Study on the synthesis and spectra of a novel kind of carbozole benzothiazole indole styryl cyanine dye with a carbazole bridged Chain

Article abstract of DOI:10.1007/s10895-013-1327-2

Based on the frequently-used cyanine dye probe thiazole orange (TO) and Cy3, a novel kind of styryl cyanine dye was designed and synthesized. Carbazole was inserted into the structures of two cyanine dyes, TO and Cy3, to act as a bridge to link the benzothiazole and indole. This modification resulted in a novel kind of carbozole benzothiazole indole cyanine dye with a carbazole-bridged chain. The dyes were characterized by HNMR and MS. The spectra of the novel dyes were also studied and the results showed that the fluorescence wavelength of novel carbazole benzothiazole indole cyanine dye shifted red, the Stokes shift and Fluorescence quantum yields increased and the fluorescence intensity was enhanced compared to that of TO. These results indicated that the novel dye could be used as an excellent fluorescent probe in biological labeling.

Full text of DOI:10.1007/s10895-013-1327-2

J Fluoresc
DOI 10.1007/s10895-013-1327-2
ORIGINAL PAPER
Study on the Synthesis and Spectra of a Novel Kind
of Carbozole Benzothiazole Indole Styryl Cyanine
Dye with a Carbazole Bridged Chain
Xuening Fei & Yachao Hao & Yingchun Gu & Chao Li &
Lu Yu
Received: 2 September 2013 /Accepted: 7 November 2013
#
Springer Science+Business Media New York 2013
Abstract Based on the frequently-used cyanine dye probe
thiazole orange (TO) and Cy3, a novel kind of styryl cyanine
dye was designed and synthesized. Carbazole was inserted
into the structures of two cyanine dyes, TO and Cy3, to act as
a bridge to link the benzothiazole and indole. This modifica-
tion resulted in a novel kind of carbozole benzothiazole indole
cyanine dye with a carbazole-bridged chain. The dyes were
characterized by HNMR and MS. The spectra of the novel
dyes were also studied and the results showed that the fluo-
rescence wavelength of novel carbazole benzothiazole indole
cyanine dye shifted red, the Stokes shift and Fluorescence
quantum yields increased and the fluorescence intensity was
enhanced compared to that of TO. These results indicated that
the novel dye could be used as an excellent fluorescent probe
in biological labeling.
strong fluorescence and easily modified by introduction of
different kinds of functions.
Thiazole Orange (TO), an embedded cyanine dye, is com-
prised of a benzothiazole ring covalently linked to a quinoline
via a monomethine bridge which has been widely used for
labeling nucleic acids for detection of DNA and RNA in gels
by flow cytometry or microscopy [6]. Although the fluores-
cence of free TO is extremely low in aqueous solutions, the
viscosity of the dye’s local environment is markedly increased
when it is bound to nucleic acids, resulting in a dramatic
increase in fluorescence intensity [7–10]. The large difference
in fluorescence between free dye and nucleic acid-bound dye
suggests an excellent way to image, label and detect cancer
cells [11–19].
The advantages of the near-IR dyes for bioanalytical appli-
cations include their excellent spectral properties in the near-
infrared region (700–1,000 nm) with minimal background
attributed to biomolecules and high sensitivity. The indole
cyanine dye Cy3, Cy5 and Cy7 dyes have been used in the
labeling of a variety of biological/biomedical macromolecules
and nanoparticles [20–22]. And also other new IR dyes were
designed and synthesized by many scientists [23–27].
The fluorescent properties of dyes mainly depend on their
chemical structures, such as conjugate system, coplanarity and
rigidity. in order to reduce the background disturbances of the
fluorescent probe and light scattering, the fluorescent dye
should be with longer conjugate system, stronger fluorescence
intensity, redshift the fluorescent emission wavelength, bigger
Stokes shift and fluorescence quantum yields. However, this
reduces photostability [28].
.
.
