Spectra, stability and labeling of a novel carbazole derivative as a fluorescent turn-on DNA probe

Article abstract of DOI:10.1007/s10895-015-1613-2

A novel fluorescent dye, 1-(6-carboxyhexyl)-4-(2-(9-ethyl-carbazole-3-yl) vinyl) quinolizinium bromide, was synthesized, and its structure was characterized by ¹H NMR, ¹³C-NMR, IR and HRMS. The spectra properties of this dye in different solvents and under different pH value were invest'igated preliminarily, and the results showed that its fluorescent properties was affected by the polarity and the dipole moment of the solvent. The photostability and thermostability test results showed that its photoreduction rate constant was 1.64×10^-5 mol/Min and its fluorescent intensity decreased little after heating at 80 °C for 6 h, suggesting the dye was quite stable. In the labeling experiment of BSA and DNA with the dye, the fluorescent all intensity increased with the addition of BSA and DNA. Specially, the dye showed an excellent turn-on effect upon binding with ctDNA.

Full text of DOI:10.1007/s10895-015-1613-2

J Fluoresc
DOI 10.1007/s10895-015-1613-2
ORIGINAL ARTICLE
Spectra, Stability and Labeling of a Novel Carbazole Derivative
as a Fluorescent Turn-on DNA Probe
Xuening Fei1,2 & Ran Li1 & Dayong Lin2 & Yingchun Gu1,3 & Lu Yu1
Received: 5 May 2015 /Accepted: 30 June 2015
#
Springer Science+Business Media New York 2015
Abstract A novel fluorescent dye, 1-(6-carboxyhexyl)-
4-(2-(9-ethyl-carbazole-3-yl) vinyl) quinolizinium bro-
mide, was synthesized, and its structure was character-
Introduction
Over the past decade, fluorescent dyes are used widely in
bioimaging, especially for DNA detection [1, 2] due to
the significant advantages of fluorescent dyes, such as
relative stability, low aggregation and high affinity for
some biological molecules [3–5]. However, in order to
decrease the fluorescent background interference caused
by the labeled organism, the stronger fluorescent intensity
is required [6, 7]. It means the dye should exhibit a dra-
matic enhancement in fluorescent intensity upon binding
to DNA and could be used as a turn-on fluorescent probe
for DNA detection. So to design novel small molecules
and study the interaction with DNA, to some extent, is
one of the most important goals in modern medicinal and
biological chemistry.
1
ized by H NMR, 13C-NMR, IR and HRMS. The spec-
tra properties of this dye in different solvents and under
different pH value were invest'igated preliminarily, and
the results showed that its fluorescent properties was
affected by the polarity and the dipole moment of the
solvent. The photostability and thermostability test re-
sults showed that its photoreduction rate constant was
1.64×10−5 mol/Min and its fluorescent intensity de-
creased little after heating at 80 °C for 6 h, suggesting
the dye was quite stable. In the labeling experiment of
BSA and DNA with the dye, the fluorescent all intensi-
ty increased with the addition of BSA and DNA.
Specially, the dye showed an excellent turn-on effect
upon binding with ctDNA.
A number of dyes have been synthesized and applied suc-
cessfully in fluorescent imaging technique and the detection of
DNA in solution [8–10]. Some investigators believe that the
positively charged heterocycles play an important role in the
enhancement of the fluorescent intensity [11–13]. Thus, lots
of positively charged dyes, such as benzooxazolinium,
benzothiazolinium and quinolizinium derivatives have been
successfully developed as fluorescent probes for DNA detec-
tion [14]. In this paper, we designed and synthesized a novel
carbazole-based quinolizinium derivative. The probe exhibit-
ed very weak fluorescence in 10 mM Tris–HCl buffer (pH=
7.4), and showed excellent turn-on effect upon binding with
calf thymus DNA hopefully. In addition, the absorption and
fluorescence spectra in different solvents and under different
pH value in aqueous solution were investigated preliminarily.
The stabilities including photostability and thermostability
were also studied. Finally, in order to assess its potential as
DNA markers, the labeling properties of the dye with BSA
and calf thymus DNA were compared.
.
.
.
Keywords Spectra properties Fluorescent probe DNA
Binding constant
* Yingchun Gu
jvgugugu@126.com; xueningfei@126.com
* Lu Yu
yulu@tcu.edu.cn
1
Tianjin Chengjian University, No. 26 Jinjing Road, Xiqing District,
Tianjin 300384, China
2
3
Tianjin University, Tianjin 300072, China
Institute of Chemistry Chinese Academy of Sciences,
Beijing 100190, China

