Synthesis of 1-phenyl-3-biphenyl-5-(N-ethylcarbazole-3-yl)-2-pyrazoline and its use as DNA probe

Article abstract of DOI:10.1016/j.saa.2009.01.019

A novel pyrazoline derivative, named 1-phenyl-3-biphenyl-5-(N-ethylcarbazole-3-yl)-2-pyrazoline, was synthesized, and its structure was confirmed by means of IR, ¹H NMR, and elementary analysis. The compound emits strong yellow fluorescence. Decrease of fluorescence intensity of the pyrazoline derivative in the presence of calf thymus DNA (ct DNA) is observed, and the quenching obey Stern-Volmer equation. There is a single quenching mechanism for the complex, which belongs to static quenching. KI quenching study shows that the magnitude of K_SV of the bound pyrazoline is lower than that of the free one. It is also found that ionic strength could affect the interaction. Binding constants for pyrazoline with ct DNA and salmon sperm DNA (ss DNA) are in the same order of 10⁴ mol L^-1, and binding site size are about 1 per base pairs. Experimental results indicate that the new compound might insert into DNA base pairs by intercalative binding mode.

Full text of DOI:10.1016/j.saa.2009.01.019

Spectrochimica Acta Part A 73 (2009) 221–225
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis of 1-phenyl-3-biphenyl-5-(N-ethylcarbazole-3-yl)-2-pyrazoline
and its use as DNA probe
Junfen Li, Duxin Li, Yuying Han, Shaomin Shuang, Chuan Dong∗
Center of Environmental Science and Engineering Research, Department of Chemistry, Shanxi University, Wucheng Street,
Taiyuan 030006, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel pyrazoline derivative, named 1-phenyl-3-biphenyl-5-(N-ethylcarbazole-3-yl)-2-pyrazoline, was
synthesized, and its structure was confirmed by means of IR, 1H NMR, and elementary analysis. The com-
pound emits strong yellow fluorescence. Decrease of fluorescence intensity of the pyrazoline derivative
in the presence of calf thymus DNA (ct DNA) is observed, and the quenching obey Stern–Volmer equation.
There is a single quenching mechanism for the complex, which belongs to static quenching. KI quenching
study shows that the magnitude of KSV of the bound pyrazoline is lower than that of the free one. It is
also found that ionic strength could affect the interaction. Binding constants for pyrazoline with ct DNA
and salmon sperm DNA (ss DNA) are in the same order of 104 mol L−1, and binding site size are about 1
per base pairs. Experimental results indicate that the new compound might insert into DNA base pairs
by intercalative binding mode.
Received 13 August 2008
Received in revised form 9 January 2009
Accepted 20 January 2009
Keywords:
Synthesis
Pyrazoline
Fluorescent probe
DNA
Interaction
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
to good photoconductivities [7–9], ready accessibility, and easily
being used as carrier transporting as well as emitting materi-
Binding studies of small molecules with deoxyribonucleic acid
(DNA) are important tools in the understanding of drug–DNA inter-
actions, and they have attracted considerable interests in chemical
biology for the design of new and efficient drugs targeted to DNA.
The binding of small molecules to DNA is also very useful in clar-
ifying the structure, function of DNA, and protein–nucleic acids
interaction. There are three primarily binding modes concerning
the interaction of small molecules with nucleic acids [1,2], inter-
calative binding, groove binding, and electrostatic interactions.
Among the three modes, the most effective mode is intercala-
tive binding. In general, planarity and non-negative charge are
suggested to be the important features needed for efficient inter-
calators.
2-Pyrazolines are well known fluorescent compounds with
high fluorescence quantum yields and widely used in industry
as whitening or brightening reagents [3–5]. They are very effi-
cient sensitizers in photography, biological stains, redox indicators,
laser dyes and fluorescence probes. Conjugation structure and elec-
tron donation from N atom make them well known intramolecular
charge transfer (ICT) compounds. An ICT process has been reported
to exist in pyrazoline moiety in the excited state [6]. Pyrazoline
derivatives have also been investigated in many other respects due
als [10]. Many 1,3,5-triaryl-2-pyrazolines have been reported as
hole transporting in organic electroluminescent devices (OELDs)
[11–17].
