Interactions of newly designed dicationic carbazole derivatives with double-stranded DNA: Syntheses, binding studies and AFM imaging

Article abstract of DOI:10.1039/c3ob40799c

The design of small molecular ligands able to bind with DNA is pivotal for the development of diagnostic agents and therapeutic drugs targeting DNA. Carbazole-derivatives are potential agents against tumors and opportunistic infections of AIDS. Here, two carbazole-derived dicationic compounds, DPDI and DPPDI, were designed, synthesized and characterized using NMR, IR and MS. The DNA binding properties of DPDI and DPPDI were sensitive to ionic strength. At low ionic strength, planar and aromatic DPDI had a strongly intercalative interaction with DNA, which was confirmed by circular dichroism (CD) and gel electrophoresis. In DPPDI, a phenyl group substituting H atom at the -NH group of DPDI destroyed molecular planarity, which resulted in no intercalative interactions between DPPDI and DNA, proved by CD. The positive enhancement of CD at 260-270 nm and Hoechst 33258 competitive binding tests indicated the strong groove interactions of both DPPDI and DPDI to DNA. The similarity and difference in the structures between DPDI and DPPDI explained different interaction preferences with DNA. In groove interactions, dications of pyridinium on either DPDI or DPPDI could interact with DNA base pairs, and -NH on DPDI or -N-Ph on DPPDI pointed out of the groove, as the classical model of DNA groove binding agents. Furthermore, AFM imaging revealed that both carbazole-derivatives drove the DNA conformation more compact. All the experimental data proved that the two dicationic carbazole-derivatives interacted with DNA strongly and might act as a novel type of DNA-binding candidate. The Royal Society of Chemistry.

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DOI: 10.1039/C3OB40799C
Chemistry
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ARTICLE TYPE
Interactions of newly designed dicationic carbazole derivatives with
double-stranded DNA: syntheses, binding studies and AFM imaging
Tao Jia a*, Jin Xiang a, Jing Wang b, Peng Guo a, Junping Yu b*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
The design of small molecular ligands able to bind with DNA is pivotal for development of diagnostic agents and therapeutic drugs
targeting DNA. Carbazole-derivatives are potential agents against tumor and opportunistic infections of AIDS. Here, two carbazole-
derived dicationic compounds, DPDI and DPPDI, were designed, synthesized and characterized by NMR, IR and MS. The DNA binding
properties of DPDI and DPPDI were sensitive to ionic strength. At low ionic strength, planar and aromatic DPDI had a strongly
10 intercalative interaction with DNA, which was confirmed by circular dichroism (CD) and gel electrophoresis. In DPPDI, a phenyl group
substituting H atom at –NH group of DPDI destroyed molecular planarity, which resulted in no intercalative interactions between DPPDI
and DNA, proved by CD. The positive enhancement of CD at 260-270nm and Hoechst 33258 competitive binding tests indicated the
strong groove interactions of both DPPDI and DPDI to DNA. The similarity and difference in the structures between DPDI and DPPDI
explained different interaction preferences with DNA. In groove interactions, dications of pyridinium on either DPDI or DPPDI could
15 interact with DNA base pairs, and –NH on DPDI or –N-Ph on DPPDI pointed out of the groove, as the classical model of DNA groove
binding agents. Furthermore, AFM imaging revealed that both carbazole-derivatives drove the DNA conformation more compact. All the
experimental data proved that the two dicationic carbazole-derivatives interacted with DNA strongly and might act as a novel type of
DNA-binding candidate.
50 derivatives, the geometry is similar to the typical groove ligands
having an extended conjugated system with crescent shape. Both
terminal cationic amidine groups and imidazoline groups engage
20 Introduction
The interactions between DNA and small molecular ligands are
of great importance in biology and medicine, due to their
foundational roles in medicinal, biochemical and biological
processes. Meanwhile, DNA remains a biological target of major
25 interests for the design of diagnostic agents and therapeutic drugs.
Small molecules can interact selectively with DNA by
intercalation, or groove binding, but the interaction preferences
depend on the structure of DNA-interacting molecules and the
nature of DNA. Small differences in the structure of a DNA-
30 interacting molecule may affect the binding types and stability of
H-bonding interactions with the bases while the carbazole –NH
points out of the groove. [6-7]
55
Another type of carbazole-derived cationic compounds (such
as BMVC, BHVC), which have extended conjugated systems
with carbazoles connected through linking groups to pyridinium,
have been successfully used as fluorescent bio-imaging probes
with high fluorescence intensity and large two-photon excited
13-15
60 fluorescence action cross-sections upon DNA binding.
The
bright fluorescence spots from these carbazole derivatives have
been observed in the nuclei of most cancer cells, and hardly
1
16
the molecule/DNA complex . A molecule containing a planar
found in the nuclei of normal cells.
