Targeting DNA with small molecules: A comparative study of a library of azonia aromatic chromophores

Article abstract of DOI:10.1039/c4ob01465k

A library of azonia aromatic cations has been studied in order to gain insights into the effect of the size, shape and charge distribution on the fluorescence, DNA interactions and DNA sequence selectivity properties. Fluorescence-based thermal denaturation experiments, spectrofluorimetric titrations, circular dichroism measurements and theoretical simulations have shown that some of the studied chromophores have interesting fluorescence properties and two of them also show a consistent DNA-binding ability by intercalation, with a potential preference for AT-rich sequences.

Full text of DOI:10.1039/c4ob01465k

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Targeting DNA with small molecules:
a comparative study of a library of azonia
Cite this: DOI: 10.1039/c4ob01465k
aromatic chromophores†
Rosa M. Suárez,‡a Pedro Bosch,‡a David Sucunza,*a Ana M. Cuadro,a
Alberto Domingo,b Francisco Mendicutic and Juan J. Vaquero*a
A library of azonia aromatic cations has been studied in order to gain insights into the effect of the size,
shape and charge distribution on the fluorescence, DNA interactions and DNA sequence selectivity pro-
perties. Fluorescence-based thermal denaturation experiments, spectrofluorimetric titrations, circular
dichroism measurements and theoretical simulations have shown that some of the studied chromophores
have interesting fluorescence properties and two of them also show a consistent DNA-binding ability by
intercalation, with a potential preference for AT-rich sequences.
Received 12th July 2014,
Accepted 20th October 2014
DOI: 10.1039/c4ob01465k
www.rsc.org/obc
Introduction
Coralyne is probably the most widely studied quinolizinium-
based alkaloid. Interest in this compound is due to several
significant properties associated with its structure, a penta-
substituted dibenzo[a,g]quinolizinium system.1 In addition to
the most commonly studied properties of this alkaloid, such
as cytotoxicity,2 intercalative DNA binding3 and potent topo-
isomerase inhibition,4 other remarkable properties have been
reported, e.g., applications as fluorescent probes,5 reagents
for the photoinduction of DNA damage,6 RNA binding,7
human telomeric quadruplex DNA,8 and hemoglobin.9
Complex formation between coralyne and c-di-AMP10 and
α-, β-, and γ-cyclodextrin11 has also been reported.
The interest in this alkaloid has led to a significant amount
of research concerned with the synthesis and study of coralyne
analogues and other related azonia cations that contain a
quinolizinium system as the core heterocycle, for example, the
recent significant contributions by Ihmels and coworkers,12
Becq and coworkers,13 inter alia (Fig. 1).
Fig. 1 Structure of coralyne and general structure of quinolizinium-
based azonia cations.
characterized by the insertion of planar aromatic rings
between the DNA base pairs through π-stacking interactions.
The positive charge enhances the propensity of these mole-
cules to bind to DNA due to attractive ionic interactions
between the cation and the phosphate backbone.14
A number of polyaromatic azonia cations are fluorescent
due to the presence of a scaffold that is related to cyanine
dyes,15 a synthetic dye family with a general polymethine struc-
ture that has been widely applied in various areas associated
with non-linear optics, photochromism, light energy trans-
formation and so on.16 For this reason, some condensed poly-
aromatic azonia cations have been used as fluorescent
probes.12b
It has been shown that fused polyaromatic azonia cations
are a representative family of DNA intercalators with fluore-
scence properties.3 The binding of these molecules to DNA is
Our recent contributions in the field of azonia heterocycles
focused on the development of general synthetic procedures
by ring-closing metathesis reactions (RCM) of azinium aza-
dienes,17 new methods for the functionalization of the quinoli-
zinium cation by palladium-promoted reactions,18 and the
investigation of quinolizinium as an acceptor unit in non-
linear optical (NLO) and two photon absorption (2PA) cationic
chromophores.19 The synthesis of some aza-quinolizinium-
aDepartamento de Química Orgánica y Química Inorgánica, Universidad de Alcalá,
28871-Alcalá de Henares, Madrid, Spain. E-mail: juanjose.vaquero@uah.es
bDepartamento de Biología de Sistemas, Universidad de Alcalá, Spain
cDepartamento de Química Analítica, Química Física e Ingeniería Química,
Universidad de Alcalá, Spain
†Electronic supplementary information (ESI) available: 1H and 13C NMR data for
all new compounds, photophysical and DNA binding experiments and MM and
MD protocols. See DOI: 10.1039/c4ob01465k
‡These authors have contributed equally to this work.
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type cations and their potential as DNA intercalators and compounds are all tetracyclic but have different ring
antiproliferative compounds have also been reported by our arrangements.
laboratory.20
Eight of the aforementioned salts were synthesized in our
We report here the results from our comparative study in laboratory either by a ring-closing metathesis (RCM) reaction
which a library of azonia cations was investigated in order to or by a Westphal condensation as key steps. Thus, quinolizi-
gain insights into the influence that the size, shape and nium-based derivatives 1, 2, 3 and 4 were obtained by the
charge distribution have on some of their properties, first strategy,17 azaquinoliziniums
5 and 8 by Westphal
with potential applications as dyes and DNA probes. The condensation with [1,4]-dioxane-2,3-diol (DODO)22 and 11 by
fluorescence, DNA interactions and DNA sequence selectivity condensation of the corresponding N-amino imidazolium salt
properties were compared.
with acenaphthenequinone.23 Finally, triazoloisoquinolinium
derivative 12 was obtained by iodination of the isoquinolinium
N-(2′-pyridyl)aminide.24
Compounds 6, 7, 9 and 10 were previously unknown and
were all prepared in this study by condensation of DODO or
acenaphthenequinone with the corresponding N-amino-
azinium or azolium salts. Thus, N-amination of 1,2-dimethyl-
1H-perimidine,25 4-methylpyrrolo[1,2-a]quinoxaline26 and 1,2-
dimethyl-1H-naphtho[2,3-d]imidazole27 with O-mesitylene-
sulfonylhydroxylamine (MSH)28 gave the corresponding salts,
which were subsequently treated with DODO in the presence
of triethylamine to afford 6, 7 or 9 in overall yields of 27–43%
(Scheme 1). The corresponding salt obtained from commer-
cially available 1,2-dimethylimidazole was reacted with
acenaphthenequinone to afford 10 in good yield (71%).
