Helical aggregates of bis(styryl) dyes formed by DNA templating

Article abstract of DOI:10.1016/j.jphotochem.2021.113378

In order to further investigate mechanism of the styryl dye – DNA interaction, three different bis(styryl)pyridinium dyes possessing OMe or/and NMe₂ substituents in phenyl ring were selected and interaction of these compounds with the calf thymus DNA (ct-DNA) was monitored by using of UV–vis and fluorescence spectroscopic techniques, circular dichroism (CD), Hoechst 33258 displacement experiments and quantum-chemical calculations. The experimental results indicated a higher fluorescence enhancement for dyes containing NMe₂ group upon binding with DNA. The results proved the interaction of the molecules with ct-DNA occurs through the formation of aggregates in minor groove at high dye concentration. Bis(styryl) dye with OMe substituent forms the helical dye aggregates of right-handed chirality, whereas, dye containing NMe₂ group demonstrates formation of left-handed chiral aggregates in minor groove of DNA. As the DNA concentration increases, the dyes begin to spread out along the minor groove. This research provided a better understanding of mechanism of styryl dye - DNA interaction and effect of dye structure on interacting mode with DNA.

Full text of DOI:10.1016/j.jphotochem.2021.113378

Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113378
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, A: Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Helical aggregates of bis(styryl) dyes formed by DNA templating
,
Maria A. Ustimovaa *, Yury V. Fedorova, Vladimir B. Tsvetkovb, Sergey D. Tokareva,
Nikolai A. Shepela, Olga A. Fedorovaa
a Laboratory of Photoactive Supramolecular Systems, A.N. Nesmeyanov Institute of Organoelement Compounds of Russian Academy of Sciences, Vavilova St. 28,
Moscow, 119991, Russia
b WCRC “Digital biodesign and personalized healthcare” , Sechenov First Moscow State Medical University, Trubetskaya St. 8/2, Moscow, 119146, Russia
A R T I C L E I N F O
A B S T R A C T
Keywords:
Bisstyryl dye
DNA
In order to further investigate mechanism of the styryl dye – DNA interaction, three different bis(styryl)pyr-
idinium dyes possessing OMe or/and NMe2 substituents in phenyl ring were selected and interaction of these
compounds with the calf thymus DNA (ct-DNA) was monitored by using of UV–vis and fluorescence spectro-
scopic techniques, circular dichroism (CD), Hoechst 33258 displacement experiments and quantum-chemical
calculations. The experimental results indicated a higher fluorescence enhancement for dyes containing NMe2
group upon binding with DNA. The results proved the interaction of the molecules with ct-DNA occurs through
the formation of aggregates in minor groove at high dye concentration. Bis(styryl) dye with OMe substituent
forms the helical dye aggregates of right-handed chirality, whereas, dye containing NMe2 group demonstrates
formation of left-handed chiral aggregates in minor groove of DNA. As the DNA concentration increases, the dyes
begin to spread out along the minor groove. This research provided a better understanding of mechanism of
styryl dye - DNA interaction and effect of dye structure on interacting mode with DNA.
Aggregate
Groove binding
FRET
1. Introduction
bis(styryl) dyes interacting with DNAs have been also investigated
[16–25]. Dyes exhibit the properties of long-wave emission, large Stokes
The spontaneous noncovalent assembly of cyanine dyes within the
minor groove of ds DNA causes the formation of helical dye aggregates
in the DNA template which differ in the size and shape [1–5]. The
formed well-defined aggregates exhibit induced chirality and interesting
optical properties. A second feature arising from the noncovalent as-
sembly is that dimerization is highly selective for A-T sequences over
G-C [6]. One important advantage of these aggregates is that their
structure and size can be controlled by the DNA template.
Binding of cyanine dyes to DNA can occur with significant fluores-
cence enhancement due to restricted internal rotation upon binding to
nucleic acid [7]. Thus, a variety of dyes based on benzoxazole, benzo-
thiazole, quinolone, pyridine heterocycles exhibits the increase of the
fluorescence intensity up to 260-fold upon binding to calf thymus DNA
[8–10].
shifts, water solubility, and large fluorescent enhancement towards
nucleic acids. The formation of aggregates within the minor groove of
double stranded DNA is less studied compared to cyanine dyes. Two
reports mentioned of styryl dye aggregates have been found in litera-
ture. Thus, for the 4-alkoxystyryl(pyridinium) dye series, the presence of
the second DNA-binding unit led to the minor groove binding accom-
panied by the aggregation [26]. Bis(styryl) dyes, in which two styr-
ylpyridine moieties are connected to each other through the oxo- or
azacrown ether unit and constructing in tail-to-tail mode, at low DNA
concentration form the aggregates located in minor groove [27]. Also
analysis of the dye-DNA binding results demonstrated that crown ether
macrocycle included in the dye is localized near G-C pairs through the
hydrogen bonds.
In this study, we investigated the effect of substituents in bis(styryl)
pyridine dyes 1a,b, 2 on the formation of aggregates in DNA template
and fluorescent response on binding with DNA. In the composition of
dyes the substituents in the phenyl ring were OMe or/and NMe2 groups.
Another well-studied example is binding of DNA to the natural
product distamycin [11–15]. This compound binds as a face-to-face
dimer in the minor groove of certain DNA sequences, including alter-
nating A-T sequences.
The selectivity, sensitivity and binding properties of the mono- and
* Corresponding author.
E-mail addresses: ustimova.maria@yandex.ru (M.A. Ustimova), fedorova@ineos.ac.ru (O.A. Fedorova).
https://doi.org/10.1016/j.jphotochem.2021.113378
Received 22 March 2021; Received in revised form 13 May 2021; Accepted 19 May 2021
Available online 21 May 2021
1010-6030/© 2021 Elsevier B.V. All rights reserved.