Meanwhile, they can
and aromatic moiety is more favorable for intercalation, in which
the moiety can slide between the adjacent base pairs and facilitate
the intercalation 2, 3. However, most organic molecules that bind
35 in the DNA grooves have extended conjugated systems with
aromatic rings that directly bonded or connected through linking
groups such as cationic amidine groups or amidine derivatives
(such as DAPI, Hoechst 33258, netropsin) 4-6. The classical
model of DNA groove binding agents is that they have an
40 extended crescent shape that closely fits the helical twist of the
groove. 7, 8
stabilize G-quadruplex DNA structure formed by human
65 telomeric sequence and inhibit telomerase activity. 16-18 However,
the binding preferences of these carbazole dications to DNA are
not clear.
A carbazole analogue, 5H-pyrrolo[3,2-c:4,5-c']dipyridine had a
characteristic structure that two C atoms at 3,6-position of
19
70 carbazole were replaced by two N atoms.
product,
Its methylated
2,8-dimethyl-5H-pyrrolo[3,2-c:4,5-c']dipyridinium
diiodide (DPDI, the structure is shown in Fig.1.), has a similar
geometry to 3,6-carbazole dicationic derivatives. To the best of
our knowledge, the interactions between DPDI or relative
75 derivatives and DNA have not been reported yet. The studies on
the interactions between this type of carbazole derivatives and
DNA would broaden the understandings and applications of 3,6-
carbazole dicationic analogues whether as DNA structural probes
or as cancer therapeutics targeted to DNA.
Carbazole derivatives have been reported to be potential agents
9
against tumor or opportunistic infections of AIDS 10, 11. The
carbazoles substituted at 3,6 or 2,7 positions with cationic
45 amidine groups or imidazoline groups have been proved to bind
10, 11
strongly in the DNA groove of AT-rich sequences.
These
carbazole derivatives look more like classical intercalators with a
planar chromophore, so they displayed an intercalating binding
mode in GC-rich sequences. 11, 12 For the 3,6-carbazole dicationic
80
Herein, DPDI have been synthesized and characterized.
Through multiple characterizations, the strong interactions with
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DNA have been confirmed. However, whether –NH on DPDI
participated in the interactions between DPDI and DNA was not
clear. In order to clarify this and the interaction preferences of
DPDI and DNA, another dicationic carbazole derivative, where a
phenyl group was utilized to block –NH of carbazole group on
DPDI, 2,8-Dimethyl-5-phenyl-5H-pyrrolo[3,2-c:4,5-c']dipyridi-
nium diiodide (DPPDI, the structure is illustrated in Fig.1.), was
synthesized and characterized. The spectral titrations of both
DPDI and DPPDI with CT-DNA including UV-Vis absorption,
5
10 fluorescence, and CD showed that both compounds had high
affinities to DNA. The differences in CD titrations, Hoechst
33258 competitions and gel electrophoresis disclosed that DPDI
interacted with DNA by mixing modes of intercalations and
groove binding, while DPPDI displayed only groove binding
15 mode. Moreover, both DPDI and DPPDI induced severe
compression of DNA conformations, as demonstrated by atomic
force microscopy (AFM).
20 Fig. 1. The structures of DPDI and DPPDI.
Fig.2-1. Fluorescence emission changes when titration of DPDI by CT-DNA. [DPDI]
60 =0.1 μM in buffer of pH=7.4 (10-4 M Tris-HCl buffer); [DNA base pairs] = 0, 0.29,
0.58, 0.87, 1.16, 1.45, 1.74, 2.03, 2.32, 2.61, 2.90 μM. (A) ionic strength of 10-1 M;
(B) ionic strength of 10-2 M; (C) ionic strength of 10-3 M; (D) ionic strength of 10-4
M; E. Relative fluorescence intensity decrease when titration of DPDI (0.1 μM) by
CT-DNA (0-2.90 μM). Black line and squares: ionic strength of 10-4 M; Red line
65 and diamonds: ionic strength of 10-3 M; Green line and triangles: ionic strength of
10-2 M; Blue line and inverted triangles: ionic strength of 10-1 M.
Results and discussion
Fluorescence titration
DPDI has strong fluorescence at Ex/Em of 266 nm/ 388 nm and
DPPDI has fluorescence at Ex/Em of 225 nm/ 395 nm. However,
25 during the experiments, fluorescence decay over time was
observed and it was different in aqueous solutions with various
ionic strengths. Therefore, aqueous solutions with four various
ionic strengths (10-4 M, 10-3 M, 10-2 M and 10-1 M) were tested to
study the fluorescence intensity changing over time as exhibited
30 in supplementary information Fig. S1. After 3 hours at room
temperature, fluorescence of both DPDI and DPPDI decayed
slowly. Therefore, DPDI or DPPDI solutions were placed at room
temperature for 3 h before fluorescence titration.
