Synthesis, absorption and fluorescence spectral characteristics of trinucleus dimethine cyanine dyes as fluorescent probes for DNA detection

Article abstract of DOI:10.1590/S0103-50532011000100009

The preparation of six trinucleus dimethine cyanine dyes with pyridine nucleus obtained by the condensation of trimethylpyridinium iodides with heterocyclic aromatic aldehyde was described. The absorption and fluorescence properties of the dyes were studied in different polarity solvents. Blue shift of the maxima absorption of the dyes was observed with the increase of solvents polarity. The fluorescence properties of the dyes in solution and in presence of DNA were studied. Significant enhancement of the fluorescent quantum yield was observed in four dyes in the presence of DNA. Specially, one of six dyes emitted weak fluorescence in Tris-HCl buffer, but displayed bright fluorescence in the presence of DNA.

Full text of DOI:10.1590/S0103-50532011000100009

J. Braz. Chem. Soc., Vol. 22, No. 1, 73-79, 2011.
Printed in Brazil - ©2011 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Article
A
Synthesis, Absorption and Fluorescence Spectral Characteristics of Trinucleus
Dimethine Cyanine Dyes as Fluorescent Probes for DNA Detection
Jun-Jie Su,a Lan-Ying Wang,*,a Xiang-Han Zhang,a Yi-Le Fu,a
Yi Huangb and Yong-Sheng Weib
aKey Laboratory of Synthetic and Natural Functional Molecule Chemistry (Ministry of Education), College
of Chemistry and Materials Science, Northwest University, Xi’an 710069, People’s Republic of China
bDepartment of Chemistry, Xianyang Normal University, Xianyang, Shaanxi 712000, China
Neste trabalho descreve-se a preparação de seis corantes cianina dimetina trinucleares com
núcleos de piridina, obtidos a partir da condensação de iodeto de trimetilpiridínio com aldeídos
aromáticos heterocíclicos. As propriedades de absorção e fluorescência dos corantes foram
estudadas em solventes com polaridades diferentes. O deslocamento hipsocrômico dos máximos
de absorção dos corantes foi observado com o aumento da polaridade do solvente. Foram estudadas
as propriedades fluorescentes dos corantes em solução e na presença de DNA. Foi observado
um aumento significativo do rendimento quântico de fluorescência na presença de DNA para
quatro corantes.. Em particular, um dos corantes emite fluorescência fraca no tampão Tris-HCl,
apresentando contudo fluorescência intensa na presença de DNA.
The preparation of six trinucleus dimethine cyanine dyes with pyridine nucleus obtained
by the condensation of trimethylpyridinium iodides with heterocyclic aromatic aldehyde was
described. The absorption and fluorescence properties of the dyes were studied in different
polarity solvents. Blue shift of the maxima absorption of the dyes was observed with the increase
of solvents polarity. The fluorescence properties of the dyes in solution and in presence of DNA
were studied. Significant enhancement of the fluorescent quantum yield was observed in four dyes
in the presence of DNA. Specially, one of six dyes emitted weak fluorescence in Tris-HCl buffer,
but displayed bright fluorescence in the presence of DNA.
Keywords: trinucleus dimethine cyanine dyes, DNA, absorption properties, fluorescence
properties, fluorescent dyes
Introduction
dimethine cyanine dyes and their applications as fluorescent
probe were rarely reported.11,12 Our previous efforts have
been devoted to developing series of cyanine dyes and
styryl dyes.13-17
Methine cyanine dyes have been widely researched
and explored as sensitizers in photography1 and as
optical recording materials in laser disk.2 Recently,
methine cyanine dyes have attracted attention owing to
their excellent fluorescence properties3-5 as well as their
potential applications in probes for DNA in living cells.6-10
From the applications of cyanine dyes it is known that the
optical properties of cyanine dyes are often influenced by
their conjugated systems and the solution environment.
Trinucleus dimethine cyanine dyes are large conjugated
systems, which contain three heteroaromatic rings joined
by two vinyl chains. However the synthesis of trinucleus
In this paper, six trinucleus dimethine cyanine dyes
with pyridine nucleus, four dyes of which were novel,
were synthesized in water or ethanol via the condensation
of appropriate heteroaromatic aldehydes with quaternary
salts of heterocyclic compounds containing active methyl
groups. The influence of different solvents on spectral
properties of the trinucleus dimethine cyanine dyes was
studied by UV-Vis and fluorescence spectroscopy.A further
objective of this study was to investigate the fluorescence
spectral properties of prepared trinucleus dimethine cyanine
dyes both in solution and in the DNA presence, exploring
their future applications as fluorescence probe.
*e-mail: wanglany@nwu.edu.cn