In recent years, pyrazolines have attracted many attentions from
medicinal chemists and clinicians because of extensive biological
activities. For example, 2-pyrazoline derivatives have been reported
to exhibit antimicrobial [18–22], anti-inflammatory [18] and anti-
hypertensive pharmacological activities [23]. Unfortunately, the
mechanism of their biological activities is not very clear. Most small
molecules studied in this field are organic dyes, metal complexes
and drugs [24–26], no attention has been paid to pyrazoline fluores-
cent dyes for the interaction with DNA. Therefore, it is necessary to
research and understand the modes and factors affected the binding
of this kind of molecules to DNA.
Carbaozole derivatives are also fluorescent compounds with
blue fluorescence. If carbazole radical is linked to pyrazoline
molecule, it would bring an excellent fluorescence efficiency.
Based on this target, a carbazole radical was introduced to 2-
pyrazoline ring on C-5 in this paper, and a new derivative of
pyrazoline was synthesized. It is named 1-phenyl-3-biphenyl-5-
(N-ethyl carbazole-3-yl)-2-pyrazoline (PBEP) (5 in Schemes 1–3),
which is a good fluorescent dye emitting strong yellow light under
ultraviolet light (fluorescent quantum yield is 0.48 in CHCl3). The
product was characterized by IR, 1H NMR, elementary analysis
and fluorescence techniques. Fluorescence analysis plays an impor-
tant role in the study of DNA-binding [27,28]. In this paper, the
∗
Corresponding author. Tel.: +86 351 7018613; fax: +86 351 7018613.
E-mail address: dc@sxu.edu.cn (C. Dong).
1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2009.01.019

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J. Li et al. / Spectrochimica Acta Part A 73 (2009) 221–225
Scheme 1. Synthesis of P-phenylacetophenone.
investigation on the binding properties of PBEP with DNA was
reported based on fluorescence spectroscopy. It is observed that
the fluorescence of PBEP is quenched dramatically and imme-
diately by the addition of DNA, and stable complex is formed.
DNA-binding affinities and binding mode are presented. Part of
PBEP’s planar molecular structure, carbazole radical, is expected to
facilitate the interaction of pyrazoline into the interior of the DNA
helix. The compound shows promising applications in the study of
DNA.
2. Experimental
Scheme 3. Synthesis of biphenyl-(N-ethylcarbazole-3-yl) ethylene ketone and 1-
phenyl-3-biphenyl-5-(N-ethylcarbazole-3-yl)-2-pyrazoline.
2.1. Reagents and materials
Pyrazoline was newly synthesized according to Schemes 1–3.
The stock standard solution with the concentration of
1 × 10−3 mol L−1 was prepared by dissolving in alcohol. ct DNA and
ssDNA were commercially obtained from Sigma and used without
further purification. They were dissolved in NaCl (0.1 mol L−1) and
stored at 4 ◦C, the concentrations had been determined by the
absorption at 260 nm after establishing the absorbance ratio of
A260/A280 in the range of 1.8–1.9, and molarities of DNA were cal-
culated based on ε260 = 6600 L mol−1 cm−1[29]. All above solutions
were further diluted as required. 0.05 M Tris–HCl buffer (pH 7.80)
was used to control the pH of the reaction system. Different pH
value of the media was controlled with Britton–Robinson buffer
solutions, which were prepared by adding appropriate amounts
of 0.2 mol L−1 NaOH into H3BO3, H3PO4 and HAc (0.04 mol L−1).
All other reagents were of analytical reagent grade and doubly
distilled water was used throughout.
2.3. Procedures
2.3.1. Synthesis of 1-phenyl-3-biphenyl-5-(N-ethylcarbazole-
3-yl)-2-pyrazoline
2.3.1.1. Synthesis of P-phenylacetophenone (1) (Scheme 1). A mixture
containing 6 g of biphemy, 28 ml of nitrobenzene, and 5 g of AlCl3
were added to a 3-necked flask and stirred with 2.8 ml of acyl chlo-
ride being dropped at ambient temperature. The reaction lasted for
4 h. Nitrobenzene was removed by steam distillation. The residue
was recrystallized from ethanol, 4 g of yellow crystals was obtained.