J Fluoresc
Experimental
General
the molar extinction coefficient ε=6600 M−1 cm−1 at 260 nm
[16, 17].
Fluorescence Quantum Yields
All chemical reagents and solvents were purchased and used
without further purification. Fluorescence spectra were
scanned on a Hitachi F-7000 spectrometer. The excitation
and emission slits were 5 nm and the PTM voltage of the
spectrometer were 700 V. The UV/Vis absorption spectra were
recorded on a Perkin Elmer LAMBDA 35 spectrometer.
1HNMR and 13CNMR spectra were recorded on a Bruker
(400 MHz) spectrometer. Chemical shifts were reported in
parts per million (ppm) down field from TMS
(tetramethylsilane) using DMSO-d6 as a solvent. HRMS
spectra were recorded on a microTOF QII ESI-Q-TOF spec-
trometer. IR (KBr pellets) spectra were recorded on a Brucker
Equinox-55 spectrometer. All chemicals were of analytical
grade. The chemical purity is analyzed by HPLC on
Laballiance PC4006A from America and the flow rate was
1.0 mL/min (4.6×250 mm, 5 μm, C18 reversed-phase
column).
The fluorescence quantum yields of the compound were de-
termined by comparison with a standard solution with the
following equation [18] to calculate relative fluorescence
quantum yields:
ꢀ
ꢁ
2
nX
nS
AS FX
⋅ ⋅
ϕX ¼ ϕS⋅
AX FS
In which the nX and nS are the refractive indexes of the
sample and reference, FX and FS are the area of fluorescence
spectra for the sample and reference respectively, and Ax and
AS are the absorbance for the sample and reference at the
excitation wavelength respectively. The standard used in this
study is Fluorescein in NaOH (0.1 mol/L) aqueous solution
with a fluorescence quantum yield of 0.90 [19].
Synthesis
Preparation of Stock Solutions
The detailed synthesis of compounds (3–5) and the illustration
were shown in Scheme 1.
For absorption or fluorescence measurements, DMSO were
used for the solvent to obtain stock solutions (10 mL), then
the stock solutions were diluted by aqueous solutions to the
desired concentration (5×10−6 mol/L).
BSA stock solutions were prepared by dissolving the sam-
ple directly into PBS (pH=7.7) and then various amounts of
stock solutions were used for dye-protein complexes. Finally,
the dye was diluted to the desired concentration.
Synthesis of compound 3. The synthesis procedure of com-
pound 3 was based on the Ref [20].
Synthesis of compound 4. Lepidine (2 g, 0.014 mol) and 6-
Bromohexanoic acid (4.1 g, 0.021 mol) were stirred 6 h at
130 °C. After cooling down to room temperature, 20 mL of
acetone was added to the reaction flask. The compound was
yielded by filtration and washed with acetone several times.
1
Yield: 85 %. Purity: 98.9 % (HPLC), H NMR (400 MHz,
Calf thymus DNA were purchased from Sigma Chemical
Co. and dissolved in 10 mM Tris–HCl buffer (pH=7.4). The
purity of DNA was checked by monitoring the absorption
spectrum and the ratio of the absorbance at 260 and 280 nm,
DMSO-d6) δ=1.39–1.44 (m, 2H), 1.52–1.57 (m, 2H), 1.92–
2.01 (m, 2H), 2.19–2.23 (t, J=9.60 Hz, 3H), 3.01 (s, 3H),
5.00–5.05 (m, 2H), 8.05–8.09 (t, J=8.0 Hz, 2H), 8.40 (d, J=
11.60 Hz, 1H), 8.60 (d, J=11.20 Hz, 2H), 9.57 (d, J=8.00 Hz,
1H). 13C-NMR (400 MHz, DMSO-d6) δ=19.98, 24.37,
25.76, 29.66, 33.89, 57.19, 119.87, 123.15, 128.37, 130.07,
A260/A280=1.83. The result was consistent with Ref [15]. The
DNA concentration in Tris–HCl buffer were determined by
Scheme 1 General procedure for
the synthesis of compounds 3–5