74
Synthesis, Absorption and Fluorescence Spectral Characteristics of Trinucleus Dimethine Cyanine Dyes
J. Braz. Chem. Soc.
Experimental
The absorption spectra were examined at room
temperature in different solvents and recorded using 1 cm
quartz cells on a Shimadzu UV-1700 UV-Vis 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 fluorescence excitation spectrum.
The absorption and fluorescence spectral date were listed
in Table 2 and Table 3.
General
All reagents were obtained from commercial sources
and used without further purification. All chemicals were
of analytical grade. Melting points were taken on a XT-4
micromelting apparatus and uncorrected. IR spectra in cm-1
were recorded on Shimadzu IRPrestige-21 spectrometer
and Bruker Equiox-55 spectrometer. 1H NMR spectra were
recorded at 400 MHz on a Varian Inova-400 spectrometer
and chemical shifts were reported relative to internal Me4Si.
13C NMR spectra were recorded at 100 MHz on a Varian
Inova-400 spectrometer and chemical shifts were reported
relative to internal Me4Si. Elemental analysis was performed
withVario EL-III instrument. The electron impact (EI) mass
spectra were recorded at 70 eV with a GCMS-QP2010
system equipped with the solid sample direct insertion
probe. The absorption spectra were recorded on a Shimadzu
UV-1700 UV-Vis spectrometer. Fluorescence measurements
were carried out on a Hitachi F-4500 spectrofluorimeter.
Preparation of trimethyl pyridine quaternary salt 2a-2b
A mixture of 3.54 g (0.033 mol) 1a or 1b with 4.71 g
(0.033 mol) CH3I was refluxed for 20 h. After cooling, the
product was filtered off and purified by recrystallization
from ethanol. Pyridine quaternary salt 2a: 5.1 g, yield 62%,
mp 130-131 °C. 2b: 5.4 g. Yield 66%, mp 235-236 °C.
Preparation of dyes 3a-3b
A mixture of 2a or 2b (0.10 g, 0.4 mmol), indole-
3-carboxaldehyde (0.15 g, 1.0 mmol) and 5 drops of
piperidine was refluxed for 24 h in 8 mL ethanol. After
cooling, ether was added and the product was filtered off,
washed with ether and recrystallized from ethanol:water =
9:1. Yield: 3a: 0.15 g (84%), 3b: 0.16 g (79%).
Measurements of the spectral properties of the dyes in
different solvents
The dye stock solutions (5.0×10-4 mol L-1 in
dimethylsulfoxide (DMSO)) were diluted with different
solvents and resulted in working solutions of dyes (2.0×10-5
mol L-1). The absorption spectra were examined at room
temperature in different solvents and recorded using 1 cm
quartz cells on a Shimadzu UV-1700 UV-Vis 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 absorption. The absorption and
fluorescence spectral data were listed in Table 1.
Preparation of dyes 3c-3d
The dyes 3c-3d were synthesized according to a
revised literature procedure.12 A mixture of 2a or 2b
(0.10 g, 0.4 mmol), 1-methylpyrrole-2-carboxaldehyde
(0.20 g, 1.0 mmol) and 5 drops of piperidine was refluxed
for 20 h in 5 mL ethanol. After cooling, ether was added
and the product was filtered off, washed with ether and
recrystallized from ethanol:water = 9:1. Yield: 3c: 0.17 g
(86%), 3d: 0.15 g (76%).
Measurements of spectral properties of the dyes in the
presence of DNA
Preparation of dyes 3e-3f
Dye stock solutions (2.0×10-4 mol L-1) were prepared
by dissolving the dyes in DMSO and further diluted with
Tris-HCl buffer (pH 7.4) to result in working solutions of
dyes (1.0×10-5 mol L-1). Stock solution of DNA was prepared
by dissolving salmon sperm DNA in 0.05 mol L-1 Tris-HCl
buffer. The concentration of DNA in stock solution was
3.1×10-3 mol L-1 base pairs (bp). The fluorescence of dyes
in the presence of DNA was tested by adding 0.5 mL DNA
solution (3.1×10-3 mol L-1 bp) into 1.0 mL 1.0×10-5 mol L-1
working solutions of dyes. All working solutions were
prepared immediately before the experiment.
Quaternary salts 2a or 2b (0.2 g, 0.8 mmol) and
furaldehyde (0.62 g, 6.4 mmol) were dissolved in 18 mL
H2O. NaOH (20%, 4.6 mL) was added with stirring over
30 min.After 1 h, the product was filtered off, washed with
cold water and recrystallized from water.Yield: 3e: 0.22 g
(69%), 3f: 0.27 g (83%).
1-Methyl-2,4-bis[2-(indole-3-yl)vinyl]pyridinium iodide
(3a)
o
Orange-red crystals with metallic luster, mp 172 C
(decomposition), 1H NMR (DMSO-d6, 400 MHz): d 4.21 (s,

