Benzothienoquinolines: New one-pot synthesis and fluorescence studies of their interaction with DNA and polynucleotides

Article abstract of DOI:10.1016/j.jphotochem.2014.08.001

In this work, we were able to obtain the benzothieno[3,2-b]quinoline 1 and benzothieno[2,3-c]quinoline 2 using a new one-pot procedure from the reaction of the commercially available 3-bromobenzo[b]thiophene-2-carbaldehyde with 2-aminophenylpinacolborane under Suzuki coupling conditions using a stereochemically hindered ligand, 2-(cyclohexylphosphane)biphenyl and Ba(OH)₂·8H₂O as the base. Fluorescence properties of the benzothieno[3,2-b]quinoline 1 and the benzothieno[2,3-c]quinoline 2 were studied in solvents of different polarity. Both compounds exhibit a solvent sensitive emission, compound 1 being less fluorescent (Φ_F < 0.05) than compound 2 (0.04 ≤& Φbsubesub ≤ 0.10). The interaction of these compounds with salmon sperm DNA and synthetic double-stranded heteropolynucleotides, poly(dA-dT)·(dA-dT) and poly(dG-dC)·(dG-dC) , was studied using spectroscopic methods, allowing the determination of the intrinsic binding constants and binding site sizes. The interaction of both compounds is stronger with adenine-thymine (A-T) base pairs. Compound 1 is the most intercalative in salmon sperm DNA (47%) and polynucleotides (46-49% of intercalated molecules), while for compound 2, 41% is intercalated in salmon sperm DNA and only 8% in poly(dG-dC)·(dG-dC). Docking studies indicate that compound 1 interacts more strongly with DNA than compound 2, with a significant value of binding free energy in the case of intercalation. Minor groove binding is also very favourable and, probably, both mechanisms occur with a preponderance of intercalation in the case of compound 1. Overall, these results indicate that both benzothienoquinolines interact with nucleic acids by both intercalation and groove binding.

Full text of DOI:10.1016/j.jphotochem.2014.08.001

Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 20–30
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Benzothienoquinolines: New one-pot synthesis and fluorescence
studies of their interaction with DNA and polynucleotides
A. Rita O. Rodriguesa, M. Solange D. Carvalhoa,b, J. André V. Cardosoa,
Ricardo C. Calhelhab, Maria-João R.P. Queirozb, Paulo J.G. Coutinhoa,
Elisabete M.S. Castanheiraa,∗
a Centro de Física (CFUM), Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
b Centro de Química, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 June 2014
Received in revised form 31 July 2014
Accepted 1 August 2014
Available online 10 August 2014
In this work, we were able to obtain the benzothieno[3,2-b]quinoline
1 and benzothieno[2,3-
c]quinoline
2 using a new one-pot procedure from the reaction of the commercially available
3-bromobenzo[b]thiophene-2-carbaldehyde with 2-aminophenylpinacolborane under Suzuki cou-
pling conditions using a stereochemically hindered ligand, 2-(cyclohexylphosphane)biphenyl and
Ba(OH)2·8H2O as the base.
Fluorescence properties of the benzothieno[3,2-b]quinoline 1 and the benzothieno[2,3-c]quinoline 2
were studied in solvents of different polarity. Both compounds exhibit a solvent sensitive emission,
compound 1 being less fluorescent (˚F < 0.05) than compound 2 (0.04 ≤ ˚F ≤ 0.10).
The interaction of these compounds with salmon sperm DNA and synthetic double-stranded het-
eropolynucleotides, poly(dA–dT)·(dA–dT) and poly(dG–dC)·(dG–dC), was studied using spectroscopic
methods, allowing the determination of the intrinsic binding constants and binding site sizes. The inter-
action of both compounds is stronger with adenine–thymine (A–T) base pairs. Compound 1 is the most
intercalative in salmon sperm DNA (47%) and polynucleotides (46–49% of intercalated molecules), while
for compound 2, 41% is intercalated in salmon sperm DNA and only 8% in poly(dG–dC)·(dG–dC). Docking
studies indicate that compound 1 interacts more strongly with DNA than compound 2, with a significant
value of binding free energy in the case of intercalation. Minor groove binding is also very favourable
and, probably, both mechanisms occur with a preponderance of intercalation in the case of compound 1.
Overall, these results indicate that both benzothienoquinolines interact with nucleic acids by both
intercalation and groove binding.
Keywords:
Benzothieno[3,2-b]quinoline
Benzothieno[2,3-c]quinoline
DNA interaction
Polynucleotides
Fluorescence
Docking studies
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
bonding, attractive van der Waals and hydrophobic interactions
[4]. DNA intercalation seems to be an essential, but not sufficient,
The investigation of the nature and dynamics of the binding
of small molecules to biomacromolecules is actually an active
area of research [1,2]. DNA interaction studies are important to
understand the mechanism of action of antitumor and antiviral
drugs and to design new DNA-targeted drugs [3,4]. Three different
modes of binding to DNA have been described: intercalation into
the base pairs, in the major or minor grooves, and outside the
double helix by electrostatic interactions. Small molecules are
stabilized in groove binding and intercalation with DNA through
a series of associative interactions such as ␲-stacking, hydrogen
step for antitumoral activity [3].