J Fluoresc
Fig. 1 The UV–vis spectra (a)
and fluorescence spectra (b) of
the dye in different solvent (5×
10−6 mol/L)
134.57, 135.59, 137.21, 148.90, 159.03, 174.74. FTIR (KBr
1 %) νmax=3433, 2939, 2563, 1724, 1616, 1602, 1529, 1406,
1371, 1232, 1209, 1161, 1055, 966, 856, 827, 763 cm−1.
Synthesis of compound 5. A solution mixture of 3 (0.2 g), 4
(0.26 g) and piperidine (2 drops) in ethanol (30 mL) was
heated to reflux overnight. After cooling down to room tem-
perature, the organic solvent was removed by rotary evapora-
tion. The residue was purified by silica-gel column chroma-
tography to get compound 5. Yield: 30 %. Purity: 98.5 %
lowest. In aprotic polar solvents, the maximum absorption
wavelength of compound 5 was red-shifted with the increas-
ing of solvent polarity. We can find that the absorbing intensity
was stronger for the solvent with higher polarity, which is
because the energy of the anti-bonding orbital and bonding
orbital of the molecular are both reduced for the solvent with
higher polarity.
The fluorescence spectra in different solvents were also
recorded at the λex of 490 nm and the results were shown in
Fig. 1b and Table 2.
1
(HPLC). H NMR (400 MHz, DMSO-d6) δ=1.39–1.52 (m,
5H), 1.60 (t, J=7.0Hz, 2H), 1.98–2.01 (m, 2H), 2.11 (t, J=
6.60 Hz, 2H), 4.53–4.58 (m, 2H), 4.99 (t, J=6.40 Hz, 2H),
7.36 (t, J=7.40 Hz, 1H), 7.58 (t, J=7.60 Hz, 1H), 7.73 (d, J=
4.20 Hz, 1H), 7.80 (d, J=4.40 Hz, 1H), 8.08 (t, J=7.60 Hz,
1H), 8.17 (d, J=4.20 Hz, 1H), 8.28 (t, J=7.80 Hz, 2H), 8.43
(q, J=24.80 Hz, 2H), 8.55 (d, J=4.60 Hz, 2H), 8.90 (s, 1H),
9.16 (d, J=4.40 Hz, 1H), 9.41 (d, J=3.00 Hz, 1H),11.80 (s,
1H). 13C-NMR (400 MHz, DMSO-d6) δ=14.28, 24.64,
25.97, 29.57, 34.53, 37.78, 56.61, 110.24, 110.40, 115.75,
116.69, 119.52, 120.25, 121.11, 122.45, 122.78, 123.37,
126.98, 127.20, 127.33, 128.11, 129.30, 135.46, 138.32,
140.69, 141.78, 145.67, 147.39, 153.70, 175.45. FTIR (KBr
1 %) νmax=3446, 2945, 2360, 1716, 1614, 1585, 1564, 1488,
1438, 1334, 1286, 1236, 1166, 1134, 960, 821, 748 cm−1.
HRMS (m/z): [M-Br]+ calcd for 463.2381, found 463.2380.
Figure 1b and Table 2 indicated that the compound pos-
sesses different maximum emission wavelength (λem) in dif-
ferent solvents, and the largest λem is obtained in CH2Cl2
solvent. In protic solvent, the λem is blue-shifted and fluores-
cent intensity decreased evidently with the increasing of the
solvent polarity and it was the lowest in water solution. The
possible reason is that the aggregation of compound 5 in water
solution, thereby resulting in the increase of the nonradiative
decay process and finally the obviously quenching occurred.
In addition, with the increasing of the dipole moment and
solvent polarity in aprotic solvents we could discover that
the λem was red-shifted. The possible reason is that the solvent
with larger polarity could result in the reduction of energy
difference between excited state and ground state, which lead
to a red shift of the dye.
We can see from Table 2 that the stokes shift in different
solvents were different. The stokes shift were influenced by
Results and Discussion
Table 1 The solvent properties and UV–vis characters of compound 5
Spectra Properties of Compound 5
Solvent
nD20 a
f(n) b
λmax/nm
ν c/10−3 cm−1
The Spectral Properties of Compound 5 in Different Solvent
CH3OH
H2O
1.33
1.34
1.36
1.37
1.42
1.48
0.169
0.173
0.181
0.184
0.202
0.221
491
461
487
484
526
490
2.037
2.169
2.053
2.066
1.901
2.041
In order to study the effect of different solvents on the UV–
visible spectra properties and fluorescence spectra properties,
the stock solutions of compound 5 were diluted to different
solvents. The spectra results were shown in Fig. 1 and the
spectral data were summarized in Table 1.
From Fig. 1a we can see the wavelength of compound 5 in
CH2Cl2 was 526 nm, while the wavelength in other solvents
were all less than 500 nm and the wavelength in H2O was the
Acetone
Ethyl acetate
CH2Cl2
DMSO
a Refractive index
b f(n)=(n 2 -1)/(2n 2 +1)
c ν=1/ λmax