Vol. 22, No. 1, 2011
Su et al.
75
3H, N+CH3), 7.20-7.32 (m, 6H,Ar-H, CH=CH), 7.52-7.54
(m, 2H, Ar-H), 7.89 (d, 1H, J 6.8 Hz, pyridine-H), 7.91-
7.92 (m, 1H,Ar-H), 8.10-8.19 (m, 2H, CH=CH), 8.19-8.25
(m, 3H, indole-H, CH=CH), 8.48 (s, 1H, pyridine-H),
8.54 (d, 1H, J 6.8 Hz, pyridine-H), 11.84 (s, 1H, indole
N-H), 11.96 (s, 1H, indole N-H). 13C NMR (DMSO-d6,
100 MHz): d 43.6, 110.2, 112.1, 113.0, 113.1, 116.7, 117.0,
117.6, 119.7, 120.0, 120.4, 120.6, 122.3, 122.4, 124.5,
131.1, 131.3, 134.3, 136.1, 136.8, 137.0, 143.5, 152.1. IR
(nC=C), 1520, 1409 (s, nC=C, nC=N), 1339, 1264, 1194, 1126,
1086 (dCH), 971, 888, 814, 710 (m, d=CH). MS: EI(70 ev)
m/z (%): 289 (45 M – CH3I), 288 (100 M – CH3I – H), 142,
127. UV-Vis λmax/nm (methanol) 473. Found: C, 53.91;
H, 3.57; N, 5.99. Calc. for C20H22N3I (431.09 g mol-1): C,
53.77; H, 3.61; N, 6.27%.
1-Methyl-2,6-bis[2-(1-methylpyrrol-2-yl)vinyl]pyridinium
iodide (3d)
o
1
n
max/cm-1 (KBr) 3400 (m, n=NH), 3068 (n=C-H), 1597, 1552
Red needle, mp 230 C (decomposition), H NMR
(CDCl3, 400 MHz): d 3.86 (s, 6H, pyrrole N-CH3), 4.28
(s, 3H, N+CH3), 6.21-6.23 (m, 2H, pyrrole-H), 6.79 (s, 2H,
pyridine-H), 6.93-6.99 (m, 4H, CH=CH, pyrrole-H), 7.50
(d, 2H, J 16.0 Hz, CH=CH), 8.08-8.10 (m, 3H, pyridine-H,
pyrrole-H). 13C NMR (DMSO-d6, 100 MHz): d 33.9,
40.9, 109.5, 112.4, 112.7, 121.2, 128.2, 130.3, 141.1,
153.2. IR nmax/cm-1 (KBr) 3446 (m, n=NH), 3073 (n=C-H),
1604, 1559 (nC=C), 1469, 1404 (s, nC=C, nC=N), 1335, 1307,
1228, 1189, 1144, 1090 (dCH), 948, 841, 802, 730 (m,
(nC=C), 1517, 1492 1423 (s, nC=C, nC=N), 1367, 1315,1238,
1128, 1107 (dCH), 957, 744 (m, d=CH). MS: EI (70 ev) m/z
(%): 361 (58 M – CH3I), 360 (100 M – CH3I – H), 142,
127. UV-Vis λmax/nm (methanol) 465. Found: C, 59.0; H,
4.43; N, 7.56. Calc. for C26H22N3I·3/2H2O (530.3 g mol-1):
C, 58.9; H, 4.72; N, 7.92%.
1-Methyl-2,6-bis[2-(indole-3-yl)vinyl]pyridinium iodide
(3b)
Orange-red crystalswith metallic luster, mp 206-207 oC,
1H NMR (DMSO-d6, 400 MHz): d 4.12 (s, 3H, N+CH3),
7.21-7.29 (m, 6H, Ar-H), 7.51-7.54 (m, 2H, CH=CH),
7.85-7.92 (m, 2H, CH=CH), 8.10-8.24 (m, 5H, Ar-H,
pyridine-H), 8.48-8.56 (m, 2H, pyridine-H), 11.86 (s,
1H, indole N-H), 11.97 (s, 1H, indole N-H). 13C NMR
(DMSO-d6, 100 MHz): d 44.0, 110.7, 112.5, 113.3, 113.4,
117.2, 117.4, 118.0, 120.1, 120.4, 120.9, 121.1, 122.7,
122.8, 124.7, 124.9, 131.3, 131.5, 131.7, 134.7, 136.4,
137.1, 137.2, 137.4, 143.9, 152.5. IR nmax/cm-1 (KBr)
3389 (m, n=NH), 3071 (n=C-H), 1598, 1551 (nC=C), 1493
1423 (s, nC=C, nC=N), 1313, 1273, 1237, 1189, 1128 (dCH),
956 (s, n=CH), 809, 745 (m, d=CH). MS: EI (70 ev) m/z (%):
361 (62 M – CH3I), 360 (100 M – CH3I – H), 142, 127.
UV-Vis λmax/nm (methanol) 464. Found: C, 61.50; H, 4.23;
N, 8.13. Calc. for C26H22N3I (503.3 g mol-1): C, 61.85; H,
4.40; N, 8.32%.
d=CH). MS: EI (70 ev) m/z (%): 289 (100 M – CH3I), 288
(58 M – CH3I – H), 142, 127. UV-Vis λmax/nm (methanol)
465. Found: C, 53.88; H, 3.65; N, 6.47. Calc. for C20H22N3I
(431.09 g mol-1): C, 53.77; H, 3.61; N, 6.27%.
1-Methyl-2,4-bis[2-(furan-2-yl)vinyl]pyridinium iodide
(3e)
o
1
Yellow powder, mp 254-255 C, H NMR (CDCl3,
400 MHz): d 4.35 (s, 3H, N+CH3), 6.54-6.55 (m, 2H,
furan-H), 6.91-6.98 (m, 4H, furan-H, CH=CH), 7.56-
7.58 (m, 2H, furan-H), 7.68-7.82 (m, 3H, CH=CH,
pyridine-H), 8.14 (s, 1H, pyridine-H), 9.10 (d, 1H, J
6.8 Hz, pyridine-H). 13C NMR (DMSO-d6, 100 MHz): d
44.4, 112.8, 115.0, 115.7, 119.8, 120.2, 126.4, 128.3, 144.9,
145.7, 146.0, 150.6, 150.8, 150.9. IR nmax/cm-1 (KBr) 3474
(m, n=NH), 3083 (n=C-H), 1608, 1558 (nC=C), 1470, 1442 (s,
nC=C, nC=N), 1386, 1330, 1301 (dCH) 1253(nasC-O-C), 1069(nsC-
O-C), 969, 880,751 (m, d=CH). MS: EI (70 ev) m/z (%): 264
(11 M – CH3I), 263 (53 M – CH3I – H), 142, 127. MS: EI
(70 ev) m/z (%): 264 (11 M – CH3I), 263 (53 M – CH3I – H),
142, 127. UV-Vis λmax/nm (methanol) 397. Found: C, 53.64;
H, 3.60; N, 3.60. Calc. for C18H16NIO2 (405.02 g mol-1): C,
53.35; H, 3.98; N, 3.46%.
1-Methyl-2,4-bis[2-(1-methylpyrrol-2-yl)vinyl]pyridinium
iodide (3c)
Red needle crystals, mp 180-181oC, 1H NMR (CDCl3,
400 MHz): d 3.70-4.01 (m, 9H, pyrrole N-CH3, N+CH3),
6.20 (s 1H, pyrrole-H), 6.24 (s 1H, pyrrole-H), 6.52 (d,
1H, J 15.2 Hz, CH=CH), 6.73 (s, 1H, pyrrole-H), 6.83
(s, 3H, pyrrole-H), 7.08 (d, 1H, J 16.0 Hz, CH=CH)
7.54 (d, 1H, J 16.0 Hz, CH=CH), 7.68 (d, 1H, J 6.8 Hz,
pyridine-H), 8.03 (d, 1H, J 15.2 Hz, CH=CH), 8.28 (d,
1H, J 6.8 Hz, pyridine-H), 8.60 (s, 1H, pyridine-H).
13C NMR (DMSO-d6, 100 MHz): d 34.0, 34.1, 44.2, 109.6,
109.7, 111.1, 111.3, 112.7, 117.6, 117.9, 119.2, 128.0,
128.1, 128.5, 130.2, 130.2, 130.4, 144.0, 151.6, 151.9.
IR nmax/cm-1 (KBr) 3440 (m, n=NH), 3031 (n=C-H), 1597, 1547
1-Methyl-2,6-bis[2-(furan-2-yl)vinyl]pyridinium iodide
(3f)
o
1
Khaki-colored powder, mp 214-215 C, H NMR
(CDCl3, 400 MHz): d 4.37 (s, 3H, N+CH3), 6.54-6.55 (m,
2H, CH=CH), 6.93-6.94 (m, 2H, furan-H), 7.21-7.27 (m,
2H, CH=CH), 7.50-7.56 (m, 4H, furan-H), 8.08 (d, 2H,
J 8.8 Hz, pyridine-H), 8.28 (t, 1H, J 8.8 Hz, pydidine-H).
13C NMR (DMSO-d6, 100 MHz): d 41.1, 112.7, 115.4,