In order to identify the azonia structures that are most
interesting as potential dyes and DNA probes, the DNA-
binding capacity was tested and the photophysical properties
of the 1–12 chromophoric derivatives were studied. DNA
binding studies were carried out by FRET-based thermal de-
naturation using custom-designed synthetic oligonucleotides
with defined AT- or GC-rich sequences. The absorption and
Results and discussion
With the aim of covering different possibilities of size, shape
and charge distribution of the chromophores, a library of
twelve aromatic azonia cations based on different heterocyclic
cores was studied. Compounds 1–4 (Fig. 2) are based on the
quinolizinium system fused with benzo-, dibenzo-, naphtho-
and indolo- moieties. Three examples (5–7) are based on an
aza-quinolizinium core and another five cations (8–12) share
an azoloazinium unit as the cationic moiety.
The role of size was studied by comparing the properties of
compounds such as 8–11 or 1 and 2 (3). Compounds 2 and 3
are appropriate to obtain data related to charge distribution
within the chromophore. In the cases of 4 and 5 the presence
of an additional nitrogen in the latter compound should
provide information about the effect of replacing a methylene
group next to the quaternary nitrogen atom of quinolizinium
with another nitrogen.3 On the basis of previous studies, the
results of which seem to support a better DNA binding profile
for non-linear chromophores,21 compounds such as 4, 6, 7
and 12 were compared to assess the role of shape. The latter
Fig. 2 Structure of the twelve selected azonia cations.
Scheme 1 Synthesis of derivatives 6, 7, 9 and 10.
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emission spectra were monitored (Fig. 2S and 3S of the ESI†). also exhibited similar features to those obtained in methanol,
In most cases the emission spectra of the derivatives, upon i.e., a single band centred around 470, 460 and 460 nm,
excitation at the maximum of the low energy absorption peak respectively. These bands were not displaced significantly
(λmax), have a single band. The former low energy bands in the upon ctDNA addition. However, rather different changes in the
absorption spectra are usually attributed to the π–π* charge fluorescence intensity were observed. Quenching of the fluo-
transfer transition, which is characteristic of high conjugated rescence intensity was observed on increasing [DNA] for 4 and
systems. The λmax location is rather sensitive to the degree of 11, whereas for 8 the fluorescence intensity remained virtually
conjugation and the electron-donating/withdrawing character- unchanged. These observations indicate a binding interaction
istic of the substituents at the quinolizinium, aza-quinolizi- for 4 and 11 with DNA that does not seem to take place for 8.
nium or azoloazinium cores. The results of DNA-binding The normalized variation of the fluorescence intensity of these
assays and the photophysical parameters for 1–12 derivatives, ligands is shown in Fig. 5 (left), (I0 − I)/I0 (I measured as the
as well as molar absorptivities, fluorescence quantum yields area under the emission spectra and subscript zero denoting
and fluorescence lifetimes, are given in Table 1. The fluo- the absence of ctDNA) against [DNA].
rescence quantum yields in methanol show rather different
The variation of the fluorescence anisotropy (r), obtained by
values and the fluorescence intensity profiles fit monoexpo- eqn (2) (ESI†), versus [DNA] at 25 °C is shown in Fig. 5 (right).
nential or biexponential decay functions depending on the The r values and signs for solutions of 4, 8 and 11 in the
nature of the system.
absence of ctDNA denote different orientations of the absorp-
The DNA-binding assays revealed that compounds 4, 10 tion and emission transition moments, molecular sizes and
and 11 have a high affinity for this biomolecule, whereas 2, 3, rotational diffusion in the low viscosity buffer solvent. The
5 and 6 have a low affinity and 1, 7, 8, 9 and 12 do not have changes in r observed upon addition of aliquots of ctDNA were
any affinity for DNA. More interestingly, all of the compounds different depending on the type of ligand. The r values for 4
that had good DNA-binding properties showed a weak inter- and 11 increased significantly with [DNA]. An increase is
action with GC-rich oligonucleotides but a detectable and barely observed for 8. This trend for increasing r is typical of
rapid increase in Tm from lower ligand concentrations in the the formation of a ligand : DNA complex whose rotational
case of AT-rich oligonucleotides.
relaxation time is higher when compared to that of the free
Several general conclusions can be drawn from the data dis- ligand.
cussed above. As one would expect, DNA binding is closely
The curves depicted in Fig. 5 (left) for 4 and 11 were
related to the size of the molecule. Pentacyclic and hexacyclic obtained by adjusting the experimental data to eqn (10) (ESI†)
compounds show good DNA-binding properties, whereas the and these show a good fit. The curves provide values for the
tricyclic compounds do not have affinity for DNA. A clear trend binding constants of ca. (2.8 1.3) × 104 and (6.9 2.6) × 104
was not observed for tetracycles as some compounds had M−1 for 4 and 11, respectively. These values are of the same
high affinity for DNA but others had none. These data are con- order of magnitude as those for some previously reported
sistent with results obtained previously by our group, which naphtho- and indolo-annelated quinolizinium DNA ligands.3
showed that
a minimum number of four fused rings The average number of nucleic base pairs per bound ligand
are needed to produce an interaction with DNA in these was 8.0 0.4 and 5.9 0.3, respectively. However, compound 8
systems.23a However, it is unclear whether, in this series, there does not seem to interact with DNA. The curves depicted in
is a clear correlation between the shape and charge distri- Fig. 5 (right) for the anisotropy changes for 4 and 11 were
bution of the molecule and its DNA binding activity. As for the obtained by fitting the experimental data to an equation
photophysical properties, quinolizinium and imidazolium similar to 9 (ESI†), but using r instead of the fluorescence
moieties generally give rise to higher fluorescence quantum intensity, I, and the K and n values obtained from fluorescence
yields, although the value for derivative 9 is surprisingly low.
