nakatsuji H, Caricato M, Li X,
Hratchian HP, Izmaylov AF, Bloino J, Zheng G, Sonnenberg GL, Hada M, Ehara
M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O,
Nakai H, Vreven T, Montgomery Jr JA, Peralta JE, Ogliaro F, Bearpark M, Heyd
JJ, Brothers E, Kudin KN, Staroverov VN, Keith T, Kobayashi R, Normand J,
Raghavachari K, Rendell A, Burant JC, Iyengar SS, Tomasi J, Cossi M, Rega N,
Millam JM, Klene M, Knox JE, Cross JB, Bakken V, Adamo C, Jaramillo J,
Gomperts R, Stratmann RE, Yazyev O, Austin AJ, Cammi R, Pomelli C, Ochterski
JW, Martin RL, Morokuma K, Zakrzewski VG, Voth GA, Salvador P, Dannenberg
JJ, Dapprich S, Daniels AD, Farkas O, Foresman JB, Ortiz JV, Cioslowski J, Fox DJ,
Gaussian, Inc, Wallingford, CT, 2010.
spectral properties and density functional theorystudies.
J Luminescence
2014;154:124e30.
[21] Coe BJ, Harris JA, Hall JJ, Brunschwig BS, Hung ST, Libaers W, et al. Syntheses
and quadratic nonlinear optical properties of salts containing benzothiazo-
lium electron-acceptor groups. Chem Mater 2006;18:5907e18.
[22] Pudhom K, Kasai K, Terauchi H, Inoue H, Kaiser M, Brun R, et al. Synthesis of
three classes of rhodacyanine dyes and evaluation of their in vitro and in vivo
antimalarial activity. Bioorg Med Chem 2006;14:8550e63.
[23] Yang M, Arai C, Md AB, Lu J, Ge JF, Pudhom K, et al. Fluorinated rhodacyanine
(SJL-01) possessing high Efficacy for visceral leishmaniasis (VL). J Med Chem
2010;53:368e73.
[24] Mastalerz H, Mohammad T, Gibson MS.
A 2-methylthio-3,5-diaryl-1,3,4-
thiadiazolim cation; its reactions, and further exploration of the chemistry