M.A. Ustimova et al.
Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113378
2. Experimental section
mixture was stirred at 80 ◦C during 4 h. After being cooled at room
temperature, 5 mL ethyl acetate was added and the precipitate was
filtered off. The precipitate was washed with cold methanol and hot
ethyl acetate and dried. Red solid, yield 25 %, m.p. 168ꢀ 172 ◦C (dec.).
2.1. General methods
1H NMR spectra were recorded at 400 MHz, and 13C NMR spectra
were recorded at 101 or 151 MHz at ambient temperature using 5 mm
tubes. Chemical shifts were determined with the accuracy of 0.01 and
0.1 ppm for 1H and 13C spectra, respectively, and are given relative to
the residual signal of the solvent that was used as an internal reference.
The coupling constants were determined with the accuracy of 0.1 Hz.
Electrospray ionization (ESI) mass spectra were detected in the mode of
full mass scanning of positive ions on a tandem dynamic mass spec-
trometer equipped with a mass analyzer with an octapole ionic trap.
Melting points were measured on Melt-temp melting point electro-
thermal apparatus and were uncorrected. Elemental analysis was per-
formed on a Carlo Erba 1108 elemental analyzer at the Laboratory of
Microanalysis of A. N. Nesmeyanov Institute of Organoelement Com-
pounds of RAS, Moscow, Russia.
1H NMR (400 MHz, CD3CN): 2.48 (m, 2H, H-
ω); 3.04 (s, 6H, CH3); 3.49
(t, 2H, H-β, J = 6.4, 6.4); 4.51 (t, 2H, H-δ, J = 7.1, 7.1); 6.79 (d, 2H, H-9’,
H-11’, J = 8.9); 7.06 (d, 1H, b’, Jtrans = 16.1); 7.58 (d, 2H, H-8’, H-12’, J
= 8.9); 7.77 (d, 1H, a’, Jtrans = 16.1); 7.88 (d, 2H, H-3’, H-5’, J = 6.9);
8.42 (d, 2H, H-2’, H-6’, J = 6.9). 13C NMR (400 MHz, CD3CN): 28.6 (C-
β), 32.6 (C-ω), 39.0 (CH3), 57.8 (C-δ), 111.7 (C-9’, C-11’), 116.3 (C-b’),
122.1 (C-7’), 122.5 (C-3’, C-5’), 130.1 (C-8’, C-12’), 142.7 (C-a’), 143.1
(C-2’, C-6’), 152.3 (C-10’), 154.7 (C-4’). Anal. calcd for C18H22Br2N2:
C, 50.73; H, 5.2; N, 6.57; Br, 37.5.; found C, 50.69; H, 5.25; N, 6.61. ESI-
MS 4 in MeCN, m/z: calcd 345.1; found 345.1 [4]+.
2.2.5. 4-(4-(dimethylamino)styryl)-1-(3-(4-(4-methoxystyryl) pyridinium-
1-yl)propyl)pyridinium iodide bromide (2)
A mixture of styrylpyridine 4 (0.14 mmol), 3a (0.14 mmol) and KI
(0.14 mmol) in MeCN (5 mL) was stirred at 80 ◦C for 100 h. After being
cooled at room temperature the solvent was evaporated and the ob-
tained residue was recrystallized from methanol, the precipitate was
filtered off. The precipitate washed with hot ethyl acetate and dried. Red
solid, yield 50 %, m.p. 195ꢀ 198 ◦C. 1H NMR (400 MHz, DMSO-d6): 2.62