Fluorescence titration by CT-DNA was applied to investigate
35 the binding properties of DPDI/DPPDI with DNA. Fig. 2-1 and
2-2 illustrated the fluorescence changing of DPDI and DPPDI,
respectively, by titration of CT-DNA at various ionic strengths.
At low ionic strength, the fluorescence of dicationic carbazoles
was quenched quickly by DNA. Especially at low ionic strength
40 of 10-4 M, dicationic carbazoles upon binding to DNA exhibited a
maximal quenching of about 67% for DPDI and about 63% for
DPPDI. With ionic strength increasing, fluorescence quenching
was lessening gradually. For DPDI, at ionic strength of 10-1 M,
fluorescence emission intensity increased at the earlier addition of
45 DNA, then decreased when further addition of DNA. While for
DPPDI at ionic strength of 10-2 M, the decrease of fluorescence
after addition of DNA was negligible (less than 10%), and then
there was no further experiments at ionic strength of 10-1 M.
Fluorescence changing (increasing or decreasing) is a criterion
50 for the interactions between small molecules and DNA, and the
extent of fluorescence quenching is corresponding to the strength
Fig. 2-2. Fluorescence emission changes when titration of DPPDI by CT-DNA.
70 [DPPDI] =0.1 μM in buffer of pH=7.4 (10-4 M Tris-HCl buffer); [DNA base pairs] =
0, 0.41, 0.82, 1.23, 1.64, 2.05, 2.46, 2.87, 3.28 μM. (A) ionic strength of 10-2 M; (B)
ionic strength of 10-3 M; (C) ionic strength of 10-4 M; (D) Relative fluorescence
intensity decrease when titration of DPPDI (0.1 μM) by CT-DNA (0-3.28 μM).
Black line and squares: ionic strength of 10-4 M; Red line and diamonds: ionic
75 strength of 10-3 M; Green line and triangles: ionic strength of 10-2 M.
UV-Vis absorption titration
The absorption spectroscopy is the most common approach to
investigate the interaction characteristics of small molecules with
20
80 DNA. Red shift and hypochromism would happen in absorption
of interactions. At low ionic strength, the fluorescence of both
21
spectrum of small molecules upon interacting with DNA.
It
dicationic carbazoles was greatly quenched by DNA, which
indicated strong interactions between dicationic carbazoles and
55 DNA. The interactions were weakening with ionic strength
increasing.
was generally accepted that the extent of the hypochromism in
absorption spectrum was consistent with the strength of the
interactions. 22
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Because the solubility of DPPDI was not good in aqueous
solutions, DMSO was used as a solvent for preparing stock
solution, but DMSO disturbed UV-Vis detection to some extent.
Therefore, DNA binding properties of DPDI not DPPDI were
studied by UV-Vis titration. Both DNA and DPDI have strong
UV-Vis absorption, so there were two sets of UV-Vis titration
experiments performed. The absorption spectra of DPDI titrated
by CT-DNA were illustrated in Fig. 3-1 and UV-Vis titrations of
CT-DNA titrated by DPDI were exhibited in Fig. 3-2. For the
5
10 first set experiment, a reference cuvette contained corresponding
CT-DNA alone to nullify the negative control absorbance. At low
ionic strength of 10-4 M, titrations of CT-DNA into DPDI caused
spectral changes obviously. Two major absorption peaks (270
and 330 nm) of DPDI exhibited apparent hypochromicity, with
15 the maxima of 73.7% at 270 nm and 75.0% at 330 nm. However,
another absorption peak of 226 nm exhibited no hypochromicity,
but a small red shift of 2 nm. With ionic strength increasing,
hypochromic effect decreased gradually and disappeared at 10-1
M. Furthermore, at low ionic strength of 10-4 M, clear isosbestic
20 points were observed at 253 nm, 278 nm and 305 nm.
For the second set experiment, a reference cuvette contained
corresponding DPDI alone to eliminate the negative control
absorbance. At low ionic strength of 10-4 M, a distinct
hypochromicity at 270 nm occurred. When ionic strength
25 increased, hypochromic effect diminished regularly. It was
indicative of considerable hypochromism in absorption spectrum
of dicationic carbazole upon binding to DNA. These phenomena
were consistent with the results obtained from Fig. 3-1.
Additionally, blue shift from 260 to 251 nm of CT-DNA
30 absorption peak was observed at ionic strength of 10-4 M, and a
new absorption peak at 233 nm was also observed. It suggested
the conformational changes of DNA in the presence of DPDI at
low ionic strength.
45
Fig. 3-2 UV-vis absorbance changing with the titration of CT-DNA by DPDI. [DNA
base pairs] =48 μM in buffer of pH=7.4; [dicationic carbazole] = 0, 3.33, 6.67, 10.00,
13.33, 16.67, 20.00, 23.33, 26.67, 30.00, 33.33 μM. (A) buffer (1.0×10-4 M Tris–
HCl, 1.0×10-4 M NaCl); (B) buffer (1.0×10-3 M Tris–HCl, 1.0×10-3 M NaCl); (C)
50 buffer (1.0×10-3 M Tris–HCl, 1.0×10-2 M NaCl); (D) buffer (1.0×10-3 M Tris–HCl,
1.0×10-1 M NaCl).