76
Synthesis, Absorption and Fluorescence Spectral Characteristics of Trinucleus Dimethine Cyanine Dyes
J. Braz. Chem. Soc.
115.5, 122.8, 128.4, 142.3, 145.7, 150.6, 152.2. IR nmax/cm-1
(KBr) 3479 (m, n=NH), 3061 (n=C-H), 1602, 1561(nC=C), 1463,
1384 (s, nC=C, nC=N), 1305 (dCH) 1247(nasC-O-C) 1068(nsC-O-C),
951, 982, 878, 757 (m, d=CH). MS: EI (70 ev) m/z (%): 264
(8 M – CH3I), 263 (39 M – CH3I – H), 142, 127. UV-Vis
furaldehyde (30 min) < 1-methylpyrrole-2-carboxaldehyde
(20 h) < indole-3-carboxaldehyde (24 h), thereby the
reaction activity of heterocyclic aromatic aldehydes was
furaldehyde > 1-methylpyrrole-2-carboxaldehyde > indole-
3-carboxaldehyde. From the structure it was also suggested
that the electron-withdrawing ability of the group attached
to aldehyde group was =C−O > =C−N > =C−C=, leading
to the positive charge density of carbon in aldehyde group
was furaldehyde > 1-methylpyrrole-2-carboxaldehyde >
indole-3-carboxaldehyde. The larger the positive charge
density of carbon in aldehyde group is, the higher the
reaction activity of heterocyclic aromatic aldehyde with
trimethylpyridinium iodides is.
λ
max/nm (methanol) 397. Found: C, 53.31; H, 3.60; N,
3.76. Calc. for C18H16NIO2 (405.02 g mol-1): C, 53.35; H,
3.46; N, 3.98%.
Results and Discussion
Synthesis
The trinucleus dimethine cyanine dye was synthesized
via the reaction of heteroaromatic aldehydes with the
quaternary ammonium salt of 1,2,4-trimethyl pyridine
quaternary salt or 1,2,6- trimethyl pyridine quaternary salt
(Scheme 1). In all cases investigated, we found that the
formation reactions of trinucleus dimethine cyanine dyes
with pyridine nucleus proceeded efficiently under catalysis
of piperidine or NaOH, and the better and purer yields
were obtained in ethanol for 3a-3d and in water for 3e-3f.
For example, the yield of 3d could reach 86% in ethanol,
and 65% in water.12 The yield of 3f could reach 83% in
water, but in ethanol 3f could hardly be obtained. Because
in organic solvents furaldehyde reacted with quaternary
salt so quickly that the reaction could not be controlled,
and there would be serious side reactions. In the synthesis
of dyes 3a-3f , the required reaction time of heterocyclic
aromatic aldehydes with trimethylpyridinium iodides was
Spectral properties of the dyes in different solvents
Figure 1 gives the absorption maxima (λmax) of six
trinucleus dimethine cyanine dyes in different solvents
(their dielectric constant: CHCl3 4.9, EtOH 24.6, MeOH
32.6, dimethylformamide 38.3, H2O 78.4). From Figure 1 it
could be found that the λmax of 3a, 3b, 3c and 3d was longer
than the λmax of 3e and 3f. The reason suggested here was
that dyes 3a-3f were all D-π-A molecules, in dyes 3a-3d
the electron donor was N of indole or pyrrole ring, and
the electron donor was O of furan ring in dyes 3e-3f, and
the electron acceptor of six dyes was all N+ of pyridinium.
The stronger was electron-donating capability of electron
donor, the longer was the λmax of dyes under the same
electron-withdrawing capability of electron acceptor. The
electron-donating capability of N situated indole or pyrrole
Ar
CH3
CH3
CHO
N
Ar
N
CH3
I
CH3
or
CH3
I
N
CH3
CHO
Ar
N
O
CH3I
H
+ 2H2O
3a, 3c, 3e
1a
2a
or
N
CHO
N
Ar
H3C
N
CH3
I
CH3
N
CH3
CH3
CH3
I
CH3
3b, 3d, 3f
2b
1b
3a, 3b: Ar = indole-3-carboxaldehyde
3c, 3d:
3e, 3f:
Ar = 1-methylpyrrole-2-carboxaldehyde
Ar = furaldehyde
Scheme 1. Synthesis of trinucleus dimethine cyanine dyes.