Keywords Carbazole bridge chain Benzothiazole Indole
.
styryl cyanine dye Fluorescent spectrum
Introduction
Because of the excellent properties as an electron rich system,
carbazole and its derivatives are widely used in the fields of
dyes, medicine[1], biology [2, 3], photoelectricity [4, 5] and
more others owing to their advantages of possessing a large
rigidity plan conjugated system, aromatic structures with
:
X. Fei Y. Hao
Recently, we studied the synthesis and properties of the
cyanine dye TO and its derivatives [29–33].
School of Chemical Engineering and Technology, Tianjin University,
No.26 Jinjing Road, Xiqing District, Tianjin 300072, China
In order to make the conjugated system lengthen with larger
Stokes shift and stronger fluorescence intensity, carbazole was
inserted into the methylidyne structure as a bridge to obtain a
novel carbazole cyanine dye with a carbazole bridged chain.
:
:
:
X. Fei Y. Gu (*) C. Li L. Yu
School of Science, Tianjin Chengjian University, Tianjin 300384,
China
e-mail: jvgugugu@126.com

J Fluoresc
Choosing the benzothiazole of TO and indole of Cy3,
carbazole was inserted into the structures of two cyanine dyes,
TO and Cy3, as a bridge to link the benzothiazole and indole
in order to generate a novel kind of carbozole benzothiazole
indole styryl cyanine dye with a carbazole bridged chain. With
the introduction of carbazole, the stability of the novel probes
increased, fluorescence wavelength shifted red, the Stokes
shift and Fluorescence quantum yields increased and the
fluorescence intensity was enhanced. The design process of
the novel probe was shown in Fig. 1.
wavelength, respectively. The standard used in this study is
rhodamine B of 5 μg/mL in absolute ethanol with a known
fluorescence quantum yield of 0.97 in this condition [35].
Synthesis
Synthesis of 3-Benzothiazole-6-Formyl Carbazole(4)
3-benzothiazole-6-formyl carbazole was synthesized by alkyl-
ation, formylation, ring closing reaction and formylation from
carbazole. The compound 4 was synthesized according to the
literature method [30].
Materials and Methods
Synthesis of N- Propionic Acid-2,3,3-Trimethyl-3H-Indole
Materials and Instruments
Derivatives of N-propionic acid-2,3,3-trimethyl-3H-indole
(6a–6c) were synthesized and the synthetic route was depicted
in Scheme 1.
Fluorescence spectra were scanned on a Cary Eclipse fluores-
cence spectrophotometer (Varian, USA). The UV–vis spectra
were recorded on a Shimazu 2550 spectrophotometer (Japan).
American Mass spectral analyses were obtained using an
1 mol Phenylhydrazinium chloride and 150 mL glacial
acetic acid were added to a flask-3-neck under a flow of
nitrogen. The solution was stirred and refluxed in the dark
and 44 mL 3-methyl-2-butanone was added to be refluxed for
another 8 h. After the solvent was removed using a rotary
evaporator, the residue was washed with saturated sodium
bicarbonate solution to adjust the pH value to a final pH
of about 7. The crude product was extracted with chloro-
form. The organic layer was then dried with anhydrous
magnesium sulfate overnight. After the solvent was evap-
orated, the remaining solid was purified by column chro-
matography with eluent of dichloromethane/petroleum
ether=1:3 (v/v) to afford product.
1
electrospray ionization (ESI) mass spectrometer. HNMR
spectra were recorded on a Bruker (300 MHz or 400 MHz)
spectrometer. Chemical shifts were reported in parts per mil-
lion (ppm) downfield from TMS (tetramethylsilane) using
CDCl3 or DMSO-d6 as a solvent.
Fluorescence Quantum Yields
Fluorescence quantum yields (Φf) were determined by com-
parison with a standard solution with the following equation
to calculate relative fluorescence quantum yields [34].
The intermediates 6a-6c were synthesized according to the
procedure as follows.