Benzothieno[3,2-b]quinoline
1 [5] and benzothieno[2,3-c]
quinoline 2 [6] are known for their anti-plasmodic and anti-
infectious activities, acting mainly through intercalation between
DNA base pairs when used in their salt form. Earlier synthesized by
separated reactions and in several steps [5,6], in this work we were
able to obtain the two compounds in a one pot procedure.
The interactions of the biologically active compounds with
nucleic acids have been studied using a variety of techniques
[7–11], including absorption and fluorescence spectroscopies. The
binding of the fluorescent polycyclic molecules to DNA can be
conveniently investigated by these methods, because their absorp-
tion and emission properties significantly change on complex
formation [7,12,13]. Fluorescence quenching experiments using
external quenchers have been used to establish the DNA binding
∗
Corresponding author. Tel.: +351 253 604321; fax: +351 253 604061.
E-mail addresses: ecoutinho@fisica.uminho.pt, emsccoutinho@gmail.com
(E.M.S. Castanheira).
http://dx.doi.org/10.1016/j.jphotochem.2014.08.001
1010-6030/© 2014 Elsevier B.V. All rights reserved.

A.R.O. Rodrigues et al. / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 20–30
21
modes, since intercalated fluorophores are less accessible to anionic
quenchers, due to electrostatic repulsion by the negatively charged
DNA backbone [13–15].
In this work, the interaction of the synthesized benzothieno-
quinolines 1 and 2 with natural double-stranded salmon sperm
DNA and with synthetic ds-polyheteronucleotides was investigated
by fluorescence emission measurements. These studies are impor-
tant due to the biological relevance of both compounds as potential
antitumorals.
performed using a Fluorolog 3 spectrofluorimeter, equipped with
double monochromators in both excitation and emission and a
temperature-controlled cuvette holder. For fluorescence quantum
yield determination, the solutions were previously bubbled for
30 min with ultrapure nitrogen. Fluorescence spectra were cor-
rected for the instrumental response of the system.
The fluorescence quantum yields (˚s) were determined using
the standard method (Eq. (1)) [16,17] and 9,10-diphenylanthracene
in ethanol as reference, ˚r = 0.95 at 25 ◦C [18].
ꢀ
ꢁ
(ArFsn2s )
(AsFrn2r )
2. Experimental
˚s =
˚
r
(1)
2.1. Synthesis
where A is the absorbance at the excitation wavelength, F the inte-
grated emission area and n the index of refraction of the solvents
used. Subscripts refer to the reference (r) or sample (s) compound.
All solutions were prepared using spectroscopic grade sol-
vents and Milli-Q grade water. Natural double-stranded salmon
sperm DNA was obtained from Invitrogen, while synthetic
double-stranded heteropolynucleotides, poly(dA–dT)·(dA–dT) and
poly(dG–dC)·(dG–dC), were obtained from Sigma-Aldrich. Salmon
sperm DNA, polynucleotides and compounds stock solutions were
prepared in 10−2 M Tris–HCl buffer (pH 7.2), with 10−3 M EDTA. The
purity of DNA was checked by monitoring the absorption spectrum
and the ratio of the absorbance at 260 and 280 nm, A260/A280 = 1.95
(good-quality DNA exhibits an A260/A280 ratio higher than 1.8
[19]). The DNA and polynucleotide concentrations in number of
bases (or phosphate groups) were determined from the molar
absorption coefficients [12], ε = 6600 M−1 cm−1 at 260 nm for
DNA, ε = 8400 M−1 cm−1 at 254 nm for poly(dG–dC)·(dG–dC) and
ε = 6600 M−1 cm−1 at 260 nm for poly(dA–dT)·(dA–dT).
2.1.1. General remarks
Melting points (◦C) were determined in a SMP3 Stuart apparatus
and are uncorrected. 1H and 13C NMR spectra were recorded on a
Varian Unity Plus at 300 and 75.4 MHz, respectively. Heteronuclear
correlations, 1H–13C, HMQC or HMBC were performed to attribute
some signals. HRMS data were recorded using a method of direct
injection by EI by the mass spectrometry service of the University of
Vigo, Spain. The reactions were monitored by thin layer chromatog-
raphy (TLC) in aluminium plates covered with a layer of silica gel 60
(Macherey-Nagel) of 0.2 mm, with UV254 fluorescence indicator.
Column chromatography was performed using silica-gel 230–400
mesh. Petroleum ether refers to the 40–60 ◦C boiling range fraction.
One pot synthesis of benzothieno[3,2-b]quinoline
1
and
benzothieno[2,3-c]quinoline 2: To a solution of 3-bromobenzo[b]
thiophene-2-carbaldehyde (150 mg, 0.600 mmol) in dioxane
(5 mL), Pd(AcO)2 (5 mol%), 2-(cyclohexylphosphane)biphenyl
(20 mol%), Ba(OH)2·8H2O (3 equiv.) and 2-aniline pinacolborane
(170 mg, 0.780 mmol). The mixture was heated at 100 ◦C for 5 h.