Yield: 52%. m.p. 120–121 ◦C. 1H NMR (CDCl3) ı: 2.66 (3H, –CH3),
7.27–8.1 (9H, diphenyl).
2.3.1.2. Synthesis of N-ethylcarbazole (2) (Scheme 2). The synthe-
sis of N-ethylcarbazole was performed as our previous work [30].
Yield: 75%. m.p. 72–74 ◦C. 1H NMR (CDCl3). ı: 1.4 (3H, –CH3), 4.4
(2H, –CH2–), 7.2–8.2 (8H, carbazole).
2.2. Apparatus
The fluorescence measurements were performed on a Cary
Eclipse fluorescence spectrophotometer, VARIAN (USA) connected
to an ultrathermostate of temperature precision 0.1 ◦C with sin-
gle cell peltier accessory. UV–Vis absorbance experiments were
conducted on a TU-1901 UV–vis spectrophotometer (Puxi Instru-
ment Factory, Beijing, China) with 2.0 nm spectrum bandwidth and
slow speed scan. The measurement of pH value of solution was
performed on a Model pHS-29A pH-meter, 2nd Analysis Instru-
mental Factory (Shanghai, China). Melting points were determined
on x-5 melting point determinter. IR spectra were recorded with
a FTIR 1730. 1H NMR spectra were obtained on a Bruker 300 MHz
instrument. Standard 1 cm quartz cells were used. Excitation and
emission bandwidths were set at 5 and 10 nm, respectively. All
experiments were performed at 20 1 ◦C.
2.3.1.3. Synthesis of N-ethyl-3-formyl carbazole (3) (Scheme 2). The
synthesis of N-ethyl-3-formyl carbazole was performed also accord-
ing to our previous work [30]. Yield: 72%. m.p. 86–88 ◦C. 1H NMR
(CDCl3). ı: 1.5 (3H, –CH3), 4.4 (2H, –CH2–), 10.1 (1H, CHO), 7.3–8.7
(7H, carbazole). IR (KBr) cm−1: 1682 (–CHO).
2.3.1.4. Biphenyl-(N-ethylcarbazole-3-yl) ethylene ketone (4)
(Scheme 3). Biphenyl (16 g), 30 ml of nitrobenzene, and 0.9 g
of p-phenylacetophenone were dissolved in 20 ml of ethanol and
1 ml of 10% NaOH aqueous solution. The reaction solution was
stirred for 24 h at ambient temperature. A yellow powder product
of 0.6 g was obtained after being filtrated and recrystallized from
ethanol. Yield: 33%. m.p. 76 ◦C. 1H NMR (CDCl3). ı: 1.49 (3H,
–CH3), 4.4 (2H, –CH2–), 6.28 (1H, –CO–CH ), 7.88 (1H, CH-(N-
Scheme 2. Synthesis of N-ethyl-3-formyl carbazole.

J. Li et al. / Spectrochimica Acta Part A 73 (2009) 221–225
223
ethylcarbazole)), 7.3–8.6 (16H, biphenyl, N-ethylcarbazole). IR
(KBr) cm−1: 1610 (–CO–).
2.3.1.5. Synthesis of 1-phenyl-3-biphenyl-5-(N-ethylcarbazole-3-
yl)-2-pyrazoline (5) (Scheme 3). Biphenyl-(N-ethylcarbazole-3-yl)
ethylene (0.6 g) was dissolved in 10 ml of ethyleneglycol monoethyl
ether, then added a little hydrochloric acid and some drops of
phenyl hydrazine. The mixture was stirred for 8 h at 110 ◦C.
The reaction liquid was poured into water, which resulted in a
greenish–black precipitate. Recrystallized from ethanol, filtrated,
and dried, 0.3 g brown powder was obtained. Yield: 47.2%. m.p.
104–106 ◦C. 1H NMR (CDCl3). ı: 1.5 (3H, –CH3). 2.4 (2H, –CH2–,
pyrazoline), 3.6 (1H, –CH , pyrazoline), 4.3 (2H, –CH2–), 7.2–8.8
(19H, ph + carbazole). IR (KBr) cm−1: 1566 (C N). Anal. calcd. for
C35H29N3 (%): C, 85.55; H, 5.91; N, 8.55. Found (%): C, 85.04; H,
5.83; N, 9.02.