Circular dichroism spectroscopy
Circular dichroism (CD) spectroscopy is a useful technique to
55 analyze interactions between small molecules and DNA, and the
observed changes of DNA CD signals are usually assigned to the
24
corresponding changes in DNA structures. Neither DPDI nor
DPPDI has intrinsic CD signals due to achirality of the structures.
Thus, the characteristic CD spectrum of CT-DNA in the region
60 220-320 nm could provide the information relevant to structural
changes on DNA upon interactions with dicationic carbazoles.
Fig. 4-1 and 4-2 depicted the CD spectra of CT-DNA with
increasing concentrations of DPDI and DPPDI, respectively, at
various ionic strengths.
The overall spectral changes with hypochromicity and
35 isosbestic point induced by binding planar polyaromatic
molecules to DNA are suggestive of strong interactions at low
ionic strengths while weak interactions at high ionic strengths. 23
65
The CD spectrum of free CT-DNA is of the typical B-form,
with a positive Cotton effect near 275 nm due to base stacking
and a negative Cotton effect near 245 nm due to right-handed
24
helicity. It is generally accepted that the classical intercalation
enhances the base stacking and stabilizes helicity, and thus
70 increases intensity of the positive band, whereas groove binding
and electrostatic interaction of small molecules show less or no
perturbation on the base stacking and helicity bands. 23
For the CD spectra of DNA with the addition of DPDI, at ionic
strength of 10-4 M as displayed in Fig. 4-1 D, there was a great
75 enhancement for the positive band at 275 nm, which was
probably due to intercalations of DPDI to DNA base pairs
enhancing the base stacking. There was another positive band at
260-270nm observed much enhancement, which was not ascribed
to intercalations. We deduced that it might be a result of groove
80 interactions. The result was in conformity with our observation of
UV-Vis titrations of CT-DNA by dicationic carbazole at low
ionic strength of 10-4 M, and during UV-Vis titrations the blue
shift from 260 to 251 nm of CT-DNA absorption peak and a new
absorption peak of 233 nm were observed. At ionic strength of
85 10-3 M, the intensity of the positive band at 275 nm got doubled
as shown in Fig. 4-1 C, while there was a little change for the
band at 260-270 nm, which indicated that there were mainly the
intercalative interactions between DPDI and CT-DNA at ionic
strength of 10-3 M. With ionic strength increasing to 10-2 M, the
90 intensity of the positive band at 275 nm increased with the
maximum of 50.8%, which turned out the weakening of
intercalative binding. At ionic strength of 10-1 M, the positive
band at 275 nm showed little change, suggesting that high ionic
strength could prevent the intercalative binding of DPDI to CT-
Fig.3-1 UV-vis absorbance changing with the titration of DPDI by CT-DNA. [DPDI]
40 =10 μM in buffer of pH=7.4; [DNA base pairs] = 0, 3.88, 7.76, 11.64, 15.52, 19.40,
23.28, 27.16, 31.04, 34.92, 38.80 μM. (A) buffer (1.0×10-4 M Tris–HCl, 1.0×10-4
NaCl); (B) buffer (1.0×10-3 M Tris–HCl, 1.0×10-3 M NaCl); (C) buffer (1.0×10-3
Tris–HCl, 1.0×10-2 M NaCl); (D) buffer (1.0×10-3 M Tris–HCl, 1.0×10-1 M NaCl).
M
M
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DNA. The intercalative binding of DPDI to CT-DNA at very low
experiments revealed only relative small decrease of original
ionic strength made CT-DNA to be a shorter, more compact
helical structure, which was consistent with the AFM results
below.
fluorescence as shown in supplementary information Fig. S2.
H33258 competitive results were shown in Fig. 5. The results
40 revealed that the fluorescence of H33258 with DNA was
decreasing rapidly with the addition of DPDI or DPPDI. 30 M
DPDI and DPPDI could quench the fluorescence for 45% and
46% at ionic strength of 10-4 M, respectively, and the
fluorescence was quenched 26% and 39% at ionic strength of 10-3
45 M, respectively. The results indicated that both DPDI and DPPDI
had strong groove interactions with DNA at ionic strength of 10-4
M, while at ionic strength of 10-3 M the groove interaction of
DPPDI with DNA was stronger than DPDI. The conclusions
accorded with the CD results of that at ionic strength of 10-4 M,
50 the great enhancement of the positive band at 260-270 nm existed
in the presence of either DPDI or DPPDI, while at ionic strength
of 10-3 M CD revealed obvious enhancement at 260-270 in the
presence of DPPDI, but little change in the presence of DPDI.