ꢀ
ꢁ
2
nX
nS
AS FX
⋅
ϕX ¼ ϕS⋅
⋅
ð1Þ
AX FS
2, 3, 3-trimethylindole derivative 5a-5c (0.02 mol) and 3-
bromopropanoic acid (0.03 mol) were refluxed in 1,2-dichlo-
robenzene for 12 h. The mixture was cooled to room temper-
ature and poured into ethyl acetate. The precipitate was col-
lected by filtration, washed with acetone and dried under
vacuum to yield compound 6.
where nX and nS are the refractive indexes of the sample and
reference, FX and FS are the integrated fluorescence spectra
for the sample and reference respectively, and Ax and AS are
the absorbance for the sample and reference at the excitation
Fig. 1 Design of novel styryl
cyanine dye with carbazole on the
methylidyne bridge chain
R2
N
R2
Br
N
HOOC
S
N
COOH
R1
N
C
H
Br
S
R2
N
N
N
N
Br
COOH

J Fluoresc
R
Scheme 1 Synthesis of N-
Hexanoic acid-2,3,3–trimethyl-
3H-indole
R
CH3CH(CH3)COCH3
R
BrCH2CH2COOH
CH3COOH, reflux
N
N
5
1,2-dichlorobenzene, reflux
Br
NHNH2 HCl
6
COOH
6a:R=H
6b:R=CH3
6c:R=Cl
5a:R=H
5b:R=CH3
5c:R=Cl
6a (300 MHz, DMSO-d6) δ: 1.53(s, 6H), 2.95(s, 3H),
2.95–3.00(t, J =6.9 Hz, 2H), 4.62–4.66(t, J =6.9 Hz, 2H),
7.58–7.61(m, 2H), 7.80–7.83(m, 1H), 7.95–7.98(m, 1H).
ESI-MS(m/z): 232.1[M+] , 233.2 [M++1].
6b (300 MHz, DMSO-d6) δ: 1.50(s, 6H), 2.50(s, 3H), 2.81
(s, 3H), 2.93–2.97(t, J =7.0 Hz, 2H), 4.58–4.63(t, J =6.9 Hz,
2H), 7.40(d, J =8.4 Hz, 1H), 7.62(s, 1H), 7.84(d, J =8.4 Hz,
1H). ESI-MS(m/z): 246.0[M+], 247.1 [M++1].
6c (300 MHz, DMSO-d6) δ: 1.53(s, 6H), 2.84(s, 3H),
2.93–2.97(t, J =6.7 Hz, 2H), 4.60–4.64(t, J =6.7 Hz, 2H),
7.67–7.71(m, 1H), 8.00–8.04(m, 2H). ESI-MS(m/z):
266.0[M+], 268.1 [M++2].
7b (300 MHz, CDCl3) δ: 0.85–0.88(t, J=7.20Hz, 3H),1.72(s,
6H), 2.43(s, 3H), 3.01–3.04(m, 2H), 4.31–4.34(m, 2H), 4.89–
4.92(m, 2H), 7.33–7.56 (m, 6H), 7.69–7.71(m, 1H), 7.82–
7.94(m, 2H), 8.02–8.20(m, 4H), 8.74(s, 1H), 8.88(s, 1H),
10.11(s, 1H). ESI-MS(m/z): 584[M+], 585[M++1].
7c (300 MHz, CDCl3) δ: 1.43–1.48(t, J =6.9 Hz, 3H),
1.56(s, 6H), 2.86(s, 2H), 4.31–4.38(q, J =6.7 Hz, 2H), 4.44–
4.54(m, 2H), 7.21(s,1H), 7.30–7.55(m, 7H), 7.74
(d, J=12.30Hz, 1H), 7.84–7.91(m, 2H),8.01(d, J=7.80Hz,
1H), 8.18(d, J =8.4 Hz, 1H), 8.68–8.70(d, J=7.20Hz, 1H),
8.86(s, 1H), 10.10(s, 1H). ESI-MS(m/z): 604.4[M+],
605.4.4[M++1].