2.3.2. Spectral behaviour studies
1.0 ml of buffer solutions with different pH and certain volume
of fluorescent dye solution were transferred to a 10 ml volumet-
ric flask, and the mixed solution was diluted to the final volume
with doubly distilled water and shaken thoroughly. Record the flu-
orescence spectra of the mixed solution and the reagent blank on
fluorescence spectrophotometer at room temperature using quartz
cell of 1 cm path.
Fig. 1. Effect of pH on the fluorescence intensity of 1-phenyl-3-biphenyl-5-(N-
ethylcarbazole-3-yl)-2-pyrazoline.
measured after mixture, and an additional incubation time was
not needed.
Effect of the concentration of pyrazoline on binding
was performed also. Remain the concentration of DNA at
1.27 × 10−5 mol L−1
, change the concentration of pyrazoline
2.3.3. Binding interaction studies of PBEP with ctDNA
from 0 to 2 × 10−5 mol L−1, the value of F0/F increases greatly and
reaches to maximum at 7.0 × 10−6 mol L−1. This concentration is
chosen as the optimum concentration.
To the solution of 2 ml PBEP (1 × 10−5 mol L−1) in Tris–HCl
buffer (pH 7.80), Spectrofluoremetric titrations were performed
with addition of aliquots of DNA. This operation ensured that the
[DNA] increased gradually. The concentration of PBEP in spectro
photomctric titration is 4 × 10−5 mol L−1. The mixing was achieved
by stirring for 3 min. Then the corresponding fluorescence and
absorption spectra were measured.
3.2.2. Absorption study of PBEP in the presence of DNA
Binding mode of PBEP to DNA was studied by spectrophotomet-
ric titration. Upon complex with DNA, characteristic changes to the
absorption spectra of PBEP are evident. A hypochromic effect in the
region of the absorption and red shifts was detected as shown in
Fig. 2. Since the hypochromicity of chromophore generally results
from p-electron transfer by stacking interaction with nucleic bases,
the spectral change appears to reflect the contribution of intercala-
tion into the nucleic base pair.
All measurements fluorescence intensity were taken at the cor-
responding maxima of excitation and emission wavelength and the
fluorescence widths of slitex and slitem were set at 5/10 nm.
3. Results and discussion
3.1. Spectral behaviour studies
3.2.3. Fluorescence study of PBEP in the presence of DNA
PBEP molecule features a planar carbozole ring, which is
expected to facilitate intercalation into the DNA helix. The interac-
Big conjugation system makes compound present strong fluo-
rescence emission in aqueous solution. It is obvious that pH value
greatly affects the luminescence emission of PBEP (Fig. 1). Under
neutral media, the fluorescence emission spectrum of the com-
pound is a single peak at 372 nm, while there are three peaks at
230, 260, and 280 nm in the excitation spectrum. Among them, the
first peak is maximum, and others are relative weak. The fluores-
cence intensity was measured at 230 nm, which is lower in acidic
and neutral medium than in basic solution. It increases sharply with
the increase of pH (<8) of the sample solution and reaches to the
maximum in weak alkaline solution of pH 8.97, and remains strong
in the whole range of pH selected. This illustrates that the pH can
affect the molecular configuration and the distribution of charges.
A pH of 7.80 is recommended in the subsequent investigations.
3.2. Interaction of PBEP with DNA
3.2.1. Study on the binding conditions
The factors affected the fluorescence of PBEP and DNA complex
were investigated in detail. The effect of incubation time shows that
upon addition of DNA into pyrozaline solution (7.5 × 10−6 mol L−1),
the maximum decrease of the fluorescence intensity can be
observed immediately and remains constant for more than 2 h.
Therefore, in this paper the fluorescence intensity was directly
Fig. 2. Absorption titration of ct DNA on 1-phenyl-3-biphenyl-5-(N-ethylcarbazole-
3-yl)-2-pyrazoline. The arrow presents concentration of DNA × 10−5 mol L−1: 0, 0.98,
2.50, 4.78, 5.76.

