Synthesis, spectral properties of rhodanine complex merocyanine dyes as well as their effect on K562 leukemia cells

Article abstract of DOI:10.1016/j.dyepig.2011.10.017

Two rhodanine complex merocyanine dyes 9a and 9b were synthesized and their structures were confirmed by ¹H NMR, IR, MS, HRMS and UV-Vis spectra. From the spectral properties of the two dyes, it could be found that the λ_max of the dyes showed hypsochromic shifts with the increase of permittivity in protonic solvents, and bathochromic shifts with the increase of refractive index in non protonic solvents. The interactions of two dyes with DNA or BSA were also studied under physiological conditions. The results showed that the quantum yield of DNA-dye 9a was up to 29.5 times compared with free dye 9a. Dyes 9a and 9b were researched in Photodynamic Therapy (PDT) as well. It was demonstrated that supplementation of dye 9a or 9b as photosensitizers for PDT in K562 cells decreases the survival rate.

Full text of DOI:10.1016/j.dyepig.2011.10.017

Dyes and Pigments 93 (2012) 1481e1487
Contents lists available at SciVerse ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Synthesis, spectral properties of rhodanine complex merocyanine dyes
as well as their effect on K562 leukemia cells
Jun-Feng Xiang , Yan-Xia Liu , Dan Sun , Su-Juan Zhang , Yi-Le Fu , Xiang-Han Zhang ,c,
Lan-Ying Wang
a
a
a
b
b
a
a
a,
*
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, Ministry of Education, College of Chemistry and Materials Science, Northwest University,
Xi’an 710069, PR China
b
Institute of Photonics & Photon-Technology, Northwest University, Xi’an 710069, PR China
School of Life Sciences and Technology, Xidian University, Xi’an 710071, PR China
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 August 2011
Received in revised form
Two rhodanine complex merocyanine dyes 9a and 9b were synthesized and their structures were
confirmed by H NMR, IR, MS, HRMS and UVeVis spectra. From the spectral properties of the two dyes, it
could be found that the lmax of the dyes showed hypsochromic shifts with the increase of permittivity in
protonic solvents, and bathochromic shifts with the increase of refractive index in non protonic solvents.
The interactions of two dyes with DNA or BSA were also studied under physiological conditions. The
results showed that the quantum yield of DNA-dye 9a was up to 29.5 times compared with free dye 9a.
Dyes 9a and 9b were researched in Photodynamic Therapy (PDT) as well. It was demonstrated that
supplementation of dye 9a or 9b as photosensitizers for PDT in K562 cells decreases the survival rate.
Ó 2011 Elsevier Ltd. All rights reserved.
1
21 October 2011
Accepted 30 October 2011
Available online 6 November 2011
Keywords:
Rhodanine complex merocyanine dyes
Spectral properties
DNA
BSA
Photosensitizers
Photodynamic therapy
1. Introduction
distinguish some certain cells and selectively enter into cancer cells
then kill it as photosensitizer directly using for PDT, or as a radiation
It is well known that several approaches have been taken for
cancer chemotherapy, and many antitumor drugs have been
applied for clinic during the past 30 years. However, in the treat-
ment of solid tumors the traditional approaches have met with only
limited success and cancer still remains as one of the most serious
diseases of human. Currently, in order to overcome the major
drawbacks of chemotherapeutic antitumor drugs, e.g. adverse
effects and drug resistance [1], photodynamic therapy (PDT) has
attracted attention [2e6]. Besides chemotherapy, surgery, radio-
therapy and immunotherapy, PDT is a promising therapeutic
modality for the treatment of a variety of premalignant and
malignant diseases [7]. Because of its low cytotoxicity, no surface
damage, the capacity of highly selective and destroying tumors
without harming normal tissues, PDT is a valuable therapy method.
Due to the strong fluorescence and absorption capacity, rhoda-
nine merocyanine dyes, as derivatives of cyanine dyes, can be used
as fluorescent labels for biomolecules [8e10]. Besides, they can
sensitizer for the treatment of solid tumors [11]. In the meanwhile,
this type of cyanine dyes used as antitumor drugs have been re-
ported [12e17]. As the affinity between cyanine dyes and tumor
cells is much higher than that between cyanine dyes and normal
cells, combining PDT with drug therapy has become a tendency and
will certainly promote the treatment of tumors. For this reason, we
designed and synthetized two rhodanine complex merocyanine
1
dyes 9a and 9b (Scheme.1), and the products were confirmed by H
NMR, IR, MS, HRMS, UVeVis. We studied the fluorescence and
UVeVis absorption spectra properties of two dyes in different
solvents, and their interaction with DNA or BSA under physiological
conditions. And using dye 9a or 9b as photosensitizers for PDT, its
effect on K562 leukemia cells survival was analyzed.
