A new family of fluorescent pyridazinobenzimidazolium cations with DNA binding properties

Article abstract of DOI:10.1016/j.dyepig.2021.109443

A series of novel azonia aromatic heterocycles formed by a pyridazinobenzimidazolium system has been synthesized. Spectrofluorimetric and circular dichroism measurements, as well as theoretical simulations for these materials, have shown their interesting fluorescence properties and DNA-binding ability. Stoichiometries and binding constants were obtained by fluorescence and the induced circular dichroism spectra analysis. Moreover, the potential of these compounds for cell staining has been investigated in living HeLa cells by confocal microscopy imaging.

Full text of DOI:10.1016/j.dyepig.2021.109443

Dyes and Pigments 192 (2021) 109443
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A new family of fluorescent pyridazinobenzimidazolium cations with DNA
binding properties
,
*
Pedro Boscha, Gema Marcelob, Alejandra Matamoros-Recioc, David Sucunzaa
,
,
**
Francisco Mendicutib, Alberto Domingoc, Juan J. Vaqueroa
a
´
´
´
Departamento de Química Organica y Química Inorganica, 28805, Alcala de Henares, Madrid, Spain
b
c
´
Departamento de Química Analítica, Química Física e Ingeniería Química, 28805, Alcala de Henares, Madrid, Spain
´
´
´
´
Departamento de Biología de Sistemas, Instituto de Investigacion Química “Andres M. Del Río” (IQAR), Universidad de Alcala, IRYCIS, 28805, Alcala de Henares,
Madrid, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of novel azonia aromatic heterocycles formed by a pyridazinobenzimidazolium system has been syn-
thesized. Spectrofluorimetric and circular dichroism measurements, as well as theoretical simulations for these
materials, have shown their interesting fluorescence properties and DNA-binding ability. Stoichiometries and
binding constants were obtained by fluorescence and the induced circular dichroism spectra analysis. Moreover,
the potential of these compounds for cell staining has been investigated in living HeLa cells by confocal mi-
croscopy imaging.
Pyridazinobenzimidazolium cation
DNA binding
Cell staining
Azonia aromatic heterocycles
1. Introduction
[10], we envisaged that more complex derivatives of this azonia system
could exhibit relatively high fluorescence quantum yields and affinity
Numerous azonia aromatic heterocycles are DNA binders with good
fluorescence properties[1]. Many of them act as intercalators as a result
of the insertion of their planar aromatic rings between the DNA base
for this biomolecule. As such, we report here the synthesis of three novel
aryl-substituted pyridazinobenzimidazolium cationic ligands (1, 2 and
3, Fig. 1), as well as an analysis of their DNA binding properties and
capacity for cell staining.
pairs via π-stacking interactions[2]. Coralyne, which is probably the
most widely studied example of this kind of cation[3], contains a qua-
ternary bridgehead nitrogen atom, and the interest in this alkaloid has
led to a significant amount of research into the properties and potential
applications of other related polycyclic heteroarenes containing a qui-
nolizinium system[4], with recent significant contributions by Ihmels
et al.[5], Vaquero et al.[6], and others[7]. [][][].
2. Methods, materials and protocols
2.1. Synthetic procedures
2.1.1. General information
Despite the fact that some other cationic systems with a quaternary
bridgehead nitrogen atom have shown similar DNA binding properties,
less attention has been paid to other azonia heterocycles[8]. In this
sense, we have recently described a new family of imidazopyridinium
derivatives with potential applications as light-up fluorescent DNA
probes [9] and, in order to gain insight into the influence that the size,
shape and charge distribution has on some of their properties, a
comparative study with a library of azonia cations[10].
Reagents of the highest commercial quality were purchased and used
without further purification, unless stated otherwise. Reactions were
monitored by thin-layer chromatography (TLC) carried out on 0.25 mm
E. Merck silica gel plates (60FS-254), using UV light to visualize the
compounds. Column chromatography was performed using silica gel (60
F254, 70–200 mm) as the stationary phase. All melting points were
determined in open capillary tubes on a Stuart Scientific SMP3 melting
point apparatus and are uncorrected. IR spectra were obtained using a
PerkinElmer FTIR spectrum 2000 spectrophotometer. 1H and 13C NMR
spectra were recorded using either Varian Mercury VX-300, Varian
Although a non-substituted tricyclic pyridazinobenzimidazolium
cation did not show a DNA binding ability in this comparative study
* Corresponding author.
** Corresponding author.
E-mail addresses: david.sucunza@uah.es (D. Sucunza), juanjose.vaquero@uah.es (J.J. Vaquero).
https://doi.org/10.1016/j.dyepig.2021.109443
Received 9 January 2021; Received in revised form 6 April 2021; Accepted 24 April 2021
Available online 13 May 2021
0143-7208/© 2021 Elsevier Ltd. All rights reserved.

P. Bosch et al.
Dyes and Pigments 192 (2021) 109443
MHz, CDCl3): δ 158.7, 152.3, 143.1, 135.1, 134.6, 128.3, 121.4, 116.9,
116.9, 114.1, 108.9, 55.3, 29.9, 13.8. HRMS (ESI-TOF) for C16H16N2O
m/z (Calc.): 253.1335, m/z (found): 253.1332.