Of the twelve selected structures, derivatives 4, 10 and 11
intensity measurements.
Fluorescence intensity decay profiles for ligands 4, 8 and 11
show the highest affinity for DNA, with 417a and 11 (Fig. 3) did not seem to be particularly sensitive to the presence of
suggesting a possible preference for AT-rich sequences. For ctDNA. The lifetimes, which are presented in Table 2, were
this reason, two of these compounds were chosen for a quite similar for all ligand solutions in the absence or in the
detailed study with DNA. Nevertheless, in order to prove the presence of ctDNA. However, whereas decay profiles for 8 and
validity of the methods and conclusions, compound 8, one of 11 were fitted to a monoexponential decay function, profiles
the fluorescent compounds that did not show affinity for DNA, for 4 were fitted to a bi-exponential function with lifetime com-
was also studied.
ponents of ∼4.5 ns and ∼15 ns. Nevertheless, <τ> barely
changed upon addition of DNA. These facts could be attribu-
ted to a rapid photoinduced electron transfer reaction where
Binding of ligands 4, 8 and 11 with ctDNA
The emission spectra obtained at 25 °C for 4, 8 and 11 in PBS DNA bases and ligand are involved. This would likely decrease
buffer solutions (pH = 7.4) during titrations are shown in DNA-bound ligand lifetimes to values shorter than even the
Fig. 4. Excitation wavelengths were fixed at 334, 310 and nanosecond detection apparatus limit.3,30
387 nm for 4, 8 and 11, respectively. The emission spectra in
The interaction of compounds 4, 8 and 11 with DNA was
the absence or in the presence of calf-thymus DNA (ctDNA) also studied by circular dichroism (CD) spectroscopy. The CD
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Table 1 DNA-binding affinities and base sequence selectivities. Some photophysical parameters: molar absorptivities (ε, M−1 cm−1) at the wave-
length used for excitation in the steady-state measurements (λmax, nm), maximum of the emission wavelength (λem, nm), fluorescence quantum
yields (ϕf), using a quinine solution in 0.1 M sulfuric acid as the standard,29 and fluorescence lifetimes (τ or <τ>, ns) monitored at the excitation/emis-
sion wavelengths of 335 nm/λem for ligands 1–12 in methanol at 25 °C
Structure
DNA binding
No
Relative GC/AT preference
ε (λmax
)
λem
ϕf
0.23
τex (<τ>) (ns)
1
2
—
3180 (338)
361
3.6
Low
Low
AT
AT
10 430 (371)
400
419
0.35
0.15
5.9
5.0
3
8240 (369)
4
5
6
Yes
AT
AT
No
5600 (334)
11 480 (331)
9400 (325)
467
424
435
0.15
0.03
11.8a
13.9
4.7a
Low
Very low
<0.01
7
No
—
14 700 (354)
472
0.02
5.3a
8
9
No
No
—
—
7210 (312)
442
437
0.33
16.7
3.6a
12 100 (351)
<0.01
10
11
12
Yes
Yes
No
AT
AT
—
13 453 (360)
11 850 (387)
7380 (320)
427
450
353
0.41
0.25
0.18
7.45
4.4
6.3
a Weighted average lifetimes using eqn (1), for those compounds whose fluorescence intensity profiles were fitted to biexponential decay functions.
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Fig. 5 (Left) Normalized variation of the fluorescence intensity for
buffer solutions (PBS buffer solutions containing 5% of DMSO) of
ligands 8, 4 and 11 ([dye] = 1.9, 1.9 and 1.94 × 10−5 mol L−1 respectively,
at these concentrations any complex precipitation was observed during
titrations) at 25 °C versus [DNA]. (Right) Fluorescence anisotropy versus
○
[DNA] for the same solutions at 25 °C. Symbols correspond to 4 ( ), 8
□
△
(
) and 11 ( ). r values were monitored at the maximum of emission
upon excitation at 334, 310 and 387 nm, respectively.
Table 2 Lifetime components, contributions and weighted averages
for the dye intercalator aqueous solutions in the absence and in the
presence of ctDNA (in parentheses) at 25 °C. The [DNA] was the highest
[DNA] used in previous experiments. A NanoLed emitting at 335 nm was
used as excitation source
Fig. 3 Melting curves (upper panels) and plot of the experimental ΔTm
versus ligand concentration on the logarithmic scale (lower panel)
obtained for reaction mixtures of compound 11 and double-stranded
DNA oligonucleotides of different base compositions and sequences.
Oligonucleotide AT (red line). -5’-CAATTAAATATAAC-3’ and its comp-
lementary. Oligonucleotide GC (blue line). -5’-GCGCGGCGTCCGGG-
CC-3’ and its complementary. Each data point is the average of
λem (nm) τ1 (ns)
%
τ2 (ns)
%
<τ> (ns)
two separate experiments. Ligand concentrations (M): 1.95 × 10–8
3.91 × 10–8 7.81 × 10–8 1.56 × 10–7 3.13 × 10–7 6.25 × 10–7
1.25 × 10–6, 2.5 × 10–6, 5.0 × 10–6, 1.0 × 10–5 and 2.0 × 10–5
,
,
4
8
465
442
4.3 (4.5) 54.2 (39.6) 14.8 (15.0) 45.8 (60.4) 9.1 (10.9)
4.1 (4.2) 100
4.3 (4.3) 100
—
—
—
—
—
—
,
,
,
,
11 460
.