2. Experimental
2.1. General
All reagents were obtained from commercial sources and used
without further purification. The solvents were of analytical grade.
*
Corresponding author. Tel.: þ86 29 88302604; fax: þ86 29 88303798.
E-mail address: wanglany@nwu.edu.cn (L.-Y. Wang).
0143-7208/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2011.10.017

1482
J.-F. Xiang et al. / Dyes and Pigments 93 (2012) 1481e1487
CH3
I
CH3
N
S
N
S
N
N
S
S
a
b
c
SH
SCH3
SCH3
N
S
S
S
Et
O
N
S
O
1
2
3
Et 4
5
CH3
I
N
S
e
CH3
7
CH
3
1
S
I
N
5
1
0
N
3
4
S 8
CH3
C9
S
2
CH3
6
H
N7
11
N
S
SCH3
O
d
Et
N
TsO
S
9a
Et
CH3
I
O
I
N
6
CH3
H C N
CH3
8
3
1
N
5
10
C 9
3
S
8
e
4
S
H
2
6
O
N7
Et
1
1
9
b
ꢁ
ꢁ
ꢁ
ꢁ
Scheme. 1. Reagents and conditions (a) (CH
3
)
2
SO
4
, 10% NaOH, 40 C (b) CH
3
I, MeOH, 43 C (c) Et
3
N, EtOH, Reflux (d) TsOMe, DMF, 130 C (e) Et
3
N, MeCN, 70 C.
ꢁ
Melting points were taken on an XT-4 micromelting apparatus and
(2.80 g, 72.2%) as colorless crystals, m.p. 142e143 C (lit, m.p.
146 C).
ꢀ
1
ꢁ
uncorrected. IR spectra in cm were recorded on Shimadzu IR
1
Prestige-21 spectrometer and Bruker Equiox-55 spectrometer. H
NMR spectra were recorded at 400 MHz on a Varian Inova-400
spectrometer and chemical shifts were reported relative to
2.2.3. 5-(3-Methyl-(3H)-benzothiazol-2-ylidene)-3-ethyl-2-thioxo-
4-thiazolidinone (5)
internal Me
4
Si. The electron impact (EI) mass spectra were recor-
A mixture of 3-methyl-2-methylthiobenzothiazolium iodide (3;
1.29 g, 4.0 mmol), 3-ethylrhodanine (4; 0.65 g, 4.0 mmol) and a few
drops of triethylamine in ethanol was refluxed for 3 h. After cooling,
the product was filtered to give (5) (1.20 g, 97.6%) as yellow powder,
ded at 70 eV with a GCMS-QP2010 system equipped with the solid
sample direct insertion probe. HRMS was recorded on a microTOF-
Q II ESI-Q-ToF LC/MS/spectrometer. The absorption spectra were
recorded on a Purkinje General UV-1900 UVeVis spectrometer.
Fluorescence measurements were carried out on a Hitachi F-4500
spectrofluorimeter.
ꢁ
ꢁ
m.p. 260e264 C (lit, m.p. 265 C).
2.2.4. 2,3-Dimethylbenzothiazolium iodide (7)
A mixture of 2-methylbenzothiazole (5.07 g, 34.0 mmol) and
methyl iodide (5.00 g, 35.2 mmol) in ethanol was stirred at room
temperature for 0.5 h, then the mixture was heated to reflux for
24 h. After cooling, the product was filtered to give (7) (8.75 g,
2
.2. Preparation of the intermediate 2e8
2
-Methylthiobenzothiazole (2)
[18],
3-methyl-2-
ꢁ
ꢁ
methylthiobenzothiazolium iodide (3) [19], 5-(3-methyl-(3H)-
benzothiazol-2-ylidene)-3-ethyl-2-thioxo-4- thiazolidinone (5)
88.4%) as white powder, m.p. 218e220 C (lit, m.p. 221e222 C).