After cooling, H2O and AcOEt were added and the phases were
separated. The organic phase was dried (MgSO4), filtered and the
removal of the solvent gave an oil which was dissolved in a small
amount of ether and filtered through silica gel column packed
and prewashed with petroleum ether. Two bands were eluted;
compound 1 with 10% ether/petroleum ether, and compound 2
using 20% ether/petroleum ether.
Compound 1 was the major product and was isolated as a colour-
less solid (45.0 mg, 30%), m.p. 173–175 ◦C [5]. 1H NMR (CDCl3,
300 MHz): ı 7.55–7.67 (3H, m, Ar-H), 7.74–7.81 (1H, m, Ar-H),
7.84–7.94 (2H, m, Ar-H), 8.31 (1H, d, J = 8.4 Hz, Ar-H), 8.59 (1H, s,
11-H), 8.66–8.71 (1H, m, Ar-H) ppm. 13C NMR (CDCl3, 75.4 MHz):
ı 123.04 (CH), 124.01 (CH), 125.08 (CH), 126.19 (CH), 126.60 (C),
127.08 (CH), 128.89 (11-CH), 129.06 (CH), 129.42 (CH), 129.90 (CH),
130.61 (C), 134.14 (C), 141.17 (C), 146.44 (C), 153.96 (C). MS (EI):
m/z (%) 235 (M+, 100). HRMS M+: Calculated for C15H9NS: 235.0456;
Found: 235.0449.
The absorption and emission spectra of several solutions with
different [nucleic acid]/[compound] ratios using the same com-
pound concentration (2 × 10−6 M) were recorded. The solutions
were left 24 h to stabilize. The absorbance at excitation wave-
lengths was always less than 0.1, in order to avoid inner filter
effects. All measurements were performed at room temperature
(25.0 0.5 ◦C). Binding analysis of the experimental data was
performed according to McGhee and von Hippel model [20] to
determine the intrinsic binding constants and the binding site
sizes (the number of base pairs between consecutive bound ligand
molecules).
3. Results and discussion
3.1. Synthesis
The reaction of the commercially available 3-bromobenzo[b]
thiophene-2-carbaldehyde with 2-aminophenylpinacolborane
Compound 2 was isolated as a colourless solid (28.0 mg, 20%),
m.p. 123–125 ◦C [6]. 1H NMR (CDCl3, 300 MHz): ı 7.61–7.70 (2H, m,
Ar-H), 7.74–7.82 (2H, m, Ar-H), 8.05–8.11 (1H, m, Ar-H), 8.30–8.36
under Suzuki coupling conditions using
a stereochemically
hindered ligand as 2-(cyclohexylphosphane)biphenyl [21] and
Ba(OH)2·8H2O as the base, gave in a one-pot procedure com-
pounds 1 and 2, which were separated by column chromatography
(Scheme 1).
Although these compounds have already been synthesized by
other authors in several steps, we were able to prepare them in a
one-pot procedure, which is very advantageous to save reagents
and time.
Using these reaction conditions, the formation of compound 1
was unexpected. It seems that it is the result of a Pd-catalyzed
C-N coupling followed by an intramolecular cyclization that may
occur by nucleophilic attack of the activated ortho position of the
diarylamine intermediate on the carbonyl of the aldehyde, after
deboronation (Scheme 2).
(1H, m, Ar-H), 8.84–8.94 (2H, m, Ar-H), 9.37 (1H, s, 6-H) ppm. 13
C
NMR (CDCl3, 75.4 MHz): ı 122.92 (CH), 123.77 (CH), 125.39 (CH + C),
126.04 (CH), 127.49 (CH), 127.51 (CH), 127.76 (CH), 130.81 (CH),
133.37 (C), 135.15 (C), 135.37 (C), 141.35 (C), 145.56 (C), 145.65 (6-
CH) [6b]. MS (EI): m/z (%) 235 (M+, 100). HRMS M+: Calculated for
C15H9NS: 235.0456; Found: 235.0457.
The data for both compounds are identical to the ones published
elsewhere [5,6], but those are not so complete as in this work.
2.2. Spectroscopic measurements
Absorption spectra were recorded in a Shimadzu UV-3101PC
UV–vis–NIR spectrophotometer. Fluorescence measurements were

22
A.R.O. Rodrigues et al. / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 20–30
4
3
5
6
N
7
2
a
3
b
2
8
1
11
S
9
10
Benzothieno[3,2-b]quinoline
1, 30%
Br
NH2
O
i
+
CHO
+
1
2
10
B
11
3
S
O
9
4
3
2
c
b
8
a
N
5
S
7
6
Benzothieno[2,3-c]quinoline
2, 20%
Scheme 1. One pot synthesis of benzothieno[3,2-b]quinoline 1 and benzothieno[2,3-c]quinoline 2.
fluorescent compound. A red-shift and loss of vibrational struc-
ture is observed for the emission in polar solvents, this effect
being more pronounced for compound 1 (red shifts between cyclo-
hexane and water are 48 nm for compound 1 and 28 nm for
compound 2). In the absorption spectra the red shifts are negli-
gible (Table 1), this behaviour indicating that solvent relaxation
after photoexcitation plays an important role, especially for the
benzothieno[3,2-b]quinoline 1. This predicts a higher ICT character
of the excited state for the latter compound.