Fig. 4 Emission spectra for solutions of ligands 8, 4 and 11 ([L] = 1.9,
1.9 and 1.94 × 10−5 mol L−1, respectively) and upon the addition of DNA
aliquots during the titration at 25 °C.
Fig. 6 CD spectra (as ellipticity, θ) in the region of DNA absorption at
25 °C for buffer solutions (PBS buffer solutions containing 5% of DMSO)
of ligand 11 in the presence of ctDNA ([DNA] = 3.57 × 10−4 M) at several
ligand/DNA molar ratios.
spectrum in the region of DNA absorption can provide infor-
mation about the changes in its secondary structure caused by
ligand–DNA interactions.31 As depicted in Fig. 6 for 11, ctDNA
shows the typical DNA B-form CD spectrum with positive and
negative Cotton effects centered at ∼278 and ∼245 nm, which
are due to base stacking and right-handed polynucleotide heli-
city, respectively. Changes in the two bands are quite sensitive
to the mode of interaction with small molecules.32 While
groove binding of small molecules with DNA shows little or no
perturbation on the base stacking and helicity bands, inter-
calation usually enhances the intensities of both bands. As
depicted in Fig. 6, the intensities of both bands increase
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significantly upon increasing the concentration of 11, thus presence of exciton coupling due to the aggregation of 4
indicating intercalative binding to DNA. These changes in the and 11, which are associated with the DNA, could be dis-
DNA CD spectra are a consequence of the increase in base counted.3,12c,33b In addition, the molar ellipticity [θ] shows an
pair–ligand interactions and also some unwinding and increase in the positive band with [ligand] that did not occur
lengthening of the DNA helix upon dye intercalation. Similar for the negative band. In analogy with other quinolizinium
changes were observed for 4.
derivatives, and based on the assumption that the transition
In contrast to the above, achiral compounds like 4, 8 and 11 moments of the bands that appear at longer wavelengths
do not have CD activity. However, a CD signal can be induced (∼335–400 nm) are almost parallel to the long ligand axis
(ICD) as a consequence of a possible non-degenerate coupling (Fig. 4S, ESI†), the sign of the bands reveals intercalation of 4
of their transition moments and the average dipole moments and 11 with their long molecular axis perpendicular to the DNA
of the nucleic bases. Consequently, the observation of a CD binding pocket.3,12c Consequently, the negative ∼295–335 nm
spectrum in the absorption region of the ligand is irrefutable band is consistent with an absorption band whose transition
evidence for the existence of ligand–DNA interaction. The moment is oriented along the short molecular axis. The rela-
intensity and the sign of the ICD depend on the position tively low quantitative ellipticity values, which are positive,
and the orientation of the chromophore relative to the DNA especially for 4, also exclude the presence of groove binding.
bases. An intercalator exhibits a weak negative ICD when
the transition moment for the corresponding absorption band
is oriented along the pseudo-long axis of the binding DNA
pocket (parallel to the bisector of the base pairs). However, a
strong positive ICD is expected when the transition moment is
polarized perpendicular to the long axis of the binding pocket.
The ICD signal for a groove-bound ligand is usually one or
two orders of magnitude larger than that for an intercalated
one. In addition, if the transition moment is oriented along
the groove that forms a 45° angle with the bases, the ICD is
expected to be positive.3,12c,32a,33
The CD spectra for intercalators 4, 8 and 11 in the presence
of DNA at a fixed ligand : DNA molar ratio of 0.4 are shown in
Fig. 7 (left). ICD was not observed for 8 at any of the molar
ratios (0.05, 0.1, 0.2 or 0.4) as interactions did not take place.
However, the CD spectra for 4 and 11, at all of the ratios used,
exhibited two regions with negative and positive Cotton effects
that extend in the ∼295–335 nm and 335–460 nm ranges,
respectively (Fig. 7, right). Nevertheless, the CD signals were
much weaker for 4 than for 11. These asymmetric negative
and positive induced bands seem to match other bands that
appear in the absorption spectra of the compounds. Thus, the
Molecular mechanics and molecular dynamics simulations
The total binding energy values are shown in Fig. 8 along with
van der Waals and electrostatics contributions as a function of
the oo′ distance along the y coordinate, for the most favourable
orientation of 11 approaching the DNA by the major or minor
groove sides. Two different approaches were considered: (i)
parallel to the planar bases as an intercalator (Fig. 8, left
panel) or (ii) parallel to the pair of helixes as a groove binder
(Fig. 8, right panel). Intercalation takes place with hardly any
potential energy barriers. However, as one would expect, in the
groove-binding approach, when guest 11 is oriented at 45°
with respect to the pair of bases, high potential barriers are
observed at y = 5 Å due to the repulsive van der Waals ligand
interaction with DNA base pairs. The results show that electro-
static interactions are mostly responsible for the stabilization
of the system. It is worth noting that for the minimum
binding structures (MBE) of 11 the intercalator ligand is
oriented in such a manner that its main axis is oriented paral-
lel to the TA and AT central base pocket axis but is slightly
■
△
Fig. 8 Total binding energies ( ), van der Waals ( ) and electrostatic
○
(
) contributions as a function of the oo’ distance (nm) measured along
the y coordinate for the ligand 11 approaching DNA as an intercalator
(left) and as a groove binder (right). Superimposed are the MBE struc-
tures, designated as 1 and 2, viewed from the positive x-axis (note the
location of the ligand). (1) and (2) MBE structures were used as starting
conformations for the MD simulations.