[20], 2,3-dimethylbenzothiazolium iodide (7) [21,22], 1,4-
2.2.5. 1,4-Dimethylquinolinium iodide (8)
dimethylquinolinium iodide (8) [21,22] were prepared according
to the literatures with some modification.
A mixture of 4-methylquinoline (2.86 g, 20 mmol) and methyl
iodide (4.26 g, 30 mmol) in ethanol was stirred at room tempera-
ture for 0.5 h, then the mixture was heated to reflux for 12 h. After
the reaction mixture was cooled to room temperature, the product
was filtered to give (8) (3.80 g, 66.7%) as yellow crystals, m.p.
2.2.1. 2-Methylthiobenzothiazole (2)
The 2-mercaptobenzothiazole (1; 3.01 g, 18.0 mmol) was dis-
ꢁ
ꢁ
solved in 7.5 mL of 10% NaOH, and dimethyl sulfate (1.7 mL,
176e177 C (lit, m.p. 174e175 C).
ꢁ
1
8.0 mmol) was added dropwise under stirring at 40 C, the
ꢁ
resulting mixture was stirred at 40 C for 0.5 h. After the reaction
2.3. Preparation of the intermediate 6 and two dyes 9a, 9b
mixture was cooled to room temperature and extracted with ether,
and the organic phase was dried over anhydrous magnesium
sulfate, filtered and evaporated off ether to give (2) (2.17 g, yield
2.3.1. Preparation of dye 9a
A mixture of (5) (0.31 g, 1.0 mmol) and methyl p-toluenesulfo-
nate (0.52 g, 3.0 mmol) in N,N-dimethylformamide (10.0 mL) was
ꢁ
ꢁ
6
6.6%) as white crystals, m.p. 51e52 C (lit, m.p. 52 C).
ꢁ
stirred at 130 C for 2.5 h. After the mixture was cooled to room
2
.2.2. 3-Methyl-2-methylthiobenzothiazolium iodide (3)
A mixture of 2-methylthiobenzothiazole (2; 2.17 g, 12.0 mmol)
temperature, ether (30 mL) was added and the product was filtered
to give yellow solid (6).
and methyl iodide (2.84 g, 20.0 mmol) in methanol was stirred at
The obtained compound (6) was dissolved in 5.0 mL acetonitrile,
and 2,3-dimethylbenzothiazolium iodide (7; 0.29 g, 1.0 mmol) was
then added, followed by a few drops of triethylamine. And the
ꢁ
4
3 C for 24 h. After cooling, excess methyl iodide and methanol
were evaporated and the product was washed with ether to give (3)

J.-F. Xiang et al. / Dyes and Pigments 93 (2012) 1481e1487
1483
ꢁ
mixture was stirred at 70 C for 1 h. After cooling, the precipitate
was collected and washed with acetonitrile and ethylacetate
respectively. Then the product was purified with methanol by
Soxhlet extraction in 48 h to give 9a (0.12 g, 21.1%, two steps).
Table 1
The physical constant and the spectral data of dye 9a, 9b in different solvents.
ꢀ
4
Dye Solvent Permittivity Refractive lmax/nm lem/nm Stockes
3
ꢂ 10
ꢀ1
shift/nm M cm
/
ꢀ
1
index
9
a
H
2
O
78.5
32.6
24.3
47.2
36.7
1.3325
1.3290
1.3614
1.4780
1.4304
1.3587
1.3325
1.3290
1.3614
1.4780
1.4304
1.3587
471.0
501.0
503.0
506.0
503.0
501.0
520.0
567.0
569.0
571.0
569.0
568.0
574.0
559.0
559.0
565.0
565.0
564.0
658.0
e
103.0
58.0
56.0
59.0
62.0
63.0
138.0
e
6.2
8.3
8.0
8.1
7.5
8.0
4.1
5.8
5.7
5.8
5.3
5.6
MeOH
EtOH
DMSO
DMF
MeCOMe 20.5
H
MeOH
EtOH
DMSO
DMF
2.3.2. Preparation of dye 9b
A mixture of (5) (0.37 g, 1.2 mmol) and methyl p-toluenesulfo-
nate (0.62 g, 3.6 mmol) in N,N-dimethylformamide (10.0 mL) was
ꢁ
stirred at 130 C for 2.5 h. After the mixture was cooled to room
temperature, ether (30 mL) was added and the product was filtered
to give yellow solid (6).