Vol. 22, No. 1, 2011
Su et al.
77
Table 1. The absorption and fluorescence spectral characteristics of dyes
3a-3f in different solvents
ring was stronger than that of O situated furan ring, so the
λmax of 3a-3d was longer than the λmax of 3e-3f. It could
be also found that with the increasing of solvent polarity
the λmax of the dyes decreased. The effect of the solvent
polarity on the absorption maximum could be illustrated by
interactions between the dye molecules and the solvents, as
the interactions made the ground state of dye more stable
by forming hydrogen bonds.18,19
λmax/nm λex/nm
λem/nm
Stokes
shift
ΦF
Methanol
3a
465
464
473
465
397
397
465
464
473
465
397
397
550
551
559
556
517
508
85
87
0.0538
0.0310
0.0031
0.0027
0.0289
0.0115
3b
3c
86
3d
91
3e
120
112
3f
Ethanol
3a
470
468
477
468
400
400
470
467
477
468
400
400
552
550
561
560
518
540
82
82
0.0781
0.0689
0.0191
0.0039
0.0029
0.0028
3b
3c
84
3d
92
3e
118
140
3f
Chloroform
3a
487
484
485
476
415
415
487
484
484
476
415
415
556
557
559
558
502
509
70
73
74
82
87
94
0.0470
0.0403
0.0098
0.0023
0.0110
0.0037
Figure 1. Solvatochromic shifts for the λmax of six trinucleus
dimethincyanine dyes in five kinds of solvents.
3b
3c
3d
The absorption, excitation maxima (λex), fluorescence
maxima emission (λem) of the dyes are summarized in
Table 1. The dyes exhibited fluorescence properties at
room temperature. Their fluorescence maxima were located
at 502-565 nm. Compared with the absorption maxima
of the dyes, the emission spectra were shifted to the red
by 69-140 nm (Stokes shift). The stokes shift of six dyes
were all large, this might be attributed to an excited-state
intramolecular charge transfer between the donor and
acceptor in the dyes. Large stokes shift could help to reduce
self-quenching and measurement error by excitation light
and scattered light.20 The fluorescence quantum yield of six
dyes was in the region 0.0001-0.0781 in different solvents.
FromTable 1 it could be found that the order of fluorescence
quantum yield was 3a, 3b > 3c, 3d. The possible reason
was that the conjugated system of 3a, 3b was larger than
that of 3c, 3d. We also found the order of fluorescence
quantum yield was 3a > 3b and 3c > 3d. The cause lay in
the fact that two D-π-A system in 3b and 3d was in opposite
orientation, which made their conjugated system became
small. It could be found that the fluorescence performance
of 3e and 3f was complex, because oxygen of furan ring
was of both electron-donating and electron-acceptor effect.
And there was also influence of solvents on the fluorescence
quantum yield of these dyes. Further investigation of the
influence of solvents on the fluorescence quantum yield of
the dyes is in progressing.
3e
3f
Dimetilformamida
3a
3b
3c
464
463
473
470
403
407
464
463
473
470
402
407
557
556
563
565
532
518
93
93
0.0298
0.0275
0.0060
0.0008
0.0148
0.0052
90
3d
3e
95
129
111
3f
Water
3a
3b
3c
447
447
456
448
402
399
447
447
456
448
402
399
554
553
562
541
529
515
107
106
106
93
0.0008
0.0008
0.0003
0.0001
0.0032
0.0016
3d
3e
127
116
3f
a The fluorescence quantum yields of the dyes were determined by the
reference standard (rhodamine B ΦF=0.49 in ethanol at 25 oC).21
Spectral properties of the dyes in the presence of DNA
The spectral properties of the dyes in the presence of
DNA are summarized in Table 2 and Table 3. The λmax of
DNA-dye solution was situated at 401-458 nm and showed
a slight red shift relative to the corresponding maxima of