2.1.4. 3-Amino-1,2-dimethyl-1H-benzo[d]imidazole-3-ium 2,4,6-trime-
thylbenzenesulfonate (8)
A solution of MSH (342 mg 1.59 mm) in anhydrous CH2Cl2 (3 ml)
was added dropwise to a solution of 1,2-dimethyl-1H-benzo[d]imid-
azole (5) (155 mg 1.06 mmol) in anhydrous CH2Cl2 (3 ml). The reaction
mixture was stirred at room temperature for 5 h, then filtered, and the
solid phase isolated and washed with CH2Cl2 and petroleum ether to
give the product in 50% yield (191 mg). M. p.: 207–209 ◦C. IR (KBr)
ν
cmꢀ 1: 3252, 3149, 1668, 1546, 1474, 1086, 1013, 754, 678.1H NMR
(300 MHz, DMSO‑d6): δ 7.91 (d, J = 4.2 Hz, 1H), 7.80 (d, J = 4.2 Hz,
1H), 7.66–7.52 (m, 2H), 6.68 (s, 4H), 3.93 (s, 3H), 2.80 (s, 3H), 2.45 (s,
6H), 2.14 (s, 3H). 13C NMR (75 MHz, DMSO‑d6): δ 151.9, 141.8, 135.6,
135.1, 131.0, 129.1, 125.0, 111.7, 39.6, 39.3, 39.0, 38.7, 38.5, 31.2,
22.2, 19.8, 9.2. HRMS (ESI-TOF) for C9H12N3+ m/z (Calc.): 162.1033,
m/z (found): 162.1035.
Fig. 1. Structure of coralyne (A), the general structure of quinolizinium-based
azonia cations (B) and pyridazinobenzimidazolium cations 1, 2 and 3.
2.1.5. 3-Amino-1,2-dimethyl-5-phenyl-1H-benzo[d]imidazole-3-ium
2,4,6-trimethylbenzenesulfonate (9)
A solution of MSH (218 mg 1.01 mmol) in anhydrous CH2Cl2 (3 ml)
was added dropwise to a solution of the starting heterocycle 6 (150 mg
0.67 mmol) in anhydrous CH2Cl2 (3 ml). The reaction mixture was
stirred at room temperature for 16 h, then filtered, and the solid phase
isolated and washed with CH2Cl2 and petroleum ether to give the
Unity 300 or 500 MHz spectrometers at room temperature. Chemical
shifts are given in ppm (δ) downfield 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 etc. High-
resolution analysis (TOF) was performed using an Agilent 6210 time-
of-flight LC/MS. O-Mesytilenesulfonylhydroxylamine (MSH) [11] was
prepared following the procedure described in the literature.
product in 93% yield (273 mg). M. p.: 221–223 ◦C. IR (KBr)
ν
cmꢀ 1
:
3511, 3270, 3044, 1655, 1479, 1216, 1179, 1087, 1016, 763, 682.1H
NMR (500 MHz, DMSO‑d6): δ 8.09 (s, 1H), 8.03 (d, J = 8,7 Hz, 1H), 7.93
(d, J = 8.7 Hz, 1H), 7.78–7.75 (m, 2H) 7.57–7.44 (m, 3H) 6.70 (s, 4H),
3.98 (s, 3H), 2.84 (s, 3H), 2.45 (s, 6H), 2.14 (s, 3H). 13C NMR (125 MHz,
DMSO‑d6): δ 152.6, 142.3, 138.5, 137.6, 135.6, 135.3, 131.8, 129.2,
128.8, 128.7, 127.6, 126.5, 124.4, 112.6, 109.6, 31.3, 22.2, 19.7, 9.2.
HRMS (ESI-TOF) for C15H16N3+ m/z (Calc.): 238.1339, m/z (found):
238.1342.
2.1.2. 1,2-Dimethyl-5-phenyl-1H-benzo[d]imidazole (6)
A solution of 5-bromo-1,2-dimethyl-1H-benzo[d]imidazole (4) (312
mg, 1.39 mmol) and Pd(PPh3)4 (80 mg, 0,07 mmol) in DMF (5 ml) was
stirred and heated under argon for 15 min. After this time, a solution of
phenylboronic acid (169 mg, 1.39 mmol) in EtOH (5 ml) was added and
the mixture was stirred and heated under reflux. After a further 15 min,
a solution of Na2CO3 in water (4.6 ml) (2 M) was added and the reaction
mixture heated under reflux for 24 h. The reaction mixture was then
cooled to room temperature and extracted with CH2Cl2. The organic
layer was washed with brine and then dried with MgSO4. The residue
was subjected to flash column chromatography (1:9 Hex/AcOEt) on
silica to give the product in 88% yield (273 mg). M. p.: 187–189 ◦C. IR
2.1.6. 3-Amino-5-(4-methoxyphenyl)-1,2-dimethyl-1H-benzo[d]
imidazole-3-ium 2,4,6-trimethylbenzenesulfonate (10)
A solution of MSH (352 mg 1.63 mmol) in anhydrous CH2Cl2 (4 ml)
was added dropwise to a solution of the starting heterocycle 7 (275 mg
1.09 mmol) in anhydrous CH2Cl2 (4 ml). The reaction mixture was
stirred at room temperature for 16 h, then filtered, and the solid phase
isolated and washed with CH2Cl2 and petroleum ether to give the
(KBr)
ν
cmꢀ 1: 3035, 1621, 1514, 1477, 1401, 1320, 752, 669.1H NMR
(300 MHz, CDCl3): δ 7.90 (s, 1H), 7.63 (d, J = 7.4 Hz, 2H), 7.50–7.41 (m,
3H), 7.34–7.27 (m, 2H), 3.70 (s, 3H), 2.61 (s, 3H). 13C NMR (75 MHz,
CDCl3): δ 142.5, 141.7, 137.4, 135.5, 128.6, 127.2, 126.5, 121.7, 117.1,
108.9, 29.9, 13.8. HRMS (ESI-TOF) for C15H14N2 m/z (Calc.): 223.1230,
m/z (found): 223.1231.