HN
S
O
+
1
+ H2O
HB
CH
O
O
Scheme 2. Proposed intermediate and products for the reaction of synthesis of
compound 1.
The fluorescence quantum yields in protic solvents tend to
decrease with increasing solvent hydrogen bonding capability (ФF
in ethanol > ФF in methanol > ФF in water). This may be due to an
increase of S → T intersystem crossing efficiency through H-bond
formation between these quinoline derivatives and protic solvents,
probably by protonation of the N atom of the pyridine moiety. A
similar behaviour was observed for several thieno[3,2-b]pyridine
derivatives synthesized in our lab [22–24]. The formation of
hydrogen bonds between chloroform and these proton acceptor
quinoline derivatives can also explain the lower fluorescence quan-
tum yield values in this solvent [25,26].
The ground state dipole moment (ꢁg) and the excited
state dipole moment (ꢁe) for benzothienoquinolines 1 and 2
(Table 2) were determined performing ab initio molecular quantum
chemistry calculations with Gaussian 09 software [27], using a 6-
311++G(d,p) basis set at the TD-SCF DFT (MPW1PW91) level of the-
ory [28] in gas phase. The optimized geometries of the ground and
excited states show that both molecules have a planar geometry
In the synthesis of the expected compound 2, a Suzuki cross-
coupling and a nucleophilic attack of the amino group on the
aldehyde occurred.
3.2. Fluorescence studies in several solvents
The absorption and fluorescence properties of compounds 1
and 2 were studied in several solvents of different polarity. The
maximum absorption (ꢀabs) and emission wavelengths (ꢀem) and
fluorescence quantum yields (˚F) of both compounds in several
solvents are presented in Table 1. The normalized fluorescence
spectra are shown in Figs. 1 and 2 (examples of absorption spectra
are also shown as insets).
Fluorescence emission was exhibited by compounds 1 and 2
in several non-polar and polar solvents, including water. Fluores-
cence quantum yield values are generally low, varying from 2 to
10% (Table 1), the benzothieno[2,3-c]quinoline 2 being the most
Table 1
Maximum absorption (ꢀabs) and emission wavelengths (ꢀem) and fluorescence quantum yields (˚F) of compounds 1 and 2 in several solvents.
a
Solvent
ꢀabs (nm)
ꢀem (nm)
˚
F
1
2
1
2
1
2
Cyclohexane
Dioxane
372, 354, 334, 272, 243
372, 354, 335, 272, 242
371, 353, 334, 271
372, 354, 336, 273, 243
374, 356, 337, 274
371, 353, 334, 270, 241
372, 355, 335
359, 342, 319, 307, 256
359, 343, 320, 308, 257
358, 342, 318, 307
360, 344, 321, 309, 257
361, 345, 322, 310, 258
358, 342, 319, 307, 255
360, 344, 320, 309
378
392
393
392
394
394
395
403
401
406
426
364
367
365
370
372
369
384
374
374
375
392
0.02
0.03
0.02
0.02
0.02
0.02
0.04
0.03
0.03
0.02
0.01
0.07
0.10
0.08
0.06
0.04
0.05
0.08
0.08
0.09
0.07
0.06
Ethylacetateb
Dichloromethane
Chloroformb
Acetonitrile
N,N-Dimethylformamideb
Dimethylsulfoxideb
Ethanol
Methanol
Water
373, 356, 337
360, 344, 322, 310
372, 336, 272, 241
371, 336, 271, 240
372, 336, 271, 240
361, 347, 321, 310, 256
361, 346, 321, 310, 256
362, 349, 321, 311, 255
a
Relative to 9,10-diphenylanthracene in ethanol (˚R = 0.95) [18]. Error about 10%.
Solvents cut-off: chloroform: 250 nm; ethyl acetate: 265 nm; N,N-dimethylformamide: 280 nm; dimethylsulfoxide: 275 nm.
b

A.R.O. Rodrigues et al. / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 20–30
23
Fig. 1. Normalized fluorescence spectra of solutions (3 × 10−6 M) of compound 1 (ꢀexc = 335 nm) in several solvents. Inset: absorption spectra of 2 × 10−5 M solutions of
compound 1 in cyclohexane and ethanol, as examples.
Fig. 2. Normalized fluorescence spectra of solutions (3 × 10−6 M) of compound 2 (ꢀexc = 325 nm) in several solvents. Inset: Absorption spectra of 10−5 M solutions of compound
2 in cyclohexane and ethanol, as examples.