J Fluoresc
Table 2 The solvent properties
and fluorescent characters of
compound 5
Solvent
nD20 a
μ b
εc
Δ f
λ
max (nm)
λ
em (nm)
Stokes shift
Φd
CH3OH
H2O
1.33
1.34
1.36
1.37
1.42
1.48
1.70
1.82
2.91
1.88
1.20
3.96
32.7
80.1
20.7
0.3080
0.3174
0.2838
0.2005
0.2183
0.2630
491
461
487
484
526
490
617
599
619
600
622
625
126
138
132
116
96
0.0069
<0.001
0.0067
0.0213
0.0193
0.0074
Acetone
Ethyl acetate
CH2Cl2
DMSO
6.02
8.9
47.2
135
a Refractive index
b Dipole moment
c Dielectric constant
d Fluorescence quantum yield
two factors, refractive index and dielectric constant. The effect
of solvents on fluorescent spectra could be stated by Lippert
equation [21].
The other reason maybe the dye was more soluble in basic
conditions, the dye aggregates can be soluble was increase so
that the fluorescence intensities will be higher than in acid
conditions.
ꢀ
ꢁ
2
ε−1
n2−1
μ*−μ
a3
Δν ¼ νem−νabs
¼
−
þ constant
hc 2ε−1 2n2 þ 1
ε−1
n2−1
The Spectral Properties of Compound 5 in Complex
with ctDNA
Δf ¼
‐
2ε−1 2n2 þ 1
Where νabs and νem are wavelengths of the excitation and
emission respectively, so νem -νabs is stokes shift. The h is
Planck constant and c is light speed. The a is the cavity radium
that encapsulate the molecule. a is a constant when there is no
hydrogen bond between the molecule and solvent. μ and μ*
are the dipole moment of ground state and excited state. The
Δf is called orientation polarizability.
The relationship between stokes shift and Δf of compound
5 in three different solvents were shown in Fig. 2. We can see
that the stokes shift is approximately proportional to Δf,
which is consistent with the Lippert equation. But for other
solvents, there is a deviation between the relationship of Δν
and Δf.
We also studied the labeling of ctDNA with the dye (5×
10−6 mol/L) in 10 mM Tris–HCl buffer (pH=7.4) and the
results were shown in Fig. 4.
Figure 4 shows that the intensity of the compound was very
weak without the addition of ctDNA. Interestingly, the inten-
sity increased remarkably with the addition of ctDNA. One
reason for this may be due to the change in the environment of
compound 5 and the shielding properties of DNA. With the
addition of ctDNA, the strong binding interaction between the
cationic N-quinolizinium unit and the anionic phosphate of
DNA were formed. The binding interaction can hinder the
rotations around various bonds and thereby decreases the pos-
sible nonradiative process.
In addition, we defined the degree of signal enhancement
as (F-F0)/F0, where F0 and F are the fluorescent intensities at
602 nm in the absence and presence of DNA respectively, and
The Spectral Properties of Compound 5 in Different pH
The spectral properties of compound 5 with the concentration
of 5×10−6 mol/L in aqueous solution were also measured in
different pH ranging from 2 to 13. The results were shown in
Fig. 3.
From Fig. 3, we can see the maximum absorption wave-
length (λmax), the maximum emission wavelength (λem) and
the shapes of the spectra change little regardless of the differ-
ent pH of the solution. But with the change of pH, the absorp-
tion and the fluorescent intensity were all changed obviously.
The fluorescent intensity in basic solution was stronger than
that in acid solution. In neutral solution, fluorescent intensity
was the strongest, which is the same to the Ref [22]. One
possible reason for this maybe due to the electronic configu-
ration of compound 5 was different under different pH values.
Fig. 2 The relationship between stokes shift and orientation
polarizability (Δ f) of compound 6 expressed by the Lippert equation