78
Synthesis, Absorption and Fluorescence Spectral Characteristics of Trinucleus Dimethine Cyanine Dyes
J. Braz. Chem. Soc.
Table 2. Spectral characteristics of dyes 3a-3f in buffer
Dye
in buffer
free
λ
max (nm)
ε
free×10-4 (dm3 mol-1 cm-1)
λex (nm)
λ
em (nm)
565
560
564
-
ΦF
447
2.2
2.1
5.5
4.8
3.8
4.2
445
445
453
448
406
406
0.0235
0.0041
0.0042
-
3a
3b
3c
3d
3e
3f
447
456
448
402
519
504
0.0557
0.0323
399
a The fluorescence quantum yields of the dyes were determined by the reference standard (rhodamine B ΦF = 0.49 in ethanol at 25oC).21
Table 3. Spectral characteristics of dyes 3a-3f in presence of DNA
in DNA presence
free
Dye
ΦFDNA/ΦF
DNA
λ
max (nm)
ε
DNA×10-4 (dm3 mol-1 cm-1)
λex (nm)
445
λ
em (nm)
545
ΦF
3a
3b
3c
3d
3e
3f
455
3.2
6.2
8.5
5.1
3.8
4.2
0.2242
0.1291
0.0791
0.0187
0.0553
0.0336
9.5
31.6
18.9
-
454
445
545
458
453
555
449
448
553
403
406
515
1.0
1.0
401
406
503
a The fluorescence quantum yields of the dyes were determined by the reference standard (rhodamine B ΦF = 0.49 in ethanol at 25oC).21
free dyes in buffer. The molar extinction coefficients for
dyes 3a-3d were increased in the presence of DNA, and
were in the range from 3.2×104 to 8.5×104 L mol-1 cm-1.
However, for dyes 3e and 3f the values of molar extinction
coefficients were unchanged relative to free dyes, which
were 3.8×104 and 4.2×104 L mol-1 cm-1, respectively.
The fluorescence emission maxima of DNA-dyes were
located at 503-555 nm, and showed a slight blue shift relative
to free dyes in solution. Stokes shift values for the DNA-dyes
were in the range of 97-109 nm. Moreover, the fluorescence
intensity of dyes 3a-3c was greatly increased in the presence
of DNA. Compared with free dyes, the quantum yields
of DNA-dyes were up to 9.5 times for dye 3a, up to 31.6
times for dye 3b (Figure 2) and up to 18.9 times for dye 3c.
Specially, thequantumyieldsofDNA-dye 3awasthehighest
in six dyes. It was noteworthy that free dye 3d could not be
detectedsignificantlyfluorescenceinbuffer, butDNA-dye3d
showed great fluorescence quantum yields. The fluorescence
enhancement of trinucleus dimethine cyanine dyes bound
to DNA was attributable to the fact that on photoexcitation
a lack of free rotation around the internuclear bridge made
isomerisation around the C-C bonds of the methine chain
difficult; and subsequently nonradiative deactivation of
the excited state was not possible, causing the dye to
fluoresce.22,23 Upon binding with DNA, 3a-3d maintained
Figure 2. Fluorescence spectra of 3b in buffer, in the presence of different
concentration of DNA.
their high Stokes shift and showed a red shift, owing to a
more efficient ICT in the excited state between the terminal
heterocyclic aromatic and the pyridimium group.24,25
Conclusions
Six trinucleus dimethine cyanine dyes with pyridine
nucleus were synthesized and isolated in 69-86% yield
with piperidine or NaOH as catalyst.