product in 62% yield (318 mg). M. p.: 233–235 ◦C. IR (KBr)
ν
cmꢀ 1
:
3253, 3145, 1671, 1607, 1487, 1204, 1188, 1087, 1015, 679.1H NMR
(500 MHz, DMSO‑d6): δ 8.03 (s, 1H), 7.98 (d, J = 8,7 Hz, 1H), 7.87 (d, J
= 8.4 Hz, 1H), 7.71 (d, J = 8.1 Hz 2H) 7.09 (d, J = 7.8 Hz 2H) 6.70 (s,
2H) 6.68 (s, 2H), 3.97 (s, 3H), 3,81 (s, 3H), 2.82 (s, 3H), 2.45 (s, 6H),
2.14 (s, 3H). 13C NMR (125 MHz, DMSO‑d6): δ 158.9, 152.3, 142.3,
137.3, 135.6, 135.3, 131.9, 130.8, 129.2, 128.3, 127.7, 124.0, 114.1,
112.5, 108.8, 54.8, 31.3, 22.2, 19.7, 9.1. HRMS (ESI-TOF) for
2.1.3. 5-(4-Methoxyphenyl)-1,2-dimethyl-1H-benzo[d]imidazole (7)
A solution of 5-bromo-1,2-dimethyl-1H-benzo[d]imidazole (4) (252
mg, 1.12 mmol) and Pd(PPh3)4 (65 mg, 0.06 mmol) in DMF (4 ml) was
stirred and heated under argon for 15 min. After this time, a solution of
p-methoxyphenylboronic acid (171 mg, 1.12 mmol) in EtOH (4 ml) was
added and the mixture was stirred and heated under reflux. After a
further 15 min, a solution of Na2CO3 in water (3.9 ml) (2 M) was added
and the reaction mixture was heated under reflux for 24 h. The reaction
mixture was then cooled to room temperature and extracted with
CH2Cl2. The organic layer was washed with brine and then dried with
MgSO4. The residue was subjected to flash column chromatography
(AcOEt) on silica to give the product in 57% yield (175 mg). M. p.:
C16H18N3O + m/z (Calc.): 268.1444, m/z (found): 268.1446.
2.1.7. 5-Methyl-2,3-diphenyl-5H-benzo [4,5]imidazo[1,2-b]pyridazin-10-
ium 2,4,6-trimethylbenzenesulfonate (1)
Diphenylethanedione (43 mg, 0.20 mmol) and sodium acetate (17
mg, 0.20 mmol) were added to a suspension of the azolium salt 8 (60 mg,
0.17 mmol) in EtOH (6 ml). The reaction mixture was stirred and heated
under reflux for 72 h, then cooled down to room temperature. Removal
of the solvent in vacuo gave a residue, which was triturated with cooled
water and filtered. The residue was washed with cooled water and
AcOEt several times to give a solid, which was dissolved in the minimum
amount of MeOH, then cool Et2O was added in an ice bath until pre-
cipitation. The solid was filtered to give the product in 62% yield (55
168–170 ◦C. IR (KBr)
ν
cmꢀ 1: 2936, 1604, 1513, 1482, 1273, 1245,
1180, 1030, 835, 800.1H NMR (300 MHz, CDCl3): δ 7.88 (s, 1H), 7.61 (d,
J = 6.7 Hz, 2H), 7.48 (d, J = 8.2 Hz, 1H), 7.31 (d, J = 7.9 Hz, 1H), 7.02
(d, J = 6.7 Hz, 2H), 3.88 (s, 3H), 3.74 (s, 3H), 2.63 (s, 3H). 13C NMR (75
mg). M. p.: 198–200 ◦C. IR (KBr)
ν
cmꢀ 1: 3440, 3054, 1638, 1604, 1466,
2

P. Bosch et al.
Dyes and Pigments 192 (2021) 109443
1379, 1196, 1083, 1016, 765, 702, 675.1H NMR (300 MHz, DMSO‑d6): δ
9.14 (s, 1H), 8.51 (d, J = 8,1 Hz, 1H) 8.27 (d, J = 8.4 Hz, 1H), 7.99 (t, J
= 7.5, 8.4 Hz, 1H), 7.85 (t, J = 8.1, 7.5, 1H) 7.51–7.36 (m, 10H), 6.70 (s,
2H), 4.34 (s, 3H), 2.46 (s, 6H), 2.14 (s, 3H). 13C NMR (75 MHz,
DMSO‑d6): δ 155.0, 142.2, 141.7, 139.1, 135.7, 135.3, 134.7, 133.6,
131.9, 129.6, 129.6, 129.3, 129.0, 128.2, 128.0, 127.0, 125.9, 120.4,
112.9, 112.8, 30.7, 22.3, 19.8. HRMS (ESI-TOF) for C23H18N3+ m/z
(Calc.): 336.1495, m/z (Found): 336.1500.
Photons were detected using a sensitive cooled photomultiplier. Data
acquisition was carried out using a multichannel analyzer (1024 chan-
nels) with a time window width of 125 ns? A total of 10,000 counts, in
the maximum peak channel, was recorded for each measurement.
Instrumental response functions were obtained regularly by measuring
the scattering of a Ludox solution. Intensity fluorescence profiles I(t),
were fitted to the usual multi-exponential decay functions,
n
∑
τi
I(t) =
Aieꢀ t/
(1)
i=1
2.1.8. 5-Methyl-2,3,8-triphenyl-5H-benzo [4,5]imidazo[1,2-b]pyridazin-
10-ium 2,4,6-trimethylbenzenesulfonate (2)
using the iterative deconvolution method[12]. The average lifetime of a
multiple-exponential decay function can be defined as[13].