J Fluoresc
Fig. 3 The UV–vis spectra (a)
and fluorescence spectra (b) of
the dye in different pH (5×
10−6 mol/L)
the fluorescence spectra of the probe in the presence of differ-
ent concentrations of DNA were shown in Fig. 5.
Due to the intensity increased remarkably with the addition
of DNA, the bonding association constant could be calculated
by Benesi-Hildebrand equation [23]:
The Spectral Properties of Compound 5 Labeled with Bovine
Serum Albumin (BSA)
We studied the labeling of BSA with the dye (5×10−6 mol/L)
and the results were shown in Fig. 7.
Different concentrations of BSA and the same concentra-
tion of dye were used to measure the fluorescence spectra. The
results were shown in Fig. 7. It indicated that the fluorescent
intensity of the compound increased with the addition of BSA,
accompanied by a blue-shift of the maximum emission wave-
length. There are two possible reasons for the increasing of
fluorescent intensity. Firstly, with the addition of BSA, the
compound was included into a hydrophobic cavity with lower
polarity inside BSA molecule. The other reason is that the
binding between BSA and the compound inhibited the rota-
tional freedom of compound 5 and caused higher intensity. All
these effects together caused the enhancement of the com-
pound’s fluorescent intensity. The blue shift of the maximum
emission wavelength can be explained by the reduction of
ground state energy of the compound when there is interaction
between the dye and BSA. These results jointly confirmed that
the compound can interact with BSA protein. But comparing
with the reaction between the dye and DNA, the effect of the
combination with BSA protein is not very satisfactory.
1
1
1
1
¼
þ
Â
Fx−F0
Fmax−F0 Fmax−F0 K½DNAŠ
Where Fx and F0 represent the fluorescent intensity of the
probe in the presence and absence of DNA, respectively. Fmax
is the maximum fluorescent intensity of the probe in the pres-
ence of DNA. [DNA] is the concentration of DNA added. The
equation could be simplified as:
Fmax−F0
Fx−F0
1
¼ 1 þ
K½DNAŠ
Plots of (Fmax−F0)/ (Fx−F0) against [DNA] for the probe
showed remarkable linear dependence (Fig. 6), justifying the
validity of the used equation and hence confirming the one-to-
one association between DNA and the probe. The binding
constant (Ka) were determined from the slope of the corre-
sponding plots, which came as 3.92×105 M−1. The value was
consistent with the previous reported [24].
Fig. 4 The fluorescence spectra of compound 5 with different
concentration of ctDNA (from 1 to 8: 0 μM, 4 μM, 8 μM, 12 μM,
16 μM, 20 μM, 24 μM, 28 μM). Excitation wavelength: 485 nm
Fig. 5 The fluorescence spectra of the probe in the presence of varying
concentrations of DNA. Inset: Linear range between (F-F0)/ F0 and the
ctDNA concentration (12–28 μM)