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J. Li et al. / Spectrochimica Acta Part A 73 (2009) 221–225
Fig. 4. Stern–Volmer plots for fluorescence quenching of 1-phenyl-3-biphenyl-5-
(N-ethylcarbazole-3-yl)-2-pyrazoline by DNA at different temperatures.
under other two temperatures 35 and 45 ◦C were investigated also
(see Fig. 4). The results show that the Stern–Volmer plots are
both linear, the Ksv decrease from 1.18 × 104 to 0.96 × 104 L mol−1
when temperature increase from 35 to 45 ◦C. It means that the
fluorescence quenching type in this experiment belongs to static
quenching.
In addition, the Stern–Volmer analysis can determine that the
quenching type should be static or dynamic. Kq is quenching rate
constant, ꢁ0 is the average lifetime of fluorophore and its value is
10−9 to 10−7 s. For PBEP, the value of Kq is about 1.85 × 10(11–13)
(at 20 ◦C), which is far greater than 2.0 × 1010 L s−1 mol−1, the
maximum diffusion collision quenching rate constant of various
quenchers with the biopolymer [31]. This also suggests that the
binding of PBEP with DNA is a static quenching process.
Fig. 3. (a) Excitation spectra of 1-phenyl-3-biphenyl-5-(N-ethylcarbazole-3-yl)-
2-pyrazoline (ꢀem = 372 nm, 5/10 nm). The arrow presents concentration of
DNA × 10−5 mol L−1
: 0, 1.27, 2.54, 3.81, 5.8, 6.35, 7.62, 8.89. (b) Fluorescence
3.2.4. Characterization of binding modes
titration of ct DNA on 1-phenyl-3-biphenyl-5-(N-ethylcarbazole-3-yl)-2-pyrazoline
(ꢀex = 260 nm, 5/10 nm). The arrow presents concentration of DNA × 10−5 mol L−1: 0,
0.633, 1.27, 1.90, 2.54, 3.17, 4.43, 5.08, 5.69, 6.35, 7.62, 8.89.
3.2.4.1. Fluorescence quenching studies. The fluorescence quench-
ing studies were performed to determine the accessibility of the
fluorescent dye to anionic quenchers and interaction pattern with
DNA. If small molecules intercalate into DNA base pairs, the double
helix of DNA would protect the bound molecules from the anionic
quencher, owing to the base above and below the intercalator [32].
On the other hand, groove binding exposes the bound molecules to
the solvent surrounding the helix [33].
Iodide ion can effectively quench the fluorescence of small
molecule. To further establish the DNA-binding affinity, the fluo-
rescence quenching experiments were chosen. I- with increasing
concentration was added into pyrazoline or pyrazoline–DNA solu-
tion, respectively. Fluorescence quenching was observed for the free
PBEP and the PBEP–DNA complex. It is found from the quench-
ing curves (not shown) that KSV values of the free pyrazoline and
the bound pyrazoline with ct DNA are 232.8 L mol−1 (r = 0.9938)
and 194.4 L mol−1 (r = 0.9906), respectively. The magnitude of KSV
of the bound small molecule is lower than that of the free one,
which suggests the intercalation of PBEP fluorophore into DNA
bases.
tion was investigated by means of fluorescence. It is observed that
the addition of DNA leads to efficient decrease in the fluorescence
intensity of pyrazoline, with almost no shifts in excitation and
emission wavelengths. All three excitation peaks can be quenched,
and the intensity at 260 nm is decreased more obviously than
the other two peaks when the concentration of DNA increasing
(Fig. 3a). Fig. 3b shows the emission spectra of fluorimetric titration
of PBEP with DNA, which strongly indicates the interaction of PBEP
with DNA.
The interaction pattern of the fluorescence probe with DNA can
be deduced from the variation of KSV according to the Stern–Volmer
equation:
I0
= 1 + KSV[Q] = 1 + Kqꢁ0[Q]
I
where I0 and I are the fluorescence intensities in the absence and in
the presence of quencher (Q), respectively. Fluorescence was mea-
sured when excited at 230 or 260 nm, and the KSV obtained are
6.57 × 103 and 1.85 × 104 L mol−1, respectively. 260 nm is chosen as
the optimum wavelength. The linearity of the Stern–Volmer depen-
dence means that there is only a single quenching mechanism for
the complex.