Diphenylethanedione (74 mg, 0.35 mmol) and sodium acetate (29
mg, 0.35 mmol) were added to a suspension of the azolium salt 9 (128
mg, 0.30 mmol) in EtOH (4 ml). The reaction mixture was stirred and
heated under reflux for 24 h, then cooled to room temperature. Removal
of the solvent in vacuo gave a residue, which was triturated with cooled
water and filtered. The resulting residue was washed with cooled water
and AcOEt several times to give a solid, which was dissolved in the
minimum amount of MeOH, then cool Et2O was added in an ice bath
until precipitation. The solid was filtered to give the product in58% yield
n
∑
A
iτ2i
〈τ
〉 = i=1
(2)
n
∑
A
iτi
i=1
where Ai is the pre-exponential factor of the component with a lifetime τi
of the decay function, assuming that each component behaves
independently.
(104 mg). M. p.: 235–237 ◦C. IR (KBr)
ν
cmꢀ 1: 3422, 2927, 1604, 1481,
1214, 1185, 1015, 767, 700, 676.1H NMR (300 MHz, DMSO‑d6): δ 9.13
(s, 1H), 8.68 (s, 1H), 8.32 (s, 2H), 7.91 (d, J = 7.2 Hz, 2H), 7.56–7.36 (m,
13H), 6.67 (s, 2H), 4.36 (s, 3H), 2.45 (s, 6H), 2.13 (s, 3H). 13C NMR (75
MHz, DMSO‑d6): δ 154.8, 142.0, 141.4, 139.1, 137.9, 137.6, 135.4,
135.1, 134.5, 133.4, 131.2, 129.4, 129.2, 129.1, 129.0, 128.9, 128.5,
128.0, 127.8, 127.7, 127.5, 126.7, 120.3, 113.3, 109.7, 30.8, 22.2, 19.8.
HRMS (ESI-TOF) for C29H22N3+ m/z (Calc.): 412.1808, m/z (Found):
412.1811.
Right angle geometry and rectangular quartz 1.0 cm path cells were
used for all measurements.
The fluorescence anisotropy r was obtained from the fluorescence
polarization measurements using the L-format spectrofluorimeter
configuration[14]. The anisotropy was defined as:
r = (IVV ꢀ GIVH) / (IVV + 2GIVH
)
(3)
where all magnitudes are well-known.
Circular dichroism measurements were performed using a JASCO-
715 spectropolarimeter. The recorded spectra were the average of
three scans taken at a speed of 50 nm minꢀ 1 with a 0.125 s time
response. The sensitivity and resolution were fixed at 20 mdeg and 0.5
nm, respectively. When recording the CD spectra for the ligands (ICD) or
DNA absorption zones, measurements were performed in 1 cm or 0.1 cm
path quartz cells, respectively, at 25 ◦C. A 2 cm path cell was used for
titration measurements. Molar ellipticity was defined as:
2.1.9. 8-(4-Methoxyphenyl)-5-methyl-2,3-diphenyl-5H-benzo [4,5]
imidazo[1,2-b]pyridazin-10-ium 2,4,6-trimethylbenzenesulfonate (3)
Diphenylethanedione (81 mg, 0.38 mmol) and sodium acetate (31
mg, 0.38 mmol) were added to a suspension of the azolium salt 10 (150
mg, 0.32 mmol) in EtOH (5 ml). The reaction mixture was stirred and
heated under reflux for 24 h, then cooled to room temperature. Removal
of the solvent in vacuo gave a residue, which was triturated with cooled
water and filtered. The residue was washed with cooled water and
AcOEt several times to give a solid, which was dissolved in the minimum
amount of MeOH, then cool Et2O was added in an ice bath until pre-
cipitation. The solid was filtered to give the product in 54% yield (111
ꢀ
)
Δ
ε
[L].l
[θ] =
m
deg cm 2m olꢀ 1
(4)
mg). M. p.: 267–269 ◦C. IR (KBr)
ν
cmꢀ 1: 3062, 2924, 1606, 1489, 1248,
where θ is the ellipticity in mdeg, [L] is the ligand concentration (mol
cmꢀ 3) and l is the optical path in cm.
1196, 1015, 829, 700, 676.1H NMR (500 MHz, DMSO‑d6): δ 9.12 (s, 1H),
8.63 (s, 1H), 8.29 (s, 2H), 7.88 (d, J = 6.3 Hz, 2H), 7.56–7.33 (m, 10H),
7.09 (d, J = 3.1 Hz, 2H), 6.69 (s, 2H), 4.34 (s, 3H), 3.83 (s, 3H), 2.45 (s,
6H), 2.13 (s, 3H). 13C NMR (125 MHz, DMSO): δ 158.9, 154.7, 141.3,
139.0, 137.8, 135.4, 135.3, 135.1, 134.9, 134.6, 134.5, 133.4, 130.7,
129.9, 129.4, 129.3, 129.0, 128.8, 128.6, 128.3, 128.0, 127.8, 127.6,
120.2, 114.0, 113.1, 108.9, 54.8, 30.7, 22.2, 19.8. HRMS (ESI-TOF) for
2.2.2. Solution preparation for titrations
Stock concentrated solutions of ligands (L) 1, 2 and 3 were dissolved
in DMSO (Aldrich, HPLC grade) and diluted with a phosphate-buffered
saline solution (PBS) at pH 7.4 to achieve a concentration of around
10ꢀ 5 M. The final solutions, which contained 5% DMSO, were used for
titrations with aqueous solutions of calf thymus DNA (ctDNA, high
molecular weight sodium salt, Aldrich). Thus, the content of the com-
mercial 2 mg/ml ctDNA vials was diluted with Milli-Q water to 2.40 ×
10ꢀ 3 mol/per base pair stock solution. This concentration was deter-
mined by UV–Vis spectroscopy (ε260nm = 13,200 Mꢀ 1cmꢀ 1)[15]. For
titrations, aliquots of this stock DNA solution were added to a 1 cm
optical path quartz cuvette containing 2.5 ml of the L solution. After
each addition during titration, the cell content was stirred for about 10
min. The effect of ligand dilution never represented an error of more
than 5% in L concentration.