J Fluoresc
Fig. 6 The linearity of (Fmax−F0)/ (Fx−F0) against [DNA]
Fig. 8 The curve of photostability
In addition, the rate of photooxidation could be calculated
based on the photooxidation mechanism and counld be
expressed as [26]:
Photostability of Compound 5
The photostability of fluorescent dyes has attracted a great
attention of experts, because it plays an important role for their
application as biomedical fluorescent labels. In this study, we
used iodine-tungsten lamp (500 W) as the illuminant. The
NaNO2 aqueous solution (50 g/L) was used as the cold trap,
which can decrease the effect on the stability caused by the
strong heat from illumination. On the other hand, the solution
could also filtrate most of the UV-lights, whose wavelength is
shorter than 400 nm. The photostability experiment was per-
formed in DMSO at room temperature without natural light
[25]. The absorption intensity of the compound was measured
after exposing to light with different time. The curve between
the intensity at λmax and the exposure time was shown in
Fig. 8.
It can be seen that there is no prominent change after dif-
ferent exposure time. After irradiation of 6 h, the dye showed
only 2.7 % photofading. It is indicated that the dye can be
exposed to the strong light for a long time and still keep its
intensity, which can make it as an excellent probe for applica-
tion in many fields.
d½DyeŠ=dt ¼ k1½Dye Š½O2Š
ð1Þ
in which [O2] is a constant of about 3×10 −4mol/ L, so the
following equation can be obtained:
−d½DyeŠ0=dt ¼ k½DyeŠ
ð2Þ
ð3Þ
ln½DyeŠ0=½DyeŠ ¼ kt
t
In the equation, [Dye]0 and [Dye]t were the original con-
centration of dye and the concentration after being illuminated
for t hours, respectively. According to Lambert-Beer’s law,
there was a linear relation between concentration and the max-
imum absorption under low concentration. So Eq. (3) could be
predigested as below:
lnð A0=AtÞ ¼ kt
ð4Þ
In the equation, A0 is the max absorbency of dye, At is the
maximum absorbency after being illuminated and k is the
photoreduction rate constant. The result was shown in
Fig. 9. The photoreduction rate constant could then be obtain-
ed from the slope, the result is 1.64×10−5 mol/Min, which is
Fig. 7 The fluorescence spectra of compound 5 with different
concentration of BSA (from 1 to 6: 0, 50, 100, 150, 200 and
250 mg/L). Excitation wavelength: 490 nm
Fig. 9 The lightfastness of the dye

J Fluoresc
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Fig. 10 The curve of thermostability
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Conclusion
In summary, a novel fluorescent dye, 1-(6-carboxyhexyl)-
4-(2-(9-ethyl-carbazole-3-yl) vinyl) quinolizinium bromide,
was synthesized and characterized by H NMR 13C-NMR,
1
IR and HRMS. The photostability and thermostability were
studied and the results showed that the compound was stable
and can meet the need of the biological labeling. The proper-
ties of the dye in different solvents and in aqueous solution
under different pH were also studied and the results showed
that the dye is both sensitive to solvent and pH value. The dye
was also used to label BSA and the results showed that the
fluorescent intensity enhanced when labeling with BSA.
Lastly, we studied the interaction of the dye with ctDNA.
The results indicated that the dye give substantial increases
in fluorescence on DNA binding and the binding constant
was 3.92×105 M−1. These jointly proved that the dye could
be used as a sensitive fluorescent turn-on probe for DNA.
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Acknowledgments This work is supported by the National Natural
Science Foundation of China (51178289, 21402139).

J Fluoresc
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