Table 2
depletion (C+) or increment (C ) [29] that occurs upon electronic
−
Ground (ꢁg) and excited state (ꢁe) dipole moments obtained from theoretical
excitation, using the lowest excited state optimized geometry
(relaxed S1 state). Alternating increases and decreases of electronic
density are observed in the ␲-electron system (Fig. 4). The main
difference between the two compounds is that for compound 1
there is no electronic density at the S atom in the LUMO orbital,
whereas for compound 2 the electronic density decreases, but does
not vanish. Also, a higher degree of charge transfer is observed
for compound 1 than for compound 2, the distance between the
calculations.
Compound
Molecular
radius (Å)
Ground state dipole
moment, ꢁg (D)
Excited state dipole
moment, ꢁe (D)
1
2
4.8
4.7
0.84
2.75
3.16
3.68
˚
barycenters of charge density increase and decrease being 1.24 A
(Fig. 3). The dipole moment vector changes upon excitation,
increasing in magnitude for both compounds, with a significant
change in direction in the case of compound 1.
Fig. 4 shows the representation of HOMO and LUMO orbitals,
as well as the corresponding centroids of either the electronic
˚
and 0.38 A, respectively. The calculations of densities of charge
increase and depletion were performed with the help of the Multi-
wfn software package [30]. These results are in accordance with the
higher solvatochromism experimentally observed for compound 1.

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A.R.O. Rodrigues et al. / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 20–30
Fig. 3. Optimized geometries of compounds 1 and 2 obtained by Gaussian 09 software (grey: C atoms; white: H atoms; blue: N atoms; yellow: S atoms). Left: ground state;
Right: lowest excited singlet state. The arrows indicate the direction of the dipole moment. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
Fig. 4. Representation of HOMO orbital (A), LUMO orbital (B) and centroids of charge density depletion (C+, red) or increment (C , green) (C) for compounds 1 and 2 (optimized
−
geometry of the lowest excited singlet state) at an iso level of 0.02 for orbitals and 0.002 for electronic density. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
The representation of the energy level diagram with the tran-
sition energies for the two quinolines is displayed in Fig. 5, in
vacuum, cyclohexane and acetonitrile, using optimized geometries
for each medium. The solvent was considered as a polarizable con-
tinuum model (PCM) using, for the definition of the molecular
shape, an united atom topological model with radii optimized for
DFT PBE1PBE/6-31G(d) level of theory, and a van der Waals sur-
face (g09 keywords: SCRF = PCM, radii = UAKSm, surface = VDW).
Comparing with the experimentally observed maxima, the calcu-
lated absorption transition energies are slightly shifted to lower
wavelengths, although there is a good agreement in the case of
acetonitrile for compound 1 (Table 1). For emission, the experi-
mental maximum wavelengths are in better accordance with the
calculated ones, these being slightly higher. Also, the higher oscilla-
tor strengths (f) calculated for compound 2 are in accordance with
its higher fluorescence quantum yields experimentally observed.
The calculated oscillator strengths show an increase with sol-
vent polarity. This is compatible with the observed quantum yield
values, if solvents for which hydrogen bonding might occur are
excluded.
The significant sensitivity of the fluorescence emission of these
two benzothienoquinolines to the fluorophore environment can
be useful when probing their interactions with DNA and polynu-
cleotides.
3.3. Interaction with salmon sperm DNA and with synthetic
double-stranded polynucleotides
The interaction of compounds 1 and 2 with natural double-
stranded salmon sperm DNA was studied by fluorescence. Changes

A.R.O. Rodrigues et al. / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 20–30
25
Fig. 5. Representation of the energy level diagram, with the transition energies for the benzothienoquinolines 1 (above) and 2 (below), obtained by molecular quantum
chemistry calculations, in vacuum, cyclohexane and acetonitrile.
in absorption spectra upon DNA interaction are negligible, as
previously observed for other neutral aromatic compounds studied
by some of us, namely tetracyclic lactams [31] and thieno[3,2-
b]pyridine derivatives [24]. Figs. 6 and 7 show the emission
spectra of the benzothienoquinolines 1 and 2 with increasing
[DNA]/[compound] ratio, where [DNA] is expressed in number of
bases or phosphate groups. For both quinoline derivatives the emis-
sion intensity reaches a limit value for [DNA]/[compound] ratios
≥100, indicating that total compound binding is achieved at this
[DNA]/[compound] ratio (spectra corresponding to ratio 100 and
120 are overlapped).
The high [DNA]/[compound] ratio needed for total binding,
together with the negligible changes observed in absorption
spectra (not shown), point to a weak interaction of these molecules
with the nucleic acid, which is also a common behaviour with tetra-
cyclic thienopyridine derivatives [24].