9b
2
O
78.5
32.6
24.3
47.2
36.7
625.0
647.0
e
56.0
76.0
e
The obtained compound (6) was dissolved in 5.0 mL acetonitrile,
1,4-dimethylquinolinium iodide (8; 0.34 g, 1.2 mmol) was then
added, followed by a few drops of triethylamine. And the mixture
MeCOMe 20.5
e
e
ꢁ
was stirred at 70 C for 1 h. After cooling, the precipitate was
collected and washed with acetonitrile and ethylacetate respec-
tively. Then the product was purified with methanol by Soxhlet
extraction in 48 h to give 9b (0.23 g, 34.3%, two steps).
absorption. The absorption and fluorescence spectral data were
listed in Table 1.
2.4. Structural confirmation
2.6. Measurements of spectral properties of the dyes in the presence
of DNA or BSA
2.4.1. 2-[3-Ethyl-5-(3-methyl-(3H)-benzothiazol-2-ylidene)-4-
oxothiazolidin-2-ylidenemethyl]-3-methyl benzothiazolium iodide
9a)
Orange-red crystals, m.p.: 248e249 C. 1H NMR (400 MHz,
ꢀ3
ꢀ1
Dyes stock solutions (5.0 ꢂ 10 mol L ) were prepared by
(
dissolving the dyes in DMSO and further diluted with TE buffer
ꢁ
ꢀ1
ꢀ1
ꢀ6
(
10 mmol L TriseHCl, 1 mmol L EDTA, pH 7.5) to result in
DMSO-d
NeCH
6
)
d
(ppm): 1.30 (t, J ¼ 6.4 Hz, 3H, eCH
3
), 4.08 (s, 3H,
e), 6.71 (s, 1H,
ꢀ1
working solutions of dyes (1.0 ꢂ 10 mol L ). Stock solutions of
DNA or BSA were prepared by dissolving DNA or BSA in TE buffer.
The concentrations of DNA or BSA in stock solutions were
þ
3
), 4.23 (s, 3H, N eCH
3
), 4.28 (b, 2H, eCH
2
eCH]), 7.38 (d, J ¼ 7.2 Hz, 1H, ArH), 7.55 (d, J ¼ 7.2 Hz, 2H, ArH),
7
.73 (d, J ¼ 7.2 Hz, 2H, ArH), 7.95e8.01 (m, 2H, ArH), 8.26 (d,
ꢀ5
ꢀ1
ꢀ1
8
ꢂ 10 mol L base pairs (bp) for DNA and 0.5 mg mL for BSA.
ꢀ1
J ¼ 7.2 Hz,1H, ArH); IR (KBr, cm ): 3020 (w,
y
]CeH), 2930 (w,
C]N), 1494 (s, CH), 1317 (s,
CH), 748 (s,
yCeH),
CeN),
Working solutions of complexes DNA-dyes and BSA-dyes were
prepared by mixing an aliquot of dyes stock solutions and an
aliquot of DNA or BSA stock solutions, and further diluted with TE
1648 (s,
yC]O), 1539 (s,
y
C]C
,
y
d
y
1268, 1200, 1065 (s,
y
CeN
,
d
d
]CeH); MS (70 eV) m/z (%):
eCH eC 5), 368
NS, 49); HRMS
4
23 (MeICH
3
eCH
eC
TOF MS ES-) calculated for C22
3
, 100), 379 (MeICH
3
3
H
2 5
,
ꢀ5
ꢀ1
ꢀ1
buffer to obtain 1.6 ꢂ 10 mol L bp for DNA-dyes or 0.1 mg mL
(
MeICH
3
eCH
3
2
H
5
eO, 45), 289 (MeICH
3
eC
8
H
7
for BSA-dyes. All working solutions were prepared immediately
before the experiment. Measurements method was mentioned as
above. The absorption and fluorescence spectral data were listed in
Table 2, Table 3 and Fig. 1.
þ
(
H
20
N
3
OS
3
: 438.0763, found:
4
38.0769; UVeVis (MeOH) lmax: 500.0 nm.