Fig. 7 CD spectra, as ellipticity, θ in mdegrees, for (left) 4, 8 and 11
ligand buffer solutions in the presence of ctDNA at [DNA] = 3.57 × 10−4
M at a [ligand]/[DNA] = 0.4; (right) 11 at several ligand/DNA molar ratios
at 25 °C. The CD spectra, as molar ellipticity, is superimposed.
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Fig. 10 Snapshots of the 1 ns MD trajectory at 300 K showing the
location of the ligand. Structures correspond to the central portion of
the 11-DNA complex: (left) at time = 0 ns, i.e., the starting minimized
MBE structure (1); (right) at time = 0.8 ns from the beginning of the
trajectory.
Fig. 9 (Upper panel) Histories of binding energies (black) and electro-
statics (grey) and van der Waals (light grey) contributions obtained from
the analysis of the 1 ns MD trajectory at 300 K starting from the mini-
mized MBE structure (1). (Bottom panel) The same for the y coordinate
(bottom black line) of the centre of the 11 molecule (o’), the end-to-end
DNA helix distance (upper black line), the average angle between the
plane of both AT and TA pairs of bases and the plane of 11 (dark grey)
and the angle between the bisector of pocket bases and the long axis of
11 (light grey).
enlargement and the base–ligand interactions are also con-
sistent with the increase in the intensity of the bands observed
in the CD spectrum at the DNA region upon 11-DNA complexa-
tion (Fig. 7, left). Details of some snapshots of the complex
structures at different times are shown in Fig. 10. At 0.8 ns, 11
is oriented as one would expect from the ICD spectrum.
outside it on the major groove side (1 in Fig. 8). However, the
When MD is performed starting from (2), i.e., the MBE
main long axis of 11 is almost parallel to both DNA chains in structure for the groove approach, the most important feature
2. The arrows in Fig. 8 indicate the ligand acenaphthene is that during the first steps of the trajectory the ligand pene-
group.
trates and prefers to intercalate between the two AT–TA base
The histories of the 11-DNA binding energies and contri- pairs. Whereas the plane of the molecule of 11 is also almost
butions and oo′ distances obtained from the analysis of the parallel to the plane of both pairs of bases (average plane
1.0 ns MD trajectories at 300 K in the water-solvated (1) MBE angle of 13
7°), in this case the angle between the long
structure are shown in Fig. 9 (upper panel). The results indi- axis and the binding pocket is around 45° and, at the end of
cate that 11-DNA interactions are favourable for intercalation the trajectory, this decreases to around 30° (average angle
throughout the whole trajectory. As with MM, the most impor- of 39 10°), which is not consistent with the experimental
tant contributions to stabilization were the electrostatics. The evidence (Fig. 11).
van der Waals interactions contributed 10% of the total
binding energy (Fig. 9, upper panel).
A similar situation occurred for DNA complexation by
ligand 4. As shown in Fig. 4S of the ESI,† intercalation of com-
The history for the y coordinate of the centre of ligand pound 4 is also favourable and electrostatic interactions are
11 (o′) is depicted in Fig. 9 (bottom panel). The intercalator mainly responsible for this stabilization. For the MBE struc-
tends to move inside the bases pocket close to the y coordinate ture the results indicate that 4 is mainly parallel to both the
value of −1 (Å). This movement also involves rotation and a central pairs of bases when it intercalates DNA, but that it
subsequent increase in the angle between the bisector of prefers to be located slightly outside the pocket by the minor
the bases pocket and the long axis of the ligand in such a groove face. Analysis of the MD trajectories also showed that
way that, after ∼0.7 ns and at the end of the trajectory, it interactions for the intercalation are favourable throughout the
is oriented almost perpendicular to the pocket and with the whole trajectory, with electrostatics being the most important
acenaphthene group lying slightly outside it by the minor contributions to the stabilization (Fig. 6S, ESI†). In this case,
groove face (see Fig. 10). The latter structure is consistent with the centre of intercalator 4 tends to stay around the y = +1 (Å)
the sign of the bands observed in the ICD spectrum for the coordinate. The average of the angle between the bisector of
region in which 11 absorbs (Fig. 7, right). In addition, the the base pocket and the long axis of the dye is 62.1 9.8°,
average of the angle between the AT and TA base pair planes which means that it deviates slightly from 90°. This behaviour
and the plane of intercalator 11 throughout the trajectory is is responsible for the weakness of the ICD signal. In addition,
close to zero, as one would expect.
the angle between the AT and TA base pair planes and the
Another characteristic observation after intercalation is that plane of intercalator 4 is close to zero, as one would expect. As
a monotonic increase of the end-to-end DNA distance (Fig. 9) in the previous study, a monotonic increase in the end-to-end
occurs with time. This change is due to the slight unwinding DNA distance occurs with time. A portion of the MBE structure
and elongation of the DNA helix upon intercalation. This helix for the 4-DNA complex used as the starting conformation for
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uncorrected. IR spectra were obtained on a Perkin-Elmer FTIR
spectrum 2000 spectrophotometer. 1H and 13C NMR spectra
were recorded with either a Varian Gemini (200 MHz), Varian
Mercury VX-300, Varian Unity 300 or 500 MHz spectrometer at
room temperature. Chemical shifts are given in ppm (d) down-
field from TMS. Coupling constants (J) are in hertz (Hz) and
signals are described as follows: s, singlet; d, doublet;
t, triplet; br, broad; m, multiplet; ap, apparent. High-resolution
analysis (TOF) was performed on an Agilent 6210 time-of-
flight LC/MS. 1,2-Dimethyl-1H-perimidine,25 4-methylpyrrolo-
[1,2-a]quinoxaline,26 1,2-dimethyl-1H-naphtho[2,3-d]imidazole,27
O-mesitylenesulfonyl-hydroxylamine (MSH),28 and [1,4]-dioxane-
2,3-diol (DODO)22 were prepared according to procedures
described in the literature.