To clarify the different behaviour of the two quinolines,
the base sequence binding preference was also investigated,
using synthetic ds-heteropolynucleotides, poly(dA–dT)·(dA–dT)
and poly(dG–dC)·(dG–dC). Fig. 9 displays the ratio of maxi-
mum emission intensities in the presence (I) and absence (I0)
of ds-heteropolynucleotides for several [nucleic acid]/[compound]
ratios, for compounds 1 and 2, respectively. The behaviour in
heteropolynucleotides is similar to that observed in salmon
sperm DNA, with a rise in fluorescence intensity with increas-
ing polynucleotide concentration for compound 1 and a decrease
for compound 2. However, the stabilization in emission intensity,
indicative of full binding, is attained at a significantly lower molar
ratio for poly(dA–dT)·(dA–dT) ([nucleotide]/[compound] = 60 and
80, respectively for compound 1 and 2). For poly(dG–dC)·(dG–dC),
An enhancement in emission intensity with increasing DNA
concentration is observed for compound 1, while the oppo-
site occurs for compound
2 (Fig. 8). This may indicate a
different type of dominating interaction of the two ben-
zothienoquinolines with DNA bases, as already observed for
other tetracyclic compounds [24,31]. Further evidence is given
by the occurrence of a slight blue shift upon DNA binding
only in the case of compound
1 (inset of Figs. 6 and 7).

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A.R.O. Rodrigues et al. / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 20–30
Fig. 6. Fluorescence spectra of the benzothienoquinoline 1 (2 × 10−6 M) in 10 mM Tris–HCl buffer (pH 7.2), with increasing DNA content. Inset: normalized spectra at
[DNA]/[1] = 0 and 120.
Table 3
Values of binding constants (Ki) and binding site sizes (n) for benzothienoquinolines
interaction with DNA and synthetic heteropolynucleotides.
Nucleic acid
Ki (M−1
)
n
Salmon sperm DNA
poly(dA–dT)·(dA–dT)
poly(dG–dC)·(dG–dC)
(2.6 0.3) × 105
(3.0 0.4) × 105
(5.9 0.6) × 104
(2.9 0.3) × 105
(3.1 0.4) × 105
(4.5 0.5) × 104
14
13
35
5
5
9
Compound 1
Compound 2
Salmon sperm DNA
poly(dA–dT)·(dA–dT)
poly(dG–dC)·(dG–dC)
16
14
25
6
5
9
and the number of base pairs between consecutive intercalated
compound molecules (n). The results are presented in Table 3.
It has already been reported that small variations in the struc-
ture of tetracyclic compounds, as differences only in the substituent
groups, influence strongly the interaction with nucleic acids
[13,24,31], either by changing the main mechanism or by affect-
ing the magnitude of interaction (binding constant and binding
site size). As both compounds exhibit a stronger interaction with
poly(dA–dT)·poly(dA–dT) than with poly(dG–dC)·(dG–dC) (higher
binding constants and lower binding site sizes in the former), it can
be concluded that the main interaction in DNA is established with
A–T base pairs. A likely mechanism is intercalation by ␲–␲ stacking.
In the present case, differently from the case of other planar aromat-
ics interacting within nucleic acids [32,33], photoinduced electron
transfer involving DNA bases is not expected to occur because the
process is thermodynamically unfavourable.
Fluorescence quenching experiments with iodide ion were
also performed for compounds 1 and 2 in the presence of DNA
and heteropolynucleotides, in the situation of total binding
([DNA]/[compound] = 100, [poly(dA–dT)·(dA–dT)]/[compound] =
80 and [poly(dG–dC)·(dG–dC)]/[compound] = 100 for both quino-
lines 1 and 2, with [compound] = 2 × 10−6 M). The quenching data
were first plotted according to the Stern–Volmer relation (Eq. (4))
[34],
Fig. 7. Fluorescence spectra of the benzothienoquinoline 2 (2 × 10−6 M) in 10 mM
Tris–HCl buffer (pH 7.2), with increasing DNA content. Inset: normalized spectra at
[DNA]/[2] = 0 and 120.
the stabilization is attained at a molar ratio of 100 for both quino-
lines, similarly to the behaviour observed with salmon sperm DNA.
The binding constants and binding site sizes were determined by
the modified Scatchard equation, given by McGhee and von Hippel
[20]
ꢂ
ꢃ
n−1
r
1 − nr
= Ki(1 − nr)
(2)
cf
1 − (n − 1)r
where Ki is the intrinsic binding constant, n is the binding site size,
r is the ratio cb/[nucleic acid], cb and cf are the concentrations of
bound and free compound, respectively, calculated by
IF,0 − IF
cb
=
× ctotal
;
ctotal = cf + cb
(3)
IF,0 − IF,b
being IF,0 the fluorescence intensity of the free compound and IF,b
the fluorescence intensity of the bound compound at total binding.
The fluorescence measurements results were fitted by least
squares methods to obtain the values of the binding constants (Ki)
I0
= 1 + KSV[Q]
(4)
I

A.R.O. Rodrigues et al. / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 20–30
27
Fig. 8. Fluorescence intensities ratio in the presence (I) and absence (I0) of DNA for compounds 1 and 2 at several [DNA]/[compound] molar ratios.
Fig. 9. Fluorescence intensities ratio in the presence (I) and absence (I0) of heteropolynucleotides for the benzothienoquinolines 1 and 2 at several [nucleotide]/[compound]
molar ratios. (A) poly(dA–dT)·(dA–dT); (B) poly(dG–dC) (dG–dC).