The dynamic quenching will be acute with the increasing of
temperature, while the static quenching will be decreased. The
Stern–Volmer quenching plots from the fluorescence titration data
3.2.4.2. Ionic strength effect on binding. DNA is an anionic poly-
electrolyte with phosphate groups. The ionic environment must
be considered in the research on DNA. In this work, NaCl is used
to control the ionic strength of the solutions. When NaCl exists
in the system, the electrostatic repulsion between the negatively
charged phosphate skeletons on adjacent nucleotides is reduced
with increasing concentration of Na+. This will tighten the DNA
chains. Therefore, it is not easy for the organic molecules to inter-

J. Li et al. / Spectrochimica Acta Part A 73 (2009) 221–225
225
(PBEP), which appears good fluorescent characteristic with fluo-
rescent quantum yields of 0.48. At pH 7.80, it has been proven to be
a DNA-binder with high affinity. Only one type of quenching pro-
cess occurs and the fluorescence quenching type belongs to static
quenching. The intercalative binding mode is proven by fluores-
cence quenching experiment in that the fluorescence is protected
from the quenching by anionic quencher after the addition of DNA.
PBEP binds to dsDNA with a binding constant of 1.13 × 104 L mol−1
and the corresponding binding site size is 1.19 at 20 ◦C. In addi-
tion, it binds to ssDNA with a binding constant of 1.45 × 104 L mol−1
and binding site size of 0.96. From the experiments, we conclude
that carbozone heteroaromatic moiety may slides into DNA base
pairs, because the planarity of carbozone group is better, and
other benzo groups could rotate. In the further study, we plan to
improve the planarity of whole pyrazoline molecule, and discuss
the effects of substituents on the pyrazoline ring to its interac-
tion affinity with DNA. Better probe with higher affinity could be
found.
Acknowledgements
Fig. 5. The effect of NaCl on the fluorescence intensity ratio (I0/I) of 1-phenyl-3-
biphenyl-5-(N-ethylcarbazole-3-yl)-2-pyrazoline: (a) in absence of DNA and (b) in
presence of DNA (2.35 × 10−5 mol L−1).
This work was supported by the National Science Foundation of
China (No. 20875059) and the Youth Science Foundation of Shanxi
Province (No. 20051005).
calate into the binding sites of DNA base pairs but to be repulsed
dissociating in the solution.
The effect of the ionic strength on the fluorescence in the pres-
ence and absence of DNA was tested (Fig. 5). In the absence of
DNA, adding of NaCl leads to the decreased fluorescence intensi-
ties of PBEP, and increased I0/I value occurs. It is apparent that in
the presence of DNA, fluorescence intensity of PBEP also decreases
gradually and I0/I values are greater than 1, which is smaller than
those in the absence of DNA at the same concentration of NaCl. This
phenomenon may be because the free of the bound PBEP from the
DNA helix with the increase of NaCl, then fluorescence intensity
increases. It suggests that high concentration of NaCl is not benefit
for the binding of PBEP with DNA.
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where r is the number of moles of bound PBEP per mol of DNA base
pair, K is the binding constant and n is the binding site size in base
pairs.
Binding constants and binding sites for PBEP with dsDNA and
ssDNA were determined. For dsDNA, the binding constant and
binding site size are 1.13 × 104 L mol−1 and 1.19 per base pair,
respectively. For ssDNA, binding constant and binding site size
are 1.45 × 104 L mol−1 and 0.96 per base pair, respectively (20 ◦C).
The binding constant values suggest that PBEP forms stable com-
plexes with both dsDNA and ssDNA with almost equal binding
affinities and similar behaviors, and they have both 1:1 binding
stoichiometries. It is expected that planar structure of carbozone
heteroaromatic moiety in pyrazoline slides into DNA base pairs due
to the twist of phenyl and biphenyl radicals.
4. Conclusions
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In this paper, we described the synthesis of a novel fluorescent
dye 1-phenyl-3-biphenyl-5-(N-ethylcarbazole-3-yl)-2-pyrazoline