7-Methyl-7H-pyridazino[1,6-a]perimidin-12-ium
tetrafluoroborate (6)
Fig. 11 (Upper panel) Histories of binding energies (black) and electro-
statics (grey) and van der Waals (light grey) contributions obtained from A solution of MSH (108 mg, 0.5 mmol) in anhydrous CH2Cl2
the analysis of the 1 ns MD trajectory at 300 K starting from the mini-
mized MBE 11-DNA groove binding structure, namely (2), obtained from
MM calculations. (Bottom panel) The same for the y coordinate (bottom
black line) for the centre of the dye molecule (o’), the averaged angle
(3 mL) was added to a solution of 1,2-dimethyl-1H-perimidine
(75 mg, 0.38 mmol) in CH2Cl2 (3 mL). The mixture was stirred
for 15 h at room temperature. The resulting N-amine salt was
isolated by filtration and washed with Et2O and petroleum
between the plane of both AT and TA pairs of bases and the plane of the
11 molecule (dark grey) and the angle between the bisector of pocket ether. A mixture of this N-amine salt (110 mg, 0.27 mmol),
bases and the long 11 axis (light grey).
DODO (32 mg, 0.27 mmol) and triethylamine (37 μL,
0.27 mmol) in anhydrous DMF (3 mL) was heated at 70 °C for
2 h. The dark red solid residue was filtered off and treated
with HBF4 (1.2 equiv., 48% w/w water) at room temperature for
the 1 ns MD trajectory at 300 K (left) and those obtained from
7 h. The crude product was purified by alumina column
the analysis of the MD trajectory (right) are represented in
chromatography (gradient from CH2Cl2 to 1 : 9 MeOH–CH2Cl2)
Fig. 7S (ESI†).
to give compound 6 as a dark red powder (53 mg, 0.17 mmol,
43%). M.p.: 199–205 °C. IR (KBr) ν cm−1: 765, 1179, 1489,
1583, 1640, 2358, 3059, 3432. 1H-NMR (200 MHz, CD3OD)
Conclusions
δ (ppm): 3.73 (s, 3H), 7.21 (dd, J = 7 Hz, J = 1 Hz, 1H),
7.54–7.68 (m, 3H), 7.83 (d, J = 8 Hz, 1H), 8.00–8.39 (m, 2H),
8.37 (dd, J = 10 Hz, J = 1 Hz, 1H), 8.84 (dd, J = 4 Hz, J = 1 Hz,
1H). 13C-NMR (50 MHz, CD3OD) δ (ppm): 35.5, 109.6, 111.9,
121.6, 124.1, 126.2, 128.1, 129.3, 129.6, 134.5, 135.1, 135.9,
146.0, 151.1. MS (ESI): [M+] 234.1; HRMS m/z calculated
for C15H12N3 [M+]: 234.1026. Found 234.1032. Anal. Calcd
for C15H12BF4N3: C, 56.11; H, 3.77; N, 13.09. Found: C, 55.94;
H, 3.92; N, 13.29.
Fluorescence, DNA interactions and DNA sequence selectivity
properties of twelve different azonia cations have been studied.
Among them, tetracyclic indoloquinolizinium 4 and hexacyclic
benzimidazolium 11 present the highest affinity for DNA, with
a potential preference for AT-rich sequences. Photophysical
and DNA binding experiments showed that both compounds
are fluorescent and bind to double-stranded DNA by intercala-
tion. The findings described here will allow further studies on
4 and 11 or its derivatives as DNA probes.
Pyridazino[1,6-a]pyrrolo[2,1-c]quinoxalin-13-ium
mesitylenesulfonate (7)
A solution of MSH (88 mg, 0.41 mmol) in anhydrous CH2Cl2
(3 mL) was added to a solution of 4-methylpyrrolo[1,2-
a]-quinoxaline (62 mg, 0.34 mmol) in CH2Cl2 (3 mL). The
Experimental
Synthetic procedures
General information. Reagents were purchased at the mixture was stirred for 5 h at room temperature. The resulting
highest quality commercially available and were used without N-amine salt was isolated by filtration and washed with Et2O
further purification, unless stated otherwise. Reactions were and petroleum ether. A mixture of this N-amine salt (100 mg,
monitored by thin-layer chromatography (TLC) carried out on 0.25 mmol), DODO (30 mg, 0.25 mmol) and triethylamine
0.25 mm E. Merck silica gel plates (60FS-254) with visualiza- (35 μL, 0.25 mmol) in EtOH (4 mL) was heated at 76 °C for
tion by UV light. Column chromatography was performed 4.5 h. The reaction mixture was concentrated under reduced
using silica gel (60 F254, 70–200 mm) as the stationary phase. pressure until dryness and triturated with acetone. The solid
All melting points were determined in open capillary tubes on was filtered off and washed with acetone and petroleum ether
a Stuart Scientific SMP3 melting point apparatus and are to give compound 7 as a dark green powder (39 mg, 0.09,
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1
mmol, 27%). M.p: 135–138 °C. IR (KBr) ν cm−1: 680, 1189, 678, 1017, 1086, 1191, 1220, 1532, 3028, 3126, 3448. H-NMR
1
1451, 1624, 3466. H-NMR (300 MHz DMSO-d6) δ (ppm): 2.15 (300 MHz, DMSO-d6) δ (ppm): 2.12 (s, 3H), 2.47 (s, 6H), 4.41
(s, 3H), 2.47 (s, 6H), 6.72 (s, 2H), 7.35 (dd, J = 2.6 Hz, J = (s, 3H), 6.69 (s, 2H), 7.98 (tap, J = 7.6 Hz, 2H), 8.31–8.47
4.2 Hz, 1H), 7.81 (tap, J = 8.4 Hz, J = 8.7 Hz, 1H), 8.02 (tap, J = (m, 1H), 8.85 (d, J = 2.1 Hz, 1H), 9.37 (s, 1H). 13C-NMR
8.4 Hz, J = 8.7 Hz, 1H), 8.25 (dd, 4.2 Hz, J = 1.2 Hz, 1H), 8.38 (50 MHz, DMSO-d6) δ (ppm): 19.8, 22.2, 33.9, 111.0, 117.9,
(dd, J = 9 Hz, J = 4.7 Hz, 1H), 8.64 (d, 8.7 Hz, 1H), 8.85 (d, J = 122.6, 123.6, 126.2, 128.1, 128.8, 128.9, 129.2, 129.6, 130.4,
8.7 Hz, 1H), 9.03 (d, J = 1.6 Hz, 1H), 9.31 (dd, J = 9.0 Hz, J = 134.0, 134.9, 135.0, 135.2, 135.5, 142.3, 156.1. MS (ESI+) m/z:
1.6 Hz 1H), 9.38 (dd, J = 4.7 Hz, J = 1.6 Hz, 1H). 13C-NMR 258.1. Anal. Calcd for C26H23N3O3S: C, 68.25; H, 5.07; N, 9.18.