Table 4
where I0 and I are, respectively, the fluorescence intensities in
Values of the accessibilities (fa) to the quencher (I−) and Stern–Volmer constants for
the absence and in the presence of quencher (I−), KSV is the
compounds 1 and 2 bound to DNA and heteropolynucleotides.
Stern–Volmer constant and [Q] is the quencher concentration.
Nucleic acid
KSV (M−1
)
fa
In all cases, Stern–Volmer plots are non-linear (Fig. 10, as an
example), with a downward curvature. This means that not all
the fluorescent molecules are accessible to the quencher. In this
case, the system contains heterogeneously emitting sites, in which
some compound molecules are accessible to the quencher and
other molecules are not accessible. Thus, the Stern–Volmer equa-
tion must be modified [35] as (5):
Salmon sperm DNA
poly(dA–dT)·(dA–dT)
poly(dG–dC)·(dG–dC)
4.65
9.33
11.1
0.53
0.54
0.51
Compound 1
Compound 2
Salmon sperm DNA
poly(dA–dT)·(dA–dT)
poly(dG–dC)·(dG–dC)
8.61
9.54
6.1
0.59
0.63
0.92
I0
1
fa
1
=
+
(5)
Anionic quenchers can be used to distinguish between DNA
binding modes [14,15,35]. Intercalated chromophores are less
accessible to quenching by iodide ion due to electrostatic repul-
sion between the negatively charged DNA and iodide anion [15].
Compounds which are bound at the DNA surface (groove binding or
ꢂI
faKSV[Q]
where ꢂI = I0 − I, fa is the accessibility to quencher. From the plots
of I0/ꢂI vs. 1/[Q], it is possible to obtain the accessibilities to the
anionic quencher. The results are summarized in Table 4.

28
A.R.O. Rodrigues et al. / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 20–30
Fig. 10. (A) Stern–Volmer plots for quenching with iodide ion (I−) for compounds 1 and 2 with salmon sperm DNA at total binding, [DNA]/[compound] = 100. (B) Corresponding
modified Stern–Volmer plots.
Fig. 11. Compound 1 – (A) docking in 1HQ7; (B) docking in 1DXA*. Compound 2 – (C) docking in 1HQ7; (D) docking in 1DXA*. Benzo[a]pyrene diol epoxide – (E) docking in
1HQ7; (F) docking in 1DXA*.

A.R.O. Rodrigues et al. / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 20–30
29
Table 5
Docking results for the interaction of the studied compounds and benzo[a]pyrene diol epoxide with model nucleic acid sequences using autodock software.a
Docking
Nucleic acid
Type of binding
Nb (%)
Lower ꢂGi
Average ꢂGi
(kcal mol−1
)
(kcal mol−1
)
Minor groove
80
20
−10.33
−9.74
–
−10.31
−9.74
1HQ7
A
Compound 1
Compound 2
Intercalation
Minor groove
Intercalation
–
B
C
1DXA*
1HQ7
100
−8.24
−8.23
Minor groove
90
10
−7.97
−7.32
–
−7.95
−7.32
Intercalation
Minor groove
Intercalation
–
D
E
1DXA*
1HQ7
100
−7.05
−7.01
Minor groove
40
10
40
10
−9.74
−9.53
−9.40
−8.95
–
−8.16
−9.84
−8.62
−9.43
−9.53
−9.03
−8.95
Benzo[a]pyrene diol epoxide
Intercalation
Minor groove
Intercalation
10
60
30
−8.16
−9.75
−8.59
F
1DXA*
a
Nb: percentage of runs in minor groove or intercalation binding mode; ꢂGi: binding free energy.
electrostatic binding) are more accessible and, therefore, emission
from these molecules can be quenched more efficiently. As these
benzothienoquinolines are neutral molecules, electrostatic binding
to nucleic acids is not involved. Therefore, the fraction of compound
molecules accessible to the external quencher (fa) should corre-
spond to bound molecules at the grooves.
stronger than that for compound 2, with the less favourable situa-
tion occurring for the intercalation of compound 2 in 1DXA*. For
BaP, intercalation in 1DXA* and minor groove binding in 1HQ7
are equally favourable. As BaP is known to interact with DNA by
intercalation, it seems that, for a sufficiently high release of
free energy as the polynucleotide structure has to accommodate
the guest molecule, intercalation is the preferred mode of bind-
ing. Thus, noting that intercalation of compound 2 is the less
favourable interaction with 1DXA* (−7 kcal mol−1), and taking also
into account both the different spectral behaviour of the com-
pounds upon DNA interaction and the higher accessibility of iodide
ion to compound 2 binding sites, the docking results support a
higher fraction of intercalation for compound 1 than for compound
2. The binding free energy of compound 1 to 1DXA* by interca-
lation is significant, as the intercalation gap corresponds to BaP
(cross-docking). In a reported docking study [39], cross-docking
of elliptine (a typical intercalating agent) with a polynucleotide in
which an intercalation gap was constructed (adapted for elliptine)
gave a binding free energy of −8.1 kcal mol−1, while the corre-
sponding self-docking showed a value of −8.7 kcal mol−1 [39].