2.4.2. 4-[3-Ethyl-5-(3-methyl-(3H)-benzothiazol-2-ylidene)-4-
oxothiazolidin-2-ylidenemethyl]-1-methyl quinolinium iodide (9b)
2
2
.7. Study on K562 cells
ꢁ
1
6
Darkviolet crystals, m.p. >298 C. H NMR (400 MHz, DMSO-d )
d
(ppm): 1.30 (t, J ¼ 6.4 Hz, 3H, eCH
3 3
), 4.17 (s, 3H, NeCH ), 4.28 (s,
e), 6.94 (s, 1H, eCH]), 7.35 (t,
.7.1. Cell culture and treatment with the dyes
K562 cells were cultured in RPMI 1640 medium supplemented
þ
3
H, N eCH
3
), 4.32 (b, 2H, eCH
2
J ¼ 7.7 Hz, 1H, ArH), 7.54 (t, J ¼ 7.7 Hz, 1H, ArH), 7.63 (d, J ¼ 7.6 Hz,
H, ArH), 7.72 (d, J ¼ 8.0 Hz, 1H, ArH), 7.85 (t, J ¼ 7.7 Hz, 1H, ArH),
.94 (d, J ¼ 8.4 Hz, 1H, ArH), 8.03 (t, J ¼ 7.6 Hz, 1H, ArH), 8.17 (d,
J ¼ 8.0 Hz, 1H, ArH), 8.72 (d, J ¼ 6.4 Hz, 1H, ArH), 8.81 (d, J ¼ 8.8 Hz,
with 10% (v/v) newborn calf serum, 1% (v/v)
units/mL antibiotics (penicillin and streptomycin) at 37 C and 5%
CO
L
-glutamine and 100
1
7
ꢁ
2
humidified incubator. The dyes were dissolved in DMSO and
ꢁ
stored at ꢀ20 C at a concentration of 1 mM. For the cell growth
ꢀ
1
ArH); IR (KBr, cm ): 3015 (w,
y
d
]CeH), 2966 (w,
CH), 1315 (s, CeN), 1228, 1200, 1071
d]CeH); MS (70 eV) m/z (%): 359
y
CeH), 1636 (s,
y
C]
5
assay, cells were seeded into 6-well plates at a density of 1 ꢂ 10
O
), 1539 (s,
s,
y
d
C]C
,
yC]N), 1494 (s,
y
cells/mL and treated with the dyes or with DMSO only (as control).
(
(
(
y
CeN
,
CH), 750 (s,
The final concentration of DMSO was kept at less than 0.05%. K562
cells were incubated with the dyes for 4 h at 37 C before PDT
irradiation.
MeICH
MeICH
3
eCH
eCH
3
3
eC
eC
2
7
H
H
5
eO, 5), 275 (MeICH
NS, 50), 246 (MeICH
3
eCH
eCH
3
eC
9
H
6
N, 48); 268
ꢁ
3
5
3
2
CH
3
eC10H10, 100);
þ
HRMS (TOF MS ES-) calculated for C24
H
22
N
3
OS
2
: 432.1199, found:
4
32.1205; UVeVis (MeOH)
lmax: 566.0 nm.
2.7.2. Photodynamic treatment
Photodynamic treatment was performed according to the
literature [23]. K562 cells in the exponential phase of growth were
harvested and suspended in RPMI 1640 medium at a density of
2
.5. Measurements of the spectral properties of the dyes in different
solvents
ꢀ
3
ꢀ1
The dye stock solutions (5.0 ꢂ 10 mol L in DMSO) were
diluted with different solvents and resulted in working solutions of
Table 2
ꢀ
5
ꢀ1
Spectral characteristics of dye 9a, 9b in buffer.
dyes (5.0 ꢂ 10 mol L ). The absorption spectra were examined
at room temperature in different solvents and recorded using 1 cm
quartz cells on a Purkinje General UV-1900 UVeVis spectrometer.
Fluorescence measurements were carried out at room temperature
on a Hitachi F-4500 spectrofluorimeter in 1 cm quartz cells.