5
For the CD spectra of DNA with DPPDI, at ionic strength of
10-4 M, there was almost no change for CD at 275nm as shown in
Fig. 4-2 C, indicating that intercalative interactions do not exist
between DPPDI and DNA, while there was a new positive band
appeared at 260-270nm, which probably manifested the groove
10 interactions between DPPDI and DNA at ionic strength of 10-4 M.
With the increase of ionic strength, the groove interactions
decreased. Because at ionic strength of 10-2 M, DPPDI had weak
effect on CD of DNA, there were no further experiments for ionic
strength of 10-1 M.
15
Fig. 4-1. CD spectra of CT-DNA (200 μM) at increasing DPDI concentration (0, 10,
20, 40 μM). (A) buffer (1.0×10-3 M Tris–HCl, 1.0×10-1 M NaCl); (B) buffer
(1.0×10-3 M Tris–HCl, 1.0×10-2 M NaCl); (C) buffer (1.0×10-3 M Tris–HCl, 1.0×10-
20 3 M NaCl); (D) buffer (1.0×10-4 M Tris–HCl, 1.0×10-4 M NaCl).
55
Fig. 5. Fluorescence spectra of H33258 (2 μM) and DNA (5 μM) with the increasing
addition of DPDI ((A) ionic strength of 10-3 M and (B) ionic strength of 10-4 M) and
DPPDI ((C) ionic strength of 10-3 M and (D) ionic strength of 10-4 M). [DPDI] or
[DPPDI]= 0, 3.33, 6.67, 10.00, 13.33, 16.67, 20.00, 23.33, 26.67, 30.00 μM.
60
Gel electrophoresis
Gel electrophoresis is a general method to evaluate the binding
26, 27
mode of small molecules and DNA base pairs.
Moreover, it
was thought that intercalative interactions result in the band lag of
65 the small molecule-DNA complex, while other binding modes do
26
not affect the speed of band migration.
By DNA gel
experiments, we tested the lpBR322 (linear pBR322) migration
rate in the presence of DPDI or DPPDI at different ratios of
dicationic carbazoles and DNA base pairs: 0, 1:1, 10:1 and 100:1
70 at low ionic strength of 10-4 M. The results were shown in Fig.
6A. Apparently, the band for high ratio of DPDI and base pairs
was slower than low ratio, and especially at the ratio of 100:1, the
band was much slower for DPDI, while for DPPDI, it seemed no
change for all band positions. We also ran the gel experiments for
75 the effects of EB and H33258 as shown in Fig. 6B. Obviously,
EB made the lpBR322 band lagging but H33258 did not affect
the rate of the band. The results indicated that DPDI, like EB, had
obvious intercalative interactions with DNA at ionic strength of
10-4 M, while DPPDI, similar to H33258, mainly had groove
80 binding with DNA at ionic strength of 10-4 M, which was
consistent with CD and H33258 competitive results.
Fig. 4-2. CD spectra of CT-DNA (200 μM) at increasing DPPDI concentration (0,
10, 20, 40 μM). (A) buffer (1.0×10-3 M Tris–HCl, 1.0×10-2 M NaCl); (B) buffer
25 (1.0×10-3 M Tris–HCl, 1.0×10-3 M NaCl); (C) buffer (1.0×10-4 M Tris–HCl, 1.0×10-
4 M NaCl).
EB and H33258 competitive experiments
EB is a classical intercalator of DNA and H33258 is a
25
30 representative DNA-groove binder.
To further confirm the
interaction modes of the dicationic carbazoles with DNA,
experiments of EB and H33258 competitive binding to DNA
were carried out by the fluorescence spectra at two low ionic
strengths (10-3 M and 10-4 M). Due to strong intercalative
35 interactions between EB and DNA, DPDI or DPPDI was hard to
replace EB from the complex of EB-DNA, so the EB competitive
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between DPDI and DNA, and also the obvious fluorescence
decay of DPDI at low ionic strength was observed. Where did the
fluorescence decay come from? Did it come from the production
35 of –N- due to the dissociation of H from –NH of DPDI and thus
aggregations due to the electrostatic interactions between –N- and
cations of pyridinium at low ionic strength? In addition, it was
worthy of exploring whether –NH participated to bind with DNA.
It was reported that –NH groups of the carbazoles substituted at
40 2,7 positions with cationic amidine groups or imidazoline groups
have been proved to bind strongly in the DNA groove of AT-rich
10, 11
sequences.
Therefore, by introducing a phenyl group
substituting H atom at –NH group of DPDI, DPPDI was designed
to clarify the questions raised above. Moreover, the investigation
45 of the interaction between DPPDI and DNA would settle another
question: whether there were any other interaction modes existing
between dicationic carbazole and DNA when benzene ring and
carbazole ring of DPPDI were not in a plane, which would affect
intercalation of dicationic carbazole to DNA.