Synthesis of Carbozole Benzothiazole Indole Cyanine Dye
7a–7c
Results and Discussion
The compounds 7a–7c were obtained by Knoecenaget con-
densation (Scheme 2).
Synthesis
The typical procedure for 7a–7c was: aldehyde 4 (0.10 g,
0.28 mmol) in 30 mL CH3CH2OH and salt 6 (0.13 g,
0.42 mmol) in 20 mL CH3CH2OH were added to a 100 mL
flask, followed by catalytic piperdine (1–3 drop). The
resulting mixture was allowed to be refluxed and stirred for
12 h. When it was cooled to room temperature, CH3COOH
was added and the mixture was stirred for an additional 2 h.
Finally, ether was added to generate a red solid, which was
then filtered, washed by ether and water to yield compound 7.
7a (300 MHz, CDCl3) δ: 0.85–0.87(t, J=6.00Hz, 3H),
1.53(s, 6H), 2.67(d, 2H), 4.19–4.37(m, 4H),
7 . 3 2 – 7 . 4 0 ( m , 2 H ) , 7 . 4 6 – 7 . 5 4 ( m , 5 H ) , 7 . 8 9
(d, J =7.5 Hz, 2H), 8.05(t, J =6.9 Hz, 2H), 8.21(s, 1H),
8.40(s, 1H), 8.85(d,J=11.1Hz, 1H), 10.10(s, 1H). ESI-
MS(m/z): 570.4[M+], 571.4[M++1].
In order to enhance the fluorescence intensity, Stokes shift and
the stability of TO, carbazole was inserted into the
methylidyne structure of TO and Cy3 as a bridge to generate
a novel carbozole benzothiazole indole cyanine dye with a
carbazole bridged chain. First, 3-substituted of carbazole was
formoxylated, reacted with aminothiopheno to afford 3-
benzothiazoleN-ethyl carbazole, which was further
formoxylated to prepare 3-benzothiazole-6- formoxyl-N-
ethyl carbazole.
During the process of formoxylation, POCl3 should be
reacted completely. However, a residual amount remained that
could not be reacted. The workup should be performed care-
fully. The reaction mixture should be poured into ice water
slowly and stirred.
Scheme 2 Benzothiazole indole
styryl cyanine dyes with
carbazole on the methylidyne
bridge chain
S
S
N
H
CHO
+
R
R
N
N
N
N
6
EtOH
Br
N
N
Br
COOH
COOH
4
7
7a:R=H
7b:R=CH3
7c:R=Cl
6a:R=H
6b:R=CH3
6c:R=Cl

J Fluoresc
The formoxyl reacted with the active methylene com-
pounds by Knoecenaget condensation to afford C=C and
the title compound with the carbazole bridged chain could
be obtained.
Effect of the Substitutional Groups of the Quinoline Side
Chain on the UV–vis Absorption Properties of Carbozole
Benzothiazole Indole
UV–vis absorption properties of carbozole benzothiazole in-
dole with a carbazole bridged chain could be affected by
substitutional groups on the quinoline side chain. In this paper,
the fluorescent spectra of 7a, 7b and 7c with the same
concentration(0.0035mmoL/L) in CH3OH were obtained
and presented in Fig. 3
There were two peaks at 310 nm and 500 nm respectively in
the UV–vis absorption figures. Due to various vibrational and
rotational states, these molecules have broad absorption peaks
[37]. Compounds 7a-7c with carbazole structure have maximum
absorption at ca. 500 nm, which was in the range of TO and its
deviratives [38]. The maximum absorption wavelength of 7c
with the -Cl group was the longest while that of 7b with the -
CH3 group was the shortest. The order of the maximum wave-
length was the same as that of maximum emission wavelengths.