From the docked structures shown in Fig. 11, it is also possible
to observe that the nitrogen atom of compound 2 is more exposed
to the medium surrounding DNA than that of compound 1. This,
along with the distinct binding modes, could be the reason for a
decrease of fluorescence intensity upon DNA binding, instead of
the observed increase in the case of compound 1.
The fraction of intercalated molecules into salmon sperm DNA
and heteropolynucleotides is higher for the benzothienoquinoline
1 (46–49%). On the contrary, compound 2 presents a very small
fraction of intercalated molecules (8%) in poly(dG–dC)·(dG–dC),
while in poly(dA–dT)·(dA–dT) the percentage is similar to the
observed in natural DNA (around 40%). As both quinolines 1 and
2 are neutral molecules, the relatively high values for fa indicate
that groove binding is an important type of interaction of these
quinoline derivatives to DNA [24,31] (electrostatic interaction is
not expected), being compound 1 the more intercalative one.
In order to further characterize the interaction of benzothieno-
quinolines 1 and 2 with DNA, docking studies using autodock [36]
were performed, for a crystallographic dodecamer B-DNA (1HQ7:
GCAAACGTTTGC sequence [37]) and a structure with nine base
pairs intercalated with benzo[a]pyrene diol epoxide (BaP) (1DXA:
GGTC[BaP]ACGAG sequence [37]) from which the intercalator was
removed (1DXA*). The setup of the autodock calculation (10 runs,
270,000 maximum number of generations, 2,500,000 maximum
number of energy evaluations, 112 × 74 × 62 grid points for 1HQ7
˚
and 76 × 72 × 62 for 1DXA* with 0.375 A spacing) and visualiza-
tion of the results were made with the help of the autodocktools
software suite (ADT) [38]. Fig. 11 shows the results of the most
stable docked structures for the studied compounds and for the
highly efficient BaP intercalator. The values of the binding energies
obtained are reported in Table 5.
4. Conclusions
A new one pot method was achieved for the synthesis of
benzothieno[3,2-b]quinoline 1 and benzothieno[2,3-c]quinoline
2 by the reaction of the commercially available 3-bromobenzo[b]
thiophene-2-carbaldehyde with 2-aminophenylpinacolborane
Two types of docking in polynucleotides are usually considered:
direct (self) docking and cross dockings [39]. Only docking F (Table 5
and Fig. 11) is a direct one, as it corresponds to the rebinding of
a molecule that was removed from the polynucleotide structure
where it was originally included. For the purpose of this work, it
serves as a control for the effectiveness of the docking procedure
and as a reference for evaluation of the other docking results. As
expected, intercalation does not occur in 1HQ7, even for a very
efficient intercalator such as BaP, because the polynucleotide struc-
ture, which is rigid during the docking procedure, does not include
an appropriate intercalating gap. From Table 5, it is possible to
conclude that the interaction of compound 1 with DNA is always
under Suzuki coupling conditions using
a
stereochemi-
cally hindered ligand, 2-(cyclohexylphosphane)biphenyl and
Ba(OH)2·8H2O as the base. Although the compounds have already
been synthesized earlier by other authors using several steps, our
methodology is advantageous saving reagents and time. Both com-
pounds are fluorescent and present a solvent sensitive emission,
despite the low fluorescence quantum yields (below 10%).
The benzothieno[3,2-b]quinoline 1 is the most intercalative
compound in DNA and synthetic heteropolynucleotides. Both com-
pounds exhibit a stronger interaction with A–T than with G–C base

30
A.R.O. Rodrigues et al. / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 20–30
pairs, exhibiting higher binding constants and smaller binding site
sizes. Fluorescence quenching measurements allowed concluding
that the interaction with DNA of these quinoline derivatives is both
intercalation and binding in the grooves.
Docking studies indicate that compound 1 interacts more
strongly with DNA than compound 2, with a significant value of
binding free energy in the case of intercalation. Minor groove bind-
ing is also very favourable and, probably, both mechanisms occur
with a preponderance of intercalation in the case of compound 1.
The docked structures showed that in compound 2 the nitrogen
atom is exposed to the medium surrounding DNA. This should also
contribute to the different spectral behaviour of both compounds
upon DNA interaction reported in this study.
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Acknowledgements
Foundation for the Science and Technology (FCT, Portugal), for
financial support to the Portuguese NMR network (PTNMR) and
also to FEDER and QREN for financial support to the Research
Centres, CFUM [Strategic Project PEst-C/FIS/UI0607/2013 (FCOMP-
01-0124-FEDER-037291)] and CQ/UM [Strategic Project PEst-
C/QUI/UI0686/2013 (FCOMP-01-0124-FEDER-037302)], and to the
research project PTDC/QUI-QUI/111060/2009 (F-COMP-01-0124-
FEDER-015603) also financed by COMPETE/QREN/EU. FCT, POPH-
QREN and FSE are acknowledged for the PhD grants of A.R.O.R.
(SFRH/BD/90949/2012) and M.S.D.C. (SFRH/BD/47052/2008), for
the Post-Doc. Grant of R.C.C. (SFRH/BPD/68344/2010) and for sup-
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