Fluorescence emission was excited at the maximum of the
Dye
In buffer
max/nm
free
ꢀ1
ꢀ1
free
F
l
3
(M cm
)
lex/nm
lem/nm
F
4
9
9
a
b
500.0
563.0
1.2 ꢂ 10
6.0 ꢂ 10
500.0
563.0
566.8
e
0.0025
e
3

1484
J.-F. Xiang et al. / Dyes and Pigments 93 (2012) 1481e1487
Table 3
Spectral characteristics of dye 9a, 9b in DNA or BSA presence.
max/nm εDNA (M cm
ꢀ1
ꢀ1
)
lex/nm
lem/nm
F
DNA
F
F
DNA
F
=
F
free
F
Dye
l
In DNA presence
4
4
9
9
a
b
504.0
569.0
3.2 ꢂ 10
1.3 ꢂ 10
480.0
550.0
550.0
642.0
0.0738 29.52
0.0020
e
BSA
ꢀ1
ꢀ1
BSA
F
BSA
F
free
F
lmax/nm
ε
(M cm
)
lex/nm
lem/nm
F
F
=
F
In BSA presence
4
9a
495.0
564.0
1.5 ꢂ 10
7.0 ꢂ 10
495.0
564.0
560.8
e
0.0027 1.08
3
9b
e
e
5
1
ꢂ 10 cells/mL. The cells were incubated for 4 h with 1 mM the
ꢁ
dyes at 37 C. Afterward the cells were seeded in 6-well plates. The
cells were illuminated with a light intensity of 350 mW/cm and
light doses of 105 J/cm . The radiation source was a xenon lamp
Fig. 2. Effects of dyes 9a and 9b on cell survival A. Cell survival rate treated with DMSO
only (as control) without dyes; B and D. Cell survival rate treated with dyes 9a and 9b,
respectively; C and E. Cell survival rate treated with dyes 9a and 9b respectively and
PDT irradiation.
2
2
emitting wavelengths over the range 400e800 nm.
2
.7.3. Measurement of cell viability
The effect of the dyes on growth of K562 cells was determined
compound (7) or (8) were prepared by N-methyl quaternization of
2-methylbenzothiazole or 4-methylquinoline. In all cases investi-
by hemacytometer. K562 cells were seeded into 6-well plates at
5
ꢁ
gated, we found that two rhodanine complex merocyanine dyes
could be purified by soxhlet extraction with methanol. And inter-
a density of 1 ꢂ10 cells/mL and incubated at 37 C in 5% CO
2
. After
experiments, the cells were stained by 0.2% trypan blue and then
counted with a hemacytometer. The percent of inhibition cell
proliferation and cell survival was calculated as follows: %
Survival ¼ (survival/control) ꢂ 100%. All experiments were per-
formed in triplicates. Data were expressed as mean þ SD. Experi-
mental results were listed in Fig. 2.
mediate
5-(3-methyl-(3H)-benzothiazol-2-ylidene)-3-ethyl-2-
thioxo-4-thiazolidinone (5) was introduced into the following reac-
tion of synthesizing rhodanine complex merocyanine dye 9a, 9b
without purification. The dyes with higher purity could be obtained,
and the procedure was simplified as well.
3.2. E,Z geometry of two dyes and DFT calculations
3
. Results and discussion
Because of C]C double bonds, there are four geometric
3
.1. Synthesis of dyes
isomers in dyes 9a and 9b, respectively. The configurations and
energies of the geometric isomers are carried out at B3LYP/6-31G*
level according to the literature [24], and their optimized config-
urations and energies are given in Fig. 3 and Fig. 4. It can be found
The rhodanine complex merocyanine dyes 9a and 9b were
synthesized via the reaction of intermediate compound (6) with 2,3-
dimethylbenzothiazole quaternary salt (7) or 1,4-dimethylquinoli-
nequaternary salt (8) (Scheme 1). The intermediate compound (6)
was easily obtained by S-methylation and N-methyl quaternization
of 2-mercaptobenzothiazole (1), the condensation of 3-methyl-2-
methylthiobenzothiazolium iodide (3) with 3-ethylrhodanine (4) in
the presence of triethylamine, and methylating the thiocarbonyl
group of the 5-(3-methyl-(3H)-benzothiazol-2-ylidene)-3-ethyl-2-
thioxo-4-thiazolidinone (5), successively. The intermediate
0
that the isomers II and II is of a minimum single point energy for
dye 9a and for dye 9b, respectively. Therefore isomer II is the most
0
stable form in all isomers for dye 9a, and isomer II is the most
stable form in all isomers for dye 9b. So the geometry of dyes 9a
0
and 9b is isomers II and II respectively, which is consistent with
the literature [12]. Dyes reported in the literature are complex
merocyanine dyes, which are similar to the dyes we synthesize in
this work.