50
Through theoretical calculations of 5-phenyl-5H-pyrrolo[3,2-
c:4,5-c’]dipyridine (2,8-dimethyl delection of DPPDI), the
structure was similar to DPPDI, as shown in Fig. 8., which
revealed that the angle between the plane of benzene ring and the
plane of carbazole was 61.2, so for DPPDI it was difficult to
Fig. 6. (A) Gel electrophoresis of DNA (lpBR322) in the presence of increasing
amount of DPDI (Lane 1-4) or DPPDI (Lane 5-8). (B) EB and H33258 acted as
controls for classic intercalative interaction and groove interaction. M is marker.
55 intercalate to DNA base pairs while there was other binding mode
between DPPDI and DNA.
5
General DNA intercalators, such as EB, acridine orange and
methylen blue, can intercalate with DNA at high ionic strength
(10-1 M). 18 These DNA-intercalators have only one cation, while
60 DPDI and DPPDI are dicationic compounds and the experimental
results proved that ionic strength had great influence on the
interactions between dicationic carbazoles and DNA. At high
ionic strength, electrostatic interactions between ions in aqueous
solutions and dications in carbazole promoted the solubility of
65 carbazoles, increased the electronic density of the molecular
plane and thus raised the fluorescence of the two carbazoles.
However, at low ionic strength, the fluorescence of carbazole
decayed greatly before stability, the reason of which was that an
electrostatic balance of dications between carbazoles and a small
70 amount of ions in aqueous solutions required time to reach and
relative less electronic density to produce fluorescence.
At the mean time, ionic strengths had great effect on the
interactions between carbazoles and DNA. At high ionic strength,
strong electrostatic interactions diminished the interactions
75 between carbazoles and DNA, while at low ionic strength,
carbazoles displayed high binding affinity to DNA. In addition,
through experiments for exploring or comparing the interactions
between DPDI/DPPDI and DNA, the questions raised above got
clear: the fluorescence decay did not come from the dissociation
80 of –NH because DPPDI with block of –NH by phenyl group had
the fluorescence decay at low ionic strength, and –NH of DPDI
did not participate to groove binding with DNA because DPPDI
had groove interaction with DNA. The phenyl ring and the
carbazole ring of DPPDI was not in a plane, affecting DPPDI
85 intercalating with DNA, while DPPDI still could bind with DNA
through groove binding. The experimental results explained that
DPDI could bind with DNA through mixing modes of
intercalation and groove binding. Since both DPDI and DPPDI
had groove binding with DNA, and the 3,6-carbazole compound
90 binds in a more “classical” model that uses both cationic
imidazoline or amidine groups for H-bonding while the carbazole
NH points out of the groove 6, 7, similarly, when groove binding
with DNA, dications of pyridinium on either DPDI or DPPDI can
interact with DNA base pairs through groove binding, and –NH
95 on DPDI or –N-Ph on DPPDI points out of the groove.
AFM imaging
AFM experiments were carried out to manifest the DNA
conformational changes induced by dicationic carbazoles at low
ionic strength of about 10-3 M. Different dicationic
10 carbazoles/pBR322 (bps) mixing ratios (0, 1:1, 10:1, 100:1) were
adopted for the AFM studies. The images were shown in Fig. 7.
With the increase of dicationic carbazoles/pBR322 ratios, the
conformations of pBR322 were changed and presented
compressing conformations, which was consistent with the results
15 of CD spectra. The phenomena observed by AFM were similar to
previously reported results 28, suggesting that both DPDI and
DPPDI strongly interacted with DNA and induced pronounced
changes in secondary structure of DNA at low ionic strength.
However, AFM results cannot provide binding mode information,
20 because either intercalation or groove binding can induce the
conformational changes of DNA. 28, 29
Fig. 7. AFM images for pBR322 with the increase of DPDI and DPPDI at the ratio
25 of DNA:carbazole=1:0 (A), 1:1 ((B) and (E)), 10:1((C) and (F)), 100:1 ((D) and (G)).
Scale bar: 500 nm.
DNA binding mode of dicationic carbazole-DNA
DPDI is a planar aromatic molecule, which contributes to the
30 intercalation of DPDI to DNA base pairs. The titrations of
fluorescence and UV-Vis confirmed the strong interactions
2
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Organic & Biomolecular Chemistry
DOI: 10.1039/C3OB40P79a9gCe 6 of 8
55 Elemental analysis was performed on VarioEL III. The synthesis
for the two dicationic carbazoles was outlined in Fig. S3 of
supplementary information. The characteristic data of 5-phenyl-
5H-pyrrolo[3,2-c:4,5-c’]dipyridine, DPDI and DPPDI have been
shown in the part of spectroscopic data in supplementary
60 information.