Vol. 22, No. 1, 2011
Su et al.
79
The absorption maxima of the dyes were located at
397-487 nm in different solvents, and with the increase
of solvents polarity the maximum absorption wavelength
of these dyes had a blue-shift. The fluorescence maxima
of the dyes in different solvents were basically located at
502-565 nm, and the fluorescence quantum yield of six
dyes was in the region 0.0001-0.0781 in different solvents.
The absorption maxima of the dyes in the presence of
DNA were situated at 401-458 nm and showed a slight
red shift relative to free dyes. The fluorescence maxima of
DNA-dyes were located at 503-555 nm, and showed a slight
blue shift relative to free dyes. The fluorescent quantum
yields of the DNA-dyes 3a-3c were up to 9.5-31.6 times
higher than that of free dyes. Specially, free dye 3d emitted
weak fluorescence in buffer, but DNA-dye 3d showed great
fluorescence quantum yields. Therefore dyes 3a-3d could
be proposed as fluorescent dyes for DNA detection.
5. Kovalska, V.; Kryvorotenko, D.; Balanda, A.; Losytskyy, M.;
Tokar, V.; Yarmoluk, S.; Dyes Pigm. 2005, 67, 47.
6. Karlsson, H.; Bergqvist, M.;, Lincoln, P.; Westman, G.; Bioorg.
Med. Chem. 2004, 12, 2369.
7. Timtcheva, I.; Maximova, V.; Deligeorgiev, T.; Zaneva, D.;
Ivanov, I.; J. Photochem. Photobiol., A 2000, 130, 7.
8. Hilal, H.; Taylor, J.; Dyes Pigm. 2007, 75, 483.
9. Rosania, G. R.; Lee, J. W.; Ding, L. Yoon, H. S.; Chang, Y. T.;
J. Am. Chem. Soc. 2003, 125, 1130.
10. Li, Q.; Kim, Y.; Namm, J.; Kulkarni, A.; Rosania, G.. R.; Ahn,
Y. H.; Chang, Y. T.; Chem. Biol. 2006, 13, 615.
11. Vincenza, B.; Daniele, F.; Condorelli, C.; Giuseppe, M.; Bioorg.
Med. Chem. 2002, 10, 2899.
12. Maria, F.; Cosimo, G. F.; Giuseppe, I.; Giuseppe, M.; Eur. J.
Org. Chem. 2002, 145.
13. Wang, L.Y.; Zhang, X. G.; Shi,Y. P.; Zhang, Z. X.; Dyes Pigm.
2004, 62, 21.
14. Wang, L. Y.; Zhang, X. G.; Li, F. M.; Zhang, Z. X.; Synth.
Commun. 2004, 34, 1.
Supplementary Information
15. Zhang, Z. X.; Zhang,Y. J.; Hao, J. X.; Li, C. E.; Sci. China, Ser.
B: Chem. 1995, 25, 689.
Supplementary data are available free of charge at
http://jbcs.sbq.org.br, as PDF file.
16. Zhang, X .H.; Wang, L. Y.; Zhai, G.. H.; Wen, Z. Y.; Zhang, Z.
X.; Bull. Korean Chem. Soc. 2007, 28, 12.
Acknowledgements
17. Huang, W.; Wang, L. Y.; Fu, Y. L.; Liu, J. Q.; Tao, Y. N.; Fan,
F. L.; Zhai, G.. H.; Wen, Z. Y.; Bull. Korean Chem. Soc. 2009,
30, 3.
We appreciate the financial support for this research by
a grant from the Natural Science Foundation of Shaanxi
Province (No. SJ08B04), the Special Science Research
Foundation of Education Committee (No. 08JK458),
NWU Excellent Doctoral Dissertation Foundation (No.
08YYB04) and NWU Graduate Cross-discipline Funds
(No. 09YJC20).
18. Pham, W.; Lai, W. F.;Weissleder, R.; Tung, C. H.; Bioconjugate
Chem. 2003, 14, 1048.
19. Mishra,A.; Behera, R. K.; Behera, P. K.; Mishra, B. K.; Behera,
G. B.; Chem. Rev. 2000, 100, 1973; Ibrahim, A. Z.; Al-Ansari,
I.; Bull. Soc. Chim. Fr. 1997, 134, 593.
20. Zhang, Z.; Achilefu, S.; Org. Lett. 2004, 6, 2067.
21. Casey, K. G.; Quitevis, E. L.; J. Phys. Chem. 1988, 92, 6590.
22. Carlsson, C.; Larsson, A.; Jonsson, M.; Albinsson, B.; J. Phys.
Chem. 1994, 98, 10313.
References
1. Karatsu, T.; Yanai, M.; Yagai, S.; Mizukami, J.; Urano, T.;
Kitamura, A.; J. Photochem. Photobiol., A 2005, 170, 123.
2. Usami, T.; Asanuma, N.;Yamakawa, K.; JP Pat 265,076 2000.
3. Kovalska, V.; Volkova, K.; Losytskyy, M.; Tolmachev, O.;
Balanda, A.; Yarmoluk, S.; Spectrochim. Acta, Part A 2006,
65, 271.
23. Anikovsky, M.Y.; Tatikolov, A. S.; Shvedove, L. A.; Kuzmin
V. A.; Russ. Chem. Bull. 2001, 50, 1190.
24. Neto, B. A. D.; Lápis, A. A. M.; Mancilha, F. S.; Vasconcelos,
I. B.; Thum, C.; Basso, L. A.; Santos, D. S.; Dupont, J.; Org.
Lett. 2007, 20, 4001.
25. Neto, B. A. D.; Lápis, A. A. M.; Molecules 2009, 14, 1725.
4. Deligeorgiev, T.; Gadjev, N.; Vasilev, A.; Maximova, V.;
Timcheva, I.; Katerinopoulos, H.; Dyes Pigm. 2007, 75, 466.
Submitted: April 17, 2010
Published online: August 10, 2010