3
.3. Spectral properties of the dyes in different solvents
200
150
100
9
9
9
a in buffer
a in BSA presence
a in DNA presence
Table 1 gives the spectral properties of the dyes in different
solvents. From Table 1, it could be found that the shortest lmax of
dye 9a or 9b was in water. The reason was that the hydrogen bonds
created between water molecules in aqueous medium and dye
molecules could reduce the delocalization of the lone pair of elec-
trons of hetrocyclic nitrogen, which lead to the observed sol-
vatochromic behavior [25]. It could be also found that the lmax of
the dyes exhibited hypsochromic shifts with the increase of the
polarity of the solvent in protonic solvents (Permittivity: EtOH 24.3,
50
MeOH 32.6, H
2 max
O 78.5), that is lmax (EtOH) > lmax (MeOH) > l
(H
2
O). The effect could be illustrated by hydrogen-bonding inter-
action between the protonic solvents and the dye molecules, which
made the ground state energy of dyes decrease, and consequently
increased the transition energy of dye molecules [26]. The greater
the polarity of protonic solvents was, and the smaller the size of
protonic solvents was, the greater was hydrogen-bonding interac-
tion between the protonic solvents and the dye molecules. It could
0
5
00
550
600
650
700
750
800
Wavelength/nm
Fig. 1. Fluorescence spectra of dye 9a in buffer and in the presence of DNA or BSA.
be also found that the lmax of the dyes exhibited bathochromic

J.-F. Xiang et al. / Dyes and Pigments 93 (2012) 1481e1487
1485
Fig. 4. The optimized configurations and energies of four geometric isomers for dye
b.
Fig. 3. The optimized configurations and energies of four geometric isomers for dye
a.
9
9
shifts with the increase of refractive index in non protonic solvent,
that is max (DMSO) > max (DMF) > max (MeCOMe); The Stokes
3.4. Spectral properties of the dyes in the presence of DNA or BSA
l
l
l
shift of two dyes in water were relatively large, which could help to
reduce self-quenching and measurement error by excitation light
and scattered light [27,28].
The detailed spectral properties of the dyes 9a and 9b, DNA-dyes
and BSA-dyes were measured in buffer (Table 2 and Table 3). From
Table 2 and Table 3, it could be found that absorption maxima of

1486
J.-F. Xiang et al. / Dyes and Pigments 93 (2012) 1481e1487
Table 4
The selected dihedral angles ( ) of dyes 9a and 9b.
ꢁ
Dyes
D(1,3,4,5)a
D(1,3,4,6)a
D(2,3,4,5)a
D(2,3,4,6)a
D(5,8,9,10)a
D(5,8,9,11)a
D(7,8,9,10)a
D(7,8,9,11)a
9
9
a
b
ꢀ7.39
ꢀ7.49
178.42
178.15
172.05
172.03
ꢀ2.13
ꢀ2.33
1.01
ꢀ11.63
ꢀ178.62
ꢀ178.58
1.78
ꢀ7.55
168.34
172.51
a
The atom labels are marked in Scheme 1.
DNA-dye 9a, 9b and BSA-dye 9b showed a slight red shift, which
were situated at 504.0, 569.0 and 564.0 nm, relative to the corre-
sponding maxima of free dyes in buffer, but BSA-dye 9a was an
exception. The molar extinction coefficients for two dyes were all
increased in the presence of DNA or BSA.
Acknowledgment
We appreciate the financial support for this research by a grant
from the Special Science Research Foundation of Education
Committee (No.11JK0558), the Natural Science Foundation of Shaanxi
Province (No. SJ08B04), Key Scientific Research Base open funds of
NWU(ZS11013), NWUGraduateCross-disciplineFunds(No. 09YJC20),
NWU Excellent Doctoral Dissertation Foundation (No. 08YYB04).
From Table 2, it could also be found that dye 9a showed
better fluorescent performance than dye 9b. The reason was that
the compound could emit fluorescence was closely related to the
ꢁ
molecular structure (The selected dihedral angles ( ) of dyes 9a
and 9b were shown in Table 4), the better planar structural
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