Additionally, the absorption intensity of 7a was the strongest and
that of 7b was the weakest, and the molar absorption values of
the compounds 7a–7c were 1.50×105 mol·L−1·cm−1, 1.08×
105 mol·L−1·cm−1 and 1.99×105 mol·L−1·cm−1 respectively.
Spectral Properties of Carbozole Benzothiazole Indole
Cyanine Dye with Carbazole Bridged Chain
Effect of the Substitutional Groups of the Quinoline Side
Chain on the Fluorescent Properties of Carbozole
Benzothiazole Indole
Fluorescent properties of carbozole benzothiazole indole with
a carbazole bridged chain could be affected by substitutional
groups on the quinoline side chain. In this paper, the fluores-
cent spectra of 7a, 7b, 7c at the same concentration
(0.002 mmol/L) in CH3OH were scaned at 490 nm and the
results were shown in Fig. 2
The Φf of 7a-7c was 0.039, 0.041 and 0.023 respectively.
The emission bands of compounds 7a, 7b and 7c were similar
in shape but with a slight different maximmu emission wave-
length in the range of 607–617 nm. The maximum emission
wavelengths of 7c with a -Cl group on the quinoline side chain
was red-shifted of 9 nm compared to that of 7b. The fluores-
cence intensity and Φf of 7b were the strongest while those of
7c were the weakest. The effect of substitutional groups on
fluorescent properties was accordance with the reference [36].
Regarding the compounds with similar structure, those with
electron-donating groups exhibited stronger fluorescence in-
tensity than those containing electron-withdrawing groups.
The nonbonding electron n on electron-donating groups -
CH3 was parallel to the π orbital of aromatic ring, which
produces the n-π conjugation and enhances the conjugation
degree, resulting in the enhancement of fluorescence intensity.
There were also a nonbonding electron n on the electron-
withdrawing group -Cl, but the n electron was not parallel to
the π orbital of the aromatic ring and the n-π conjugation did
not exit. The n-π*transition was a kind of forbidden transition,
whose molar absorptivity was low, leading to the fluorescence
intensity decreased.
Effect of Concentration on the Fluorescence of 7a
Concentration was another factor that affected fluorescence. In
this paper, the fluorescent spectra of 7a samples with the con-
centrations from 0.17×10−5 mol/L to 20×10−5 mol/L in CH3OH
were excitated at 490 nm and the results were shown in Fig. 4.
The fluorescent spectra were shown in Fig. 4. Peak charac-
teristics of fluorescence and the maximum emission wavelength
of samples with different concentrations revealed little differ-
ence. The florescence intensity increased with the increasing of
concentration between 0.17×10−5 mol/L and 2.5×10−5 mol/L,
but decreased when the concentration exceeded 2.5×10−5 mol/
L. With the increasing of the concentration, the absorbent excit-
ed quantum number increased while the emissive quantum
number was also raised, leading to the enhancement of the
0.5
60
7a (494nm)
7b (607nm,53)
50
0.4
7c(502nm)
7a (613nm,41)
7b(491nm)
40
0.3
7c (617nm,25)
30
20
10
0
0.2
0.1
500
600
700
800
300
400
500
600
Wavelength(nm)
wavelength(nm)
Fig. 2 Fluorescent spectra of benzothiazole indole styryl cyanine dye
Fig. 3 UV–vis spectra of benzothiazole indole styryl cyanine dye bear-
bearing different substitutional groups on indole. (c=7×10−6 mol/L)
ing different substitutional groups on indole. (c=7×10−6 mol/L)

J Fluoresc
60
50
40
30
20
10
0
Table 1 The fluorescent data of 7a compared with TO
Dyes Emission
Stocks
Intensity Fluorescence
wavelength (nm) shift (nm) (a.u.)
quantum yields (Φf)
TO
7a
530
615
29 [38]
119
7
0.003
0.039
98
The fluorescent emission wavelength of the novel com-
0
5
10
15
20
pound 7a was at 615 nm, which had a shift of 85 nm [41] and
fluorescence intensity was enhanced about 14 fold compared
to that of TO. The Stokes shift increased about 100 nm com-
pared to that of TO and the fluorescence quantum yields
increased about 10 times and Cy3 [42].