Fig. 8. Theoretical calculations on optimized molecular structure of 5-phenyl-5H-
pyrrolo[3,2-c:4,5-c’]dipyridine (2,8-dimethyl delection of DPPDI) were carried out
at the DFT//B3LYP/6-31G level in the Amsterdam Density Functional (ADF)
5 2009.01 program.
Materials
Calf thymus DNA (CT-DNA), Ethidium Bromide (EB) and
Hoechst 33258 was purchased from Sigma-Aldrich. Plasmid
65 pBR322 were purchased from Toyobo Biochemicals. Reagents
for synthesis were of chemical grade from Sinopharm Chemical
Reagent Co, Ltd. Other reagents were of analytical grade from
Sinopharm Chemical Reagent Co,Ltd unless specified otherwise.
Double distilled deionized water was used to prepare buffers, and
70 ionic strength in buffers was adjusted with NaCl. The CT-DNA
concentration in base pairs was determined by an extinction
Experimental Section
Synthesis
5-phenyl-5H-pyrrolo[3,2-c:4,5-c’]dipyridine
10 A mixture of phenyl bromide (190 μL, 1.8 mmol), 5H-
pyrrolo[3,2-c:4,5-c’]dipyridine (100 mg, 0.59 mmol), CuI (12 mg,
0.06 mmol), 18-crown-6 (16 mg, 0.06 mmol), K2CO3 (165 mg,
1.2 mmol), and DMPU (1.0 mL) was stirred and heated to 190 oC
in a sealed tube for 72 h. After cooling, the mixture was extracted
15 with CH2Cl2, and the organic layer was dried over anhydrous
Na2SO4. After removal of the solvent, the residue was purified by
column chromatography on silica gel using CH2Cl2/MeOH (20:1,
V:V) as the eluent to give a white powder, Yield: 66 %. 1H NMR
(DMSO-d6) δ: 7.41 (d, J = 5.6 Hz, 2H, H1, H8), 7.60-7.74 (m, 5H,
20 Hph), 8.57 (d, J = 4.7 Hz, 2H, H2, H7), 9.58 (s, 2H, H4, H5); 13C
NMR (DMSO-d6) δ: 106.01, 118.35, 126.92, 129.30, 130.89,
135.30, 144.06, 144.88, 146.94; IR (KBr) ν: 3035, 2923, 1644,
1623, 1595 (C=C, C=N), 1502, 1470, 1384, 1232, 1192, 1166,
821, 757, 698 cm-1; HRMS (ESI) m/z calcd for C16H12N3 [M+H]+
25 246.1031, found 246.1023.
coefficient of 1.32  104 M-1 cm-1 at 260 nm. The ratio A260/A280
1.80 was used to indicate the high DNA purity. The DNA
>
o
solution was stored for a short period of time at 4 C if not used
75 immediately. A DPDI stock solution (210-3 M) was prepared by
dissolving an appropriate amount of DPDI in deionized water,
and a DPPDI stock solution (210-3 M) was in deionized
water/DMSO (1:1).
Fluorescence experiments
80 Fluorescence spectra and titrations were measured on a Perkin-
Elmer LS-55 spectrometer (Perkin–Elmer Co., USA) with Ex/Em
slit 10 nm/10 nm. Fluorescence decay was performed at a freshly
diluted dicationic carbazoles (0.1 μM) in aqueous solutions with
various ionic strengths. Fluorescence titrations were performed at
85
a
fixed dicationic carbazole concentration with various
concentrations of CT-DNA. The fluorescence from 300 to 500
nm was recorded with Ex of 266 nm for DPDI, and fluorescence
from 300 to 440 nm was recorded with Ex 225 for DPPDI.
2,8-Dimethyl-5H-pyrrolo[3,2-c:4,5-c’]dipyridinium diiodide
(DPDI)
Methylation of 5H-pyrrolo[3,2-c:4,5-c’]dipyridine by an excess
amount of methyl iodide in MeOH afforded a yellow powder. 1H
30 NMR (DMSO-d6) δ: 4.52 (s, 6H, CH3), 8.41 (d, J = 7.0 Hz, 2H,
H1, H8), 9.02 (d, J = 7.0 Hz, 2H, H2, H7), 10.03 (s, 2H, H4, H5);
13C NMR (DMSO-d6) δ: 48.34, 111.85, 118.82, 142.11, 143.48,
149.08; IR (KBr) ν: 2936, 2786, 2637 (N-H), 1630, 1593 (C=C,
C=N), 1472, 1271, 1246, 1183, 830 cm-1; MS (ESI) m/z: 452 [M-
35 H]-; Anal. calcd for C12H13 I2N3: C 31.81, H 2.89, N 9.27; found
C 31.90, H 2.90, N 9.29.