C
30H24N3O + m/z (Calc.): 442.1914, m/z (Found): 442.1918.
2.2. Ligand-DNA binding
2.2.1. Instrumentation
Absorption spectra were recorded using a UV–Vis Uvikon 941
(Kontron Instruments) spectrophotometer. Steady-state fluorescence
measurements were carried out using a PTI Quanta Master spectroflu-
orimeter equipped with a Xenon flash lamp as light source, with single
concave grating monochromators and Glan-Thompson polarizers in the
excitation and emission paths. Detection was performed using a pho-
tomultiplier cooled by a Peltier system. Slit widths were selected at 8 nm
for both excitation and emission paths and polarizers were fixed at the
“magic angle” condition. Fluorescence decay measurements were per-
formed using a PTI time-correlated single-photon-counting (TCSPC)
spectrometer upgraded to use monochromatic nanoleds (Horiba). A
nanoled emitting at 335 nm was employed as the excitation source.
2.3. Live-cell studies
HeLa cells were maintained at 37 ◦C and 5% CO2 in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal calf
serum and 100 units/mL penicillin and streptomycin.
For live-cell staining, cells were grown on coverslip-bottom dishes
3

P. Bosch et al.
Dyes and Pigments 192 (2021) 109443
(Nunc™, Thermo Fisher Scientific) and stained by addition of the
quinine sulfate in water as standard[17], and weighted average fluo-
rescence lifetimes for the isolated ligands studied. Ligand 1 exhibits two
bands in the 250–550 nm range. Similar features were found for ligands
2 and 3, although the latter band appeared shifted to the red for com-
pound 2 and even more so for compound 3. Bands placed at the smallest
compound solution (50 mM in DMSO), diluted 1:400 in culture medium
(125 μM final). Micrographs were obtained using a Leica SP5 confocal
fluorescence microscope equipped with a thermostatic chamber and the
following excitation-emission channels (named by the emission colour).
Blue: excitation UV diode laser 405 nm, emission filter 420–460 nm.
Green: excitation argon laser 488 nm, emission filter 500–570 nm. Red:
He/Ne laser 536 nm, emission filter 575–625. Infrared: excitation He/
Ne laser 633 nm, emission filter 650–700 nm.
energy for these compounds are attributable to the transition to a π–π*
excited state (locally excited) which precedes the formation, if this is the
case, of a stable intramolecular charge transfer (ICT) excited state
complex from which the emission occurred[18]. The bathochromic ef-
fect and widening to the red of the absorption bands are due to the
different character of the aryl donor substituents (H < Ph < p-MeOPh).
The emission spectra of ligands in 5% DMSO/water dilute solutions
were monitored upon excitation at the absorption maxima for the lower
energy bands (Fig. 2). All isolated ligands showed single emission bands
at around 455, 520 and 464 nm, respectively. The emission of 1 and 2
free ligands in water showed analogous features as the absorption
spectra for the correlation between the electron-donating character of H
and Ph-substituents. However, emission of 3, whose maximum appeared
at 464 nm, did not exhibit that correlation. In addition, a decrease in
fluorescence and minimal displacement of the emission maximum upon
addition of DNA aliquots during titration experiments with derivatives 1
and 2 was observed (Fig. 3). However, derivative 3 again behaved in a
different way. Thus, DNA addition was accompanied by the appearance
of another emission band centered at around 550 nm. Initially, the
fluorescence intensity (area under both bands) increased for the first
additions (0–0.35 DNA/3 M ratios, R), whereas from R = 0.35 to 11.5,
this intensity decreased. Additionally, the ratios of fluorescence in-
Cytotoxicity experiments were carried out using the standard 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
[16]. The cells were seeded into a 24-well plate at a density of 1.5 × 104
cells per well and incubated in medium containing the compound under
study at different concentrations for 24 h. A 100 μL aliquot of MTT was
added to each well, and the plates were incubated at 37 ◦C for 4 h to
allow MTT to form formazan crystals upon reaction with metabolically
active cells. The MTT-containing medium was then removed from the
wells. Intracellular formazan crystals were dissolved by adding 100 μL of
DMSO to each well and the plates were shaken for 10 min. The absor-
bance was recorded using a multiwell plate reader.
3. Results and discussion
3.1. Synthesis of cations 1, 2 and 3
Compounds 1, 2 and 3 were readily synthesized from the commer-
cially available benzimidazoles 4 or 5 in 31%, 47% and 19% overall
yields, respectively. Thus, amination of 5, by treatment with O-(mesi-
tylsulfonyl)hydroxylamine (MSH)[11], and subsequent reaction of in-
termediate 8 with benzil and a base in a Westphal condensation,
afforded the simpler chromophore 1 in only two steps. Compounds 2
and 3 were obtained in a similar manner, in three steps, using the bromo
derivative 4 as starting material, adding a previous step under standard
Suzuki reaction conditions to introduce the corresponding aryl substi-
tution at the 8-position (Scheme 1).
tensities of both bands measured at 550 nm and 464 nm (I550/I464
)
increased significantly in the first range of molar ratios, and more
smoothly in the second one (Fig. 1S of the SI). The appearance of the
band at 550 nm, whose emission was shifted to the red relative to the
presumable emission from the named locally excited state of 3 (464 nm)
[18], as well as the I550/I464 ratio increase upon DNA addition, may arise
from the formation of a stable ICT complex. This complex, in contrast to
ligand 2, is strongly favored at restricted conformations that occurred
when 3 associated to DNA. A similar dual emission was previously
observed for a dibenzopyridoimidazocinnolinium cation which con-
tained the same p-MeOPh donor substituent[9]. [][][].