(75 MHz DMSO-d6) δ (ppm): 19.8, 22.3, 115.8, 116.3, 117.1, Found: C, 67.96; H, 5.11; N, 9.36.
119.7, 120.5, 122.6, 126.8, 127.2, 129.4, 130.4, 132.0, 132.1,
Photophysical properties
135.4, 135.7, 139.4, 142.4, 146.1, 148.9. MS (ESI): [M+] 220.1;
HRMS m/z calculated for C14H10N3 [M+]: 220.0869. Found Absorption spectra were recorded on
a
UV-Vis Uvikon
220.0859. Anal. Calcd for C23H21N3O3S: C, 65.85; H, 5.05; 941 (Kontron Instruments) spectrophotometer. Steady-state
N, 10.02. Found: C, 65.66; H, 5.12; N, 9.91.
fluorescence measurements were carried out using a PTI
Quanta Master spectrofluorimeter equipped with a xenon flash
lamp as a light source, with single concave grating monochro-
mators and Glan–Thompson polarizers in the excitation and
5-Methyl-5H-naphtho[2′,3′:4,5]imidazo[1,2-b]pyridazin-12-ium
mesitylenesulfonate (9)
A solution of MSH (174 mg, 0.81 mmol) in anhydrous CH2Cl2 emission paths. Detection was carried out with a photomulti-
(2 mL) was added to a solution of 1,2-dimethyl-1H-naphtho- plier cooled by a Peltier system. Slit widths were selected at
[2,3-d]imidazole (106 mg, 0.54 mmol) in CH2Cl2 (2 mL). The 6 nm for both excitation and emission paths and polarizers
mixture was stirred for 1 h at room temperature. The resulting were fixed under the ‘magic angle’ conditions. Fluorescence
N-amine salt was isolated by filtration and washed with Et2O decay measurements were performed on a time-correlated
and petroleum ether. A mixture of the N-amine salt (206 mg, single-photon-counting
FL900
Edinburgh
Instruments
0.50 mmol), DODO (60 mg, 0.50 mmol) and triethylamine Spectrometer. A monochromatic NanoLed (Horiba) emitting at
(70 μL, 0.50 mmol) in anhydrous DMF (3 mL) was heated at 335 nm was used as the excitation source. Double concave
70 °C for 1 h. The reaction mixture was concentrated under grating monochromators were employed at both excitation and
reduced pressure until dryness and triturated with Et2O. The emission paths. Photons were detected by a red-sensitive
solid was filtered off and washed with acetone to give com- cooled (by a Peltier system) photomultiplier. The data acqui-
pound 9 as a yellow powder (99.7 mg, 0.23 mmol, 43%). M.p: sition was carried out using 1024 channels of a multichannel
233–235 °C (decomposition). IR (KBr) ν cm−1: 677, 1015, 1085, analyser with a time window width of 200 ns. A total of 10 000
1191, 1522, 1622, 2365, 3045. 1H-NMR (300 MHz, MeOD) counts in the maximum peak channel were taken for each
δ (ppm): 2.05 (s, 3H), 2.41 (s, 6H), 4.20 (s, 3H), 6.75 (s, 2H), measurement. The instrumental response function was regu-
7.61–7.70 (m, 2H), 8.15–8.26 (m, 3H), 8.55 (s, 1H), 8.74 (d, J = larly achieved by measuring the scattering of a Ludox solution.
8.2 Hz, 1H), 8.99 (s, 1H), 9.07 (d, J = 4.6 Hz, 1H). 13C-NMR Intensity fluorescence profiles were fitted to multi-exponential
(75 MHz, MeOD) δ (ppm): 20.8, 23.2, 31.3, 110.9, 112.9, 121.4, decay functions using the iterative deconvolution method.34
128.0, 128.8, 129.1, 129.4, 130.0, 131.6, 131.9, 132.2, 132.5, The equations used are given in the ESI.†
135.3, 138.1, 140.0, 140.8, 144.5, 147.3. MS (ESI): [M+] 234.1;
Theoretical protocols for molecular mechanics and molecular
dynamics calculations
HRMS m/z calculated for C15H12N3 [M+]: 234.1026. Found
234.1027. Anal. Calcd for C24H23N3O3S: C, 66.49; H, 5.35;
N, 9.69. Found: C, 66.70; H, 5.20; N, 9.56.