UV-Vis absorption spectra
90 UV-Vis absorption spectra and titrations were recorded on a
Perkin-Elmer Lambda 35 UV-Vis spectrometer (Perkin–Elmer
Co., USA). The spectrometric measurements were performed at
room temperature in a quartz cuvette of 1 cm path length, and the
sample solution was stirred for 3 min before measurement. UV-
95 Vis absorption titration experiments were performed at a fixed
dicationic carbazole concentration (10 μM) in buffers with
various ionic strengths and different CT-DNA concentrations (0-
2,8-Dimethyl-5-phenyl-5H-pyrrolo[3,2-c:4,5-c’]dipyridinium
diiodide (DPPDI)
38.8 μM).
A reference cuvette contained corresponding
concentration of CT-DNA alone to nullify the absorbance of CT-
100 DNA. Absorption titrations were performed at a fixed CT-DNA
concentration (48 μM) in buffers of various ionic strengths with
various concentrations (0-33.3 μM) of dicationic carbazole, and a
reference cuvette contained corresponding concentrations of
dicationic carbazoles alone to nullify the absorbance of dicationic
105 carbazole.
Methylation of 5-pheny-5H-pyrrolo[3,2-c:4,5-c’]dipyridine by an
40 excess amount of methyl iodide in MeOH afforded a brown
powder. 1H NMR (DMSO-d6) δ: 4.56 (s, 6H, CH3), 7.77-7.88 (m,
5H, Hph), 8.21 (d, J = 7.0 Hz, 2H, H1, H8), 9.06 (d, J = 7.1 Hz, 2H,
H2, H7), 10.14 (s, 2H, H4, H5); 13C NMR (DMSO-d6) δ: 48.04,
110.14, 118.27, 126.86, 130.91, 132.28, 141.76, 144.02, 148.95;
45 IR (KBr) ν: 3014, 1668, 1633(C=C, C=N), 1590, 1500, 1471,
1337, 1258, 1186, 811, 770, 701 cm-1; HRMS (ESI) m/z calcd for
C18H17IN3 [M-I]+ 402.0467, found 402.0464.
Circular Dichroism
CD spectra were recorded on a Jasco J-810 spectrometer (Tokyo,
Japan). The CD spectra of CT-DNA (200 μM) in buffers with
various ionic strengths and different concentrations (0-40 μM) of
110 DPDI and DPPDI were measured using a 1 cm path length
cylindrical cell in the 220-320 nm wavelength region at room
temperature. The bandwidth was 1 nm, and the response time was
1 s. Each spectrum is an average of two different scans obtained
5H-Pyrrolo[3,2-c:4,5-c’]dipyridine was prepared as described
19
previously.
The NMR spectrum was recorded on Bruker
50 AV400 spectrometer. Infrared spectrum was obtained on a
Thermo Nexus 470 spectrometer. Low resolution electrospray
ionization (ESI) mass spectrum was obtained on AB SCIEX
API4000 LC/MS. High resolution electrospray ionization (ESI)
mass spectra were obtained on Thermo LTQ-Obitrap XL.
6
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DOI: 10.1039/C3OB40799C
Page 7 of 8
Organic & Biomolecular Chemistry
by collecting data at 0.5 nm intervals with a scan speed of 200
nm/min.
Acknowledgements
The authors are grateful for the financial supports of National
Natural Science Foundation of China (NO. 21002074, 21105114).
H33258/EB competitive experiments
CT-DNA solutions with H33258 in different buffers were titrated
by dicationic carbazoles of increasing concentration (0-30 μM),
with the constant concentrations of CT-DNA (5 μM) and H33258
(2 μM). After each addition of dicationic carbazoles, the
fluorescent emission spectra of H33258 from 400 to 600 nm were
recorded with Ex of 380 nm on a Perkin-Elmer LS-55
Notes and references
5
65 a School of Pharmaceutical Sciences, Wuhan University, Wuhan, Hubei
430072, PR China Fax: +86-27-68759850, E-mail: jiat@whu.edu.cn
b State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese
Academy of Sciences, Wuhan, Hubei 430071, PR China Fax:+86-27-
87199492, Email yujp@wh.iov.cn
10 spectrometer with Ex/Em slit 10 nm/5 nm.
A CT-DNA solution with EB (CT-DNA/EB 5 μM : 2 μM) in
different buffer was prepared. The titration was performed similar
to H33258 competitive experiments, where the fluorescent
emission spectra of EB from 550 to 650 nm were recorded with
15 Ex of 510 nm with Ex/Em slit 10 nm/10 nm.
70 † Electronic Supplementary Information (ESI) available: Fluorescence
decay, synthesis and characterization of DPDI and DPPDI were included.
See DOI: 10.1039/b000000x/
‡ Footnotes should appear here. These might include comments relevant
to but not central to the matter under discussion, limited experimental and
75 spectral data, and crystallographic data.
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40 studies showed the interactions between dicationic carbazoles and
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This journal is © The Royal Society of Chemistry [year]