3.2. Ligand-DNA binding
Titration experiments for ligands 1, 2 and 3 were also performed by
monitoring the excitation spectra at 25 ◦C (Fig. 2S of the SI). A decrease
in the fluorescence intensity upon addition of DNA was obtained for the
titration of derivatives 1 and 2. The monotonic decrease in fluorescence
Fig. 2 shows the absorption and emission spectra for dilute solutions
of 1, 2 and 3 in a 5% DMSO/water mixture at 25 ◦C. Table 1 summarizes
the positions of the absorption and emission bands, molar absorptivities
at the excitation wavelength (λmax), fluorescence quantum yields using
Scheme 1. Synthesis of cations 1, 2 and 3.
4

P. Bosch et al.
Dyes and Pigments 192 (2021) 109443
Fig. 2. Absorption and normalized (at the maxima) emission spectra for dilute solutions of ligands 1, 2 and 3 in 5% DMSO/water at 25 ◦C.
Table 1
Some photophysical parameters for ligands 1, 2 and 3 in 5% DMSO/water at 25 ◦C. Fluorescence quantum yields were obtained using quinine sulfate in water as
standard.
Structure
Absorption spectra (peaks and shoulders), λex (black)
264(p), 345(p)
λem, max (nm)
ε
(Mꢀ 1cmꢀ 1) in 5%DMSO/water at λex
φf at λex
<
τ> (ns)
#
1
455
11,510
17,080
11,040
0.34
5.8
4.0
4.8
2
3
250(p), 352(p),
520
464
0.13
0.01
378(s)
263(p), 275(s),
352(p), 388 (s)
intensity for 3, however, was detected at DNA/3 M ratios (R) higher
than 0.35, with hardly any variation being observed at lower ratios. In
addition, the relative variation in fluorescence anisotropy (r/r0) was
examined at the emission maximum upon excitation at the wavelengths
at which each fluorescence spectra was collected. Fig. 3S in the SI de-
picts this ratio versus R molar ratios at 25 ◦C. The r/r0 values increased
significantly with [DNA] for all titrations, which is compatible, as in the
previous experiments, with the formation of DNA-ligand complexes
whose rotational relaxation times are larger than those for the free li-
gands. This variation depends on the nature of the ligand, which is
smaller for 1 than for 3. The largest variation was observed for ligand 2.
Fig. 4 depicts the normalized variation in ligand fluorescence in-
tensity as (I–I0)/I0 (I measured as the area under emission spectra and
subscript zero means in the absence of DNA) versus R. Similar repre-
sentations were obtained from the intensities measured from the exci-
tation spectra depicted in the supporting information (Fig. 2S).
the binding constant of (1.7 ± 0.4) × 105 and (1.5 ± 0.1) × 105 Mꢀ 1 for 1
and 2, respectively. The average number of nucleic base pairs per bound
ligand n was 6.9 for ligand 1 but, surprisingly, 0.1 only for ligand 2.
Given the shape of the curve, the data for compound 3 could not be fitted
to equation (10S). However, from the ratio of the intensities of the bands
located at 550 and 464 nm, the values of K and n were (5.4 ± 0.8) × 105
Mꢀ 1 and 0.01, respectively, for ligand 3.
The fluorescence intensity decay profiles for isolated ligands 1, 2 and
3 were fitted to double exponential functions with lifetime components
of 1.0, 1.8 and 2.4 ns and 6.2, 4.10 and 6.6 ns for ligands 1, 2 and 3,
respectively As a result, weighted average lifetimes (<τ>) of 5.8, 4.0
and 4.8 ns, respectively, were obtained (eq. (2)).
The circular dichroism (CD) spectrum in the region of the ctDNA
absorption shows positive and negative bands centered at around 278
and 245 nm, which is characteristic of the secondary structure of a DNA
B-form. These bands are usually attributed to base stacking and right-
handed polynucleotide helicity, respectively. Their intensity and loca-
tion are dependent on the mode and degree of interaction with the
The curves depicted in Fig. 4 for titrations 1 and 2 are the result of
fitting the experimental data to equation 10S (SI), which gave values for
5

P. Bosch et al.
Dyes and Pigments 192 (2021) 109443
Fig. 3. Titration experiments: Emission spectra for buffer solutions of ligands 1, 2 and 3 ([1] = 9.71 × 10ꢀ 6 M, [2] = 1.28 × 10ꢀ 5 M and [3] = 1.37 × 10ꢀ 5
respectively) at 25 ◦C upon addition of DNA aliquots [DNA] = 2.40 × 10ꢀ 5 M. Each spectrum was recorded at different DNA/ligand molar ratios (R).
M
Fig. 4. Normalized changes in fluorescence intensity for ligands 1, 2 and 3 in 5% DMSO and 95% buffer solutions ([1] = 9.71 × 10ꢀ 6 M, [2] = 1.28 × 10ꢀ 5 M and
[3] = 1.37 × 10ꢀ 5 M respectively) at 25 ◦C versus DNA/ligand molar ratios (R).
ligands[19]. Groove binding and the electrostatic interaction of ligands
with DNA usually result in little or no perturbations in either band,
whereas intercalation via strong base stacking interactions and stable
DNA conformations frequently enhances their intensities. In contrast,
other authors have asserted that the intercalation of some ligands pro-
duces a weakening of base stacking, thus resulting in a decrease in band
intensities[20]. The CD spectrum for ligand 1 (Fig. 5) shows that the
intensity of the higher energy band (245 nm) decreases slightly while
Fig. 5. Circular dichroism spectra as molar ellipticity [θ], in the region of DNA absorption, for ligands 1, 2 and 3 in 5% DMSO and 95% buffer solutions in the
presence ctDNA at several ligand/DNA molar ratios monitored at 25 ◦C. Optical path: 0.1 cm.