Molecular mechanics (MM) and molecular dynamics (MD)
calculations were performed to study the forces responsible for
the binding of quinolizinium dyes to DNA and to assess the
stability of the system. Calculations were performed with Sybyl
11-Methyl-11H-acenaphtho[1,2-e]imidazo[1,2-b]pyridazin-
8-ium mesitylenesulfonate (10)
A solution of MSH (0.61 g, 2.83 mmol) in anhydrous CH2Cl2 X-2.035 and the Tripos Force Field.36 B-DNA helical fragments,
(3 mL) was added to a solution of commercially available 1,2- whose charges were derived using the Gasteiger and Marsili
dimethylimidazole (0.18 g, 1.89 mmol) in CH2Cl2 (3 mL). The method, contained twelve nucleotides with a CGCGAATTCGCG
mixture was stirred for 1 h at room temperature. The resulting sequence (approximately the percentage of the ctDNA bases).
N-amine salt was isolated by filtration and washed with Et2O Charges for ligands were obtained using the Gaussian suite
and petroleum ether. A mixture of the N-amine salt (0.49 g, of quantum chemical programs at the HF/6-31G(d) level of
1.59 mmol), acenaphthoquinone (0.29 g, 1.59 mmol) and theory.37 A relative permittivity ε = 1 was used for electrostatic
sodium acetate (0.13 g, 1.59 mmol) in ethanol (12 mL) was contributions in the presence of explicit water (ε = 3.5 for
heated under reflux for 5 h. The reaction mixture was concen- some preliminary calculations under vacuum). Water solvation
trated under reduced pressure until dryness and triturated was performed using the Molecular Silverware (MS) algorithm
with water. The solid was filtered off and washed with and periodic boundary conditions (PBC).38 Non-bonded cut-off
water and acetone to give compound 10 as a pale brown solid distances for MM and for MD were set at 12 Å. Optimizations
(0.61 g, 1.33 mmol, 71%). M.p: 260–261 °C. IR (KBr) ν cm−1
:
were carried out using the simplex algorithm and the
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conjugate gradient was used as a termination method, with the midpoint of the transition (Tm) was calculated. This allowed
gradients of 3.0 kcal (mol Å)−1 for the calculations carried out us to estimate the increases in melting temperatures (ΔTm)
in water.39 For further information and figures, see ESI.†
brought about by ligand binding. A series of custom-designed
oligonucleotides were designed to provide at least a single
occurrence of all possible 3-base pair long and all possible
DNA binding experiments
DNA calf thymus binding. Concentrated solutions of palindromic 4-base pair long binding sites composed of either
ligands 4, 8 and 11 in DMSO (Aldrich, HPLC grade) were A and T or G and C bases. All measurements were carried out at
diluted with a phosphate buffered saline solution (PBS) at least in duplicate. The raw data recovered from the instrument
pH = 7.4 containing 5% DMSO to give concentrations of 1.90 × for each plate consisted of an array of FAM fluorescence values
10−5, 1.90 × 10−5 and 1.94 × 10−5 mol L−1, respectively. Calf for each of the 96 wells at each temperature. These data were
thymus DNA (high molecular weight sodium salt, ctDNA) was analysed to estimate the increases in melting temperatures
purchased from Aldrich and dissolved in Milli-Q water to (ΔTm) brought about by ligand binding at the different concen-
obtain a ∼2 mg mL−1 solution. The ctDNA concentration (per trations analyzed. The numeric analysis was carried out
base pair) of this solution, determined by UV-Vis using using an in-house developed Visual Basic application running
ε (260 nm) = 13 200 M−1 cm−1, was 4.1 × 10−3 mol L−1 40
For on Microsoft Excel (Microsoft, Redmond, WA, USA).
.
titrations, 10 μL aliquots of this ctDNA solution were added
to a 10 mm quartz cell containing 3 mL of the above ligand
solutions and the samples were stirred prior to measurement
to ensure temperature equilibration (10 min). At the end of the
Acknowledgements
titration 170 μL of ctDNA was added and the ligand/DNA Financial support from the Spanish Ministerio de Economía
molar ratios reached values of approximately 1 : 12. The Competitividad (project CTQ2011-24715), Instituto de
y
effect of ligand dilution after the addition of each aliquot did Salud Carlos III (REDinREN, RD12/0021/0014) and University
not give an error of more than 5% for the ligand concen- of Alcalá (project CCG2013/EXP-036) and grants for P. B. and
tration. Nevertheless, the fluorescence emission intensities R. M. S. are gratefully acknowledged.
were corrected due to this dilution. The derivation of the
equations for the ligand–macromolecule binding is described
in the ESI.†
Notes and references
DNA melting assay. Synthetic oligodeoxynucleotides
with one strand 5′-end-labelled with the fluorophore
6-carboxyfluorescein (6-FAM, F) and the complementary strand
3′-end-labelled with the quencher tetramethylrhodamine
(TAMRA, Q) were synthesized and purified by high-performance
liquid chromatography as a single peak by biomers.net GmbH
(Ulm, Germany). Annealing of each F-oligonucleotide with its
complementary Q-oligonucleotide at a final duplex concen-
tration of 0.1 μmol L−1 in Dulbecco’s PBS buffer [137 mM NaCl,
2.7 mM KCl, 8.06 mM Na2HPO4, 1.47 mM KH2PO4 (pH 7.4),
ionic strength 999999] was accomplished on a PTC-100 thermo-
cycler (MJ Research) by first heating to 95 °C for 5 min and
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