J. Braz. Chem. Soc., Vol. 22, No. 1, S1-S11, 2011.
Printed in Brazil - ©2011 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Supplementary Information
SI
Synthesis, Absorption and Fluorescence Spectral Characteristics of Trinucleus
Dimethine Cyanine Dyes as Fluorescent Probes for DNA Detection
Jun-Jie Su,a Lan-Ying Wang,*,a Xiang-Han Zhang,a Yi-Le Fu,a
Yi Huangb and Yong-Sheng Weib
aKey Laboratory of Synthetic and Natural Functional Molecule Chemistry (Ministry of Education), College
of Chemistry and Materials Science, Northwest University, Xi’an 710069, People’s Republic of China
bDepartment of Chemistry, Xianyang Normal University, Xianyang, Shaanxi 712000, China
For Fluorescence emission of 3a-3f in buffer, in the presence of DNA see Figure S1-S6. For 1H NMR, 13C NMR and
IR see Figure S6-S24.
Figure S3. Fluorescence emission of 3c in buffer, in the presence of DNA.
a: 3c in buffer. b: 3c in the presence of DNA.
Figure S1. Fluorescence emission of 3a in buffer, in the presence of DNA.
a: 3a in buffer. b: 3a in the presence of DNA.
Figure S2. Fluorescence emission of 3b in buffer, in the presence of DNA.
a: 3b in buffer. b: 3b in the presence of DNA.
Figure S4. Fluorescence emission of 3d in buffer, in the presence of DNA.
a: 3d in buffer. b: 3d in the presence of DNA.
*e-mail: wanglany@nwu.edu.cn

S2
Synthesis, Absorption and Fluorescence Spectral Characteristics of Trinucleus Dimethine Cyanine Dyes
J. Braz. Chem. Soc.
Figure S6. Fluorescence emission of 3f in buffer, in the presence of DNA.
a: 3f in buffer. b: 3f in the presence of DNA.
Figure S5. Fluorescence emission of 3e in buffer, in the presence of DNA.
a: 3e in buffer. b: 3e in the presence of DNA.
Trinucleus dimethine cyanine dye 3a
Figure S7. 1H NMR spectrum of 3a.

Vol. 22, No. 1, 2011
Su et al.
S3
Figure S8. 13C NMR spectrum of 3a.
Figure S9. IR spectrum of 3a.

S4
Synthesis, Absorption and Fluorescence Spectral Characteristics of Trinucleus Dimethine Cyanine Dyes
J. Braz. Chem. Soc.
Trinucleus dimethine cyanine dye 3b
Figure S10. 1H NMR spectrum of 3b.
Figure S11. 13C NMR spectrum of 3b.

Vol. 22, No. 1, 2011
Su et al.
S5
Figure S12. IR spectrum of 3b.
Trinucleus dimethine cyanine dye 3c
Figure S13. 1H NMR spectrum of 3c.

S6
Synthesis, Absorption and Fluorescence Spectral Characteristics of Trinucleus Dimethine Cyanine Dyes
J. Braz. Chem. Soc.
Figure S14. 13C NMR spectrum of 3c.
Figure S15. IR spectrum of 3c.

Vol. 22, No. 1, 2011
Su et al.
S7
Trinucleus dimethine cyanine dye 3d
Figure S16. 1H NMR spectrum of 3d.
Figure S17. 13C NMR spectrum of 3d.

S8
Synthesis, Absorption and Fluorescence Spectral Characteristics of Trinucleus Dimethine Cyanine Dyes
J. Braz. Chem. Soc.
Figure S18. IR spectrum of 3d.
Trinucleus dimethine cyanine dye 3e
Figure S19. 1H NMR spectrum of 3e.

Vol. 22, No. 1, 2011
Su et al.
S9
Figure S20. 13C NMR spectrum of 3e.
Figure S21. IR spectrum of 3e.

S10
Synthesis, Absorption and Fluorescence Spectral Characteristics of Trinucleus Dimethine Cyanine Dyes
J. Braz. Chem. Soc.
Trinucleus dimethine cyanine dye 3f
Figure S22. 1H NMR spectrum of 3f.
Figure S23. 13C NMR spectrum of 3f.

Vol. 22, No. 1, 2011
Su et al.
S11
Figure S24. IR spectrum of 3f.