6

P. Bosch et al.
Dyes and Pigments 192 (2021) 109443
that for the low energy band (278 nm) increases with the ligand/DNA
ratio (R). In contrast, ligands 2 and 3 exhibit an increase in the negative
intensity of the band located at 245 nm and an increase in the band at
278 nm. Both bands are shifted to the red, and two isoelliptic points
were observed near 248-50 nm and 277-80 nm respectively.
positive ones and are one order of magnitude stronger. The bands match
with those observed in the absorption and excitation spectra. These re-
sults agree with the probable intercalation of ligand 1 and the groove
binding of ligands 2 and 3, which contain an extra aromatic ring, to
DNA. It is remarkable that none of the CD spectra in Fig. 6 exhibit
exciton coupling or any signs of ligand aggregation[ [2,21a,21b]].
ICD monitoring is also useful for obtaining ligand to DNA binding
constants upon titration with DNA. Fig. 4S (SI) shows ICD spectra, as
The achiral ligands 1, 2 and 3 can couple their transition moments
and the average dipole moment of the bases when interacting with DNA,
thus resulting in an induced circular dichroism (ICD) with a signal in the
regions where the chromophores absorb. The absorption bands for all
three ligands, especially 2 and 3, overlap with the low energy region in
which DNA also absorbs. As such, the ICD signal for DNA/ligands at 278
nm, which is responsible for the CD increase, cannot be interpreted as
previously[9].
ellipticity (Δε), for ligands in the presence of DNA at different DNA
concentrations while maintaining the ligand concentration constant. As
expected (not shown), no signal was obtained for isolated achiral ligands
1, 2 and 3, whereas, in the presence of DNA, the signal observed
increased with [DNA]. Fig. 7 depicts a plot of Δε versus R during ligand
In addition to the evidence for complexation, the magnitude and sign
of the ICD provides information regarding the mode of interaction
(groove or intercalation) and the relative orientation of the ligand with
respect to the pair of bases in the pocket. Thus, an intercalator exhibits a
weak, negative ICD when the transition moment for the corresponding
absorption band is oriented along the long axis of the binding DNA
pocket (perpendicular to the pseudo-dyad axis), whereas a positive ICD
is expected when the transition moment is perpendicular to the long axis
of the binding pocket. If the transition moment of the ligand is oriented
to form an angle of 45◦ with the bases (in the case of groove binding), the
ICD is expected to be positive and one or two orders of magnitude larger
than for an intercalated ligand[ [2,19g,21]].
titration. Fitting of the experimental data to an equation similar to eq. 9S
(SI), where I is Δε and the ellipticity for the ligand in the absence of DNA
(Δε0) is zero, provides K and n. The values of K=(1.7 ± 0.9) × 105 Mꢀ 1
and n = 6.4 ± 0.4 obtained for 1 are rather similar to the ones obtained
from fluorescence intensity measurements. However, the binding con-
stants for ligands 2 and 3 were (1.4 ± 0.6) × 104 Mꢀ 1 and (7.7 ± 01.7) ×
105 Mꢀ 1, respectively, with calculated n values of 2.0 ± 0.3 and 2.1 ±
0.2 respectively.
Despite this, the K and n values obtained from ICD measurements
(see Fig. 5S and SI) also reproduce the experimental variation of the
fluorescence anisotropies for the complete curve in 1 and for the first
portion of the curves for ligands 2 and 3 (Fig. 5S). The discrepancies
between the values of K and/or n obtained from the fluorescence in-
tensity for 2 and 3 are probably a consequence of the overlap of the
absorption and emission bands and the derived strong self-absorption of
the emission and/or to the dual emission that occurred for ligand 3 upon
DNA complexation. Assuming that the transition moment of the bands
that appear at wavelengths longer than 300 nm is nearly parallel to the
long axis in ligand 1, the negative value for the ICD reveals that 1 is
oriented in the DNA/1 complex with its long molecular axis parallel to
the DNA binding pocket. As stated above, the ICD spectra for ligands 2
and 3 suggest a groove binding to DNA mode.
Fig. 6 shows ICD spectra, as molar ellipticity, for isolated ligands and
in the presence of DNA at identical DNA and ligand concentrations
(molar ratio R = 1.0) in the region where the ligands absorb but DNA
does not. As expected, no signal was obtained for isolated achiral ligands
1, 2 and 3. However, CD spectra for 1/, 2/and 3/DNA solutions
exhibited Cotton effects, the intensities (and band signs) of which
depend on the nature of the ligand. The ICD spectra of 1/DNA solutions
exhibit weakly negative bands, whereas 2/and 3/DNA solutions exhibit
To get an insight into the interaction responsible for the association
of ligands 1 and 3 to a DNA fragment and the structure of the L-DNA
complexes, molecular mechanics (MM) and molecular dynamics (MD)
calculations were performed. For MM calculations three possible L to
DNA approaching modes were considered: intercalation, and binding to
the major and to the minor grooves (Fig. 6S). The calculated total
binding energy and the contribution changes along the L-to-DNA dis-
tance (Fig. 7S) allowed us to obtain the minima binding energy (MBE)
structures (Fig. 8S). Electrostatic interactions were mostly responsible
for reaching stabilization. In the case of 1/DNA, MBE complex structures
occurred at similar binding energy values for intercalation and binding
via the minor groove, the approach via the major groove being less
favourable. However, 3/DNA complexation seemed to prefer a ligand
approach via any of the grooves to binding by intercalation. Analysis of
short MD simulations starting from the corresponding optimized MBE
structures of Fig. 8S indicated that L-DNA interactions are favourable to
a greater or lesser degree for all of the binding modes (Figs. 9S and 10S).