In comparison with the structure of TO, the novel carbozole
benzothiazole indole has an extended conjugated system. The
π electrons are more easily excited, resulting in enhanced
fluorescence intensity and red-shifted wavelength. The novel
carbazole benzothiazole indole also has a large rigidity plan,
which makes the reciprocity and conjugation of π electrons
increase. Consequently, the wavelength of the novel carbazole
carbozole benzothiazole indole was red-shifted to be near the
near-infrared region, the fluorescence intensity was enhanced
and the Stokes shift also increased. The novel dyes displayed a
better performance than TO, which can be used as an excellent
cyanine dye probe in biological labeling.
C (10-5 mol/L)
Fig. 4 Fluorescent spectra and tendency chart of 7a with different con-
centrations in CH3OH
fluorescence intensity of the fluorescent compound. When the
fluorescence intensity was enhanced to its maximum, it then
decreased possibly due to fluorescence self-quenching.
Solvent Effect on the Fluorescent Spectra of 7a
Solution obviously affects the fluorescent properties. The fluo-
rescent spectra of 7a in different solutions were shown in Fig. 5.
The fluorescence intensity of 7a was the strongest in CHCl3
and weakest in acetone , being about 2.5 times higher in
CHCl3 than in acetone. In aprotic polar solvents, the maxi-
mum fluorescent emission wavelength was red-shifted with
the solvent polarity increased, which is accordance with the
squarylium indocyanine dyes [39].
The maximal emission wavelength in DMSO was at
622 nm, which was shifted 9 nm, 4 nm and 17 nm compared
to those in CH3OH, CH3COCH3 and CHCl3, respectively.
The reason may be that when the dye molecule is excited,
the electronic excited state has a greater polarity than the
ground state. The increased polarity makes the electronic
excited states much more stable than the ground states [40],
resulting in the red shift of wavelength.
Conclusion
In summary, we showed the synthesis of a novel kind of
carbozole benzothiazole indole styryl cyanine dye with a
carbazole bridged chain, based on the structures and proper-
ties of the two excellent nucleic acid molecular marker cya-
nine dye probes, TO and Cy3. The compounds were charac-
terized by 1HNMR and MS.
The novel carbazole benzothiazole indole styryl cyanine
dye displayed excellent fluorescent properties. Fluorescent
results showed that the fluorescence wavelength of the novel
carbazole benzothiazole indole cyanine dye was red-shifted
ca. 85 nm compared to that of TO, the Stokes shift was
increased ca. 100 nm, and the fluorescence intensity was
enhanced 15 fold and the fluorescence quantum yields in-
creased about 10 fold. The novel carbozole benzothiazole
indole maintained the same conjugated plane structure as its
TO precursor but possessed a better fluorescence performance
than TO. Therefore it can be used as an excellent cyanine dye
probe in biological labeling. The possible application in biol-
ogy of this dye is ongoing.
Properties Compared with TO
The novel carbazole dyes have better fluorescent properties com-
pared to those of TO. The fluorescent data were shown in Table 1.
CHCl3 (616nm,1183)
DMSO (622nm,61)
120
100
80
60
40
20
0
CH3OH (613nm,53)
CH3COCH3 (619nm,48)
Acknowledgments This work was financially supported by the
National Natural Science Foundation of China (51178289), Tech-
nology Development Foundation Plan Project of Tianjin Colleges
(No. 20110509 20120506).
500
550
600
650
700
750
800
850
Wavelength(nm)
Fig. 5 Fluorescent spectra of 7a in different solvents. (c=2.5×10−5 mol/L)

J Fluoresc
a water-soluble near-infrared dye for cancer detection using optical
imaging. Bioconjug Chem 16(3):735–740
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