The most important contributions to stabilization are electrostatic, with
van der Waals interactions scarcely contributing. It is interesting that the
ligand in 1/DNA in intercalation mode (Fig. 11S), in agreement with the
negative sign of the ICD band which extends above 315 nm, was ori-
ented essentially with its main axis (long axis) nearly parallel to the base
pocket throughout the trajectory (Fig. 9S,a).
The structure of the 3/DNA complex in which 3 approaches the DNA
via minor groove is the most favourable at the beginning and at the end
of the trajectory (Fig. 13S). Nevertheless, other modes of ligand
approach are also plausible (Figs. 10S and 14S). It should be noted that
the initial structure obtained in the intercalation mode at the end of the
trajectory suggests a similar structure to that obtained in the minor
groove mode (Fig. 14S). These structures at the end of the trajectory for
any of the approaching modes agree with the strong positive Cotton
Fig. 6. Molar ellipticity [θ] (eq. (4)) for isolated ligands 1, 2 and 3 in 5% DMSO
and 95% buffer solutions ([L] = 9.64 × 10ꢀ 5 M) concentration and in the
presence of DNA ([DNA] = 9.64 × 10ꢀ 5 M) at a ligand/DNA molar ratio of 1.0
at 25 ◦C. Optical path: 1 cm.
7

P. Bosch et al.
Dyes and Pigments 192 (2021) 109443
Fig. 7. Ellipticity, Δ
ε
changes for buffer solutions of ligands 1, 2 and 3 ([1] = 1.55 × 10ꢀ 4 M, [2] = 7.74 × 10ꢀ 5 M and [3] = 7.75 × 10ꢀ 5 M respectively) upon
addition of DNA aliquots [DNA] = 2.40 × 10ꢀ 5 M versus molar ratios R during titration experiments. Measurements were obtained from ICD spectra depicted in
Fig. 4S at 360, 400 and 414 nm respectively.
Fig. 8. Confocal fluorescence microscopy of
living HeLa cells cultured in the presence of
compounds 1 and 3 (50 mM in DMSO,
diluted 1:400 in culture medium (125
μM
final)) for 2 h. Scale bar: 25 μm in all panels.
(1) Compound 1 shows fluorescence in the
blue channel. The corner marks in the left
image indicate the area shown enlarged in
the right. (3) Compound 3 showing fluores-
cence in the green (a) and blue (b) channels,
and the overlay composition (c) with a
phase-contrast micrograph. The corner
marks in the upper images indicate the area
shown enlarged in the lower ones.
8

P. Bosch et al.
Dyes and Pigments 192 (2021) 109443
effects observed in the ICD spectra for 3 in the presence of DNA. Full
details of the computational protocols for the application to 1- and 3-to-
DNA complexation and additional results, are provided in the SI.
Conceptualization, Resources, Writing – original draft, Supervision,
Funding acquisition.
Declaration of competing interest
3.3. Live-cell studies
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Cytotoxicity studies show apparent lethal dose 50% (LD50) >10ꢀ 4
M
for compound 1 and 8.3 × 10ꢀ 6 M for compound 3 (Fig. 15S and SI).
Compound 2 could not be assayed due to its complete insolubility in the
cell culture medium.
Acknowledgments
Live-cell staining was studied by confocal fluorescence microscopy.
The results for compounds 1 and 3 (Fig. 8) show a pattern of accumu-
lation in localized intracellular structures, apparently membranous
vesicles and organelles, compatible with active uptake by endocytic
mechanisms.
Financial support from the Spanish Ministerio de Ciencia
e
´
Innovacion (projects CTQ2017-85263-R and RTI2018-097609-B-C21),
Instituto de Salud Carlos III (ISCIII RETIC REDINREN RD16/0009/015
FEDER FUNDS) and UAH (project CCG19/CC-033) is gratefully
Compound 3 shows dual spectral properties, with fluorescence
excitation and emission in two different channels (blue and green) from
the same compound, which are not co-localized (Fig. 8, panel 3c).
The vesicle-like cytoplasmic distribution of fluorescence shown by 1
(and by 3 in the blue channel) is compatible with an endoplasmic re-
ticulum localization. The fluorescence pattern shown by 3 in the green
channel may resemble a mitochondrial localization.
´
acknowledged. P. B. thanks the University of Alcala for his grant. Live-
cell studies were performed by ICTS “NANBIOSIS” and its Confocal
Microscopy Service: Ciber in Bioengineering, Biomaterials & Nano-
medicine (CIBER-BBN), University of Alcala (CAI Medicine Biology).
Appendix A. Supplementary data
The reduced staining of the cell nucleus in the case of 1, or the
speckle-like intranuclear fluorescence of compound 3 observed in the
blue channel, is remarkable considering the DNA-binding capacity
assessed for both compounds. Entry via endocytic mechanisms could
confine the compounds to vesicles or organelles, thereby preventing
diffusion to the nucleoplasm and binding to chromosomal DNA. The
observed decrease in fluorescence emission caused by DNA binding
could also explain or contribute to the lack of apparent nuclear staining.
The speckle-like intranuclear pattern shown by 3 in the blue channel
suggests that this compound enters the nucleoplasm to some extent.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.dyepig.2021.109443.
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