2.2. Synthesis of the compounds 1a,b, 2, 5a,b
4-[4-(Methoxy)styryl]pyridine 3a [28], 4-[4-(dimethylamino)styryl]
pyridine 3b [29], (E)-4-[4-(methoxy)styryl]-1-methylpyridinium iodide
5a and (E)-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide 5b
[30] were prepared by literature procedures. All other reagents and
solvents were obtained from commercial sources and used as received.
(m, 2H, H-ω); 3.02 (s, 6H, N(CH3)2); 3.83 (s, 3H, OCH3); 4.58 (t, 2H, H-δ,
J = 7.6, 7.6); 4.65 (t, 2H, H-β, J = 7.3, 7.3); 6.78 (d, 2H, H-9’, H-11’ J =
9.0); 7.06 (d, 2H, H-9, H-11, J = 8.4); 7.2 (d, 1H, b’, Jtrans = 16.0); 7.39
(d, 1H, b, Jtrans = 16.2); 7.59 (d, 2H, H-8, H-12, J = 8.9); 7.71 (d, 2H, H-
8’, H-12’, J = 8.1); 7.95 (d, 1H, a’, Jtrans = 16.0); 8.03 (d, 1H, a, Jtrans
=
2.2.1. General procedure for the synthesis of bis-styryl 1a and 1b
A mixture of styrylpyridine 3a or 3b (0.47 mmol) and dibromopro-
pane (0.235 mmol) in DMF (5 mL) was stirred at 80 ◦C for 24 h. After
being cooled at room temperature, 10 mL ethyl acetate was added and
the precipitate was filtered off. The precipitate was washed with hexanes
and ethyl acetate and dried.
16.2); 8.09 (d, 2H, H-3’, H-5’, J = 5.8); 8.22 (d, 2H, H-3, H-5, J = 5.4);
8.84 (d, 2H, H-2’, H-6’, J = 5.8); 8.98 (d, 2H, H-2, H-6, J = 5.4). 13
C
NMR (400 MHz, DMSO-d6): 31.3 (C-ω), 39.5 (N(CH3)2), 55.4 (OCH3)
56.0 (C-δ), 56.4 (C-β), 111.9 (C-9’, C-11’), 114.7 (C-9, C-11), 117.1 (C-
b’), 120.7 (C-b), 122.4 (C-3’, C-5’), 123.3 (C-7’), 123.4 (C-3, C-5), 127.8
(C-7), 130.1 (C-8, C-12), 130.3 (C-8’, C-12’), 141.2 (C-a), 142.5 (C-a’),
143.6 (C-2’, C-6’), 144.2 (C-2, C-6), 152.0 (C-10’), 153.6 (C-4), 154.0 (C-
4’), 161.3 (C-10). Anal. calcd for C32H35BrIN3O: C, 56.15; H, 5.15; N,
6.14; found C, 56.25; H, 5.11; N, 6.1. ESI-MS 2 in MeCN, m/z: calcd
2.2.2. (E)-1,1’-(propane-1,3-diyl)bis(4-(4-methoxystyryl)pyridinium)
bromide (1a)
Yellow solid, yield 36 %, m.p. 256–258 ◦C. 1H NMR (400 MHz,
238.8; found 238.9 [2]2+
.
CD3CN): 2.67 (m, 1H, H-ω); 3.84 (s, 3H, CH3); 4.68 (t, 2H, H-β, J = 6.9,
6.8); 7.01 (d, 2H, H-9, H-11, J = 8.8); 7.21 (d, 1H, b, Jtrans = 16.2); 7.66
(d, 2H, H-8, H-12, J = 8.8); 7.81 (d, 1H, a, Jtrans = 16.2); 7.98 (d, 2H, H-
3, H-5, J = 6.9); 8.77 (d, 2H, H-2, H-6, J = 6.9). 13C NMR (400 MHz,
2.3. Preparation and handling of DNA solutions
Calf thymus DNA (type I; highly polymerized sodium salt, Sigma)
was dissolved without further purification in 10 mM sodium phosphate
buffer at a concentration of 1–2 mg mLꢀ 1 and kept at 4 ◦C for at least 16
h. After that the solution was filtered through a PVDF membrane filter
CD3CN): 32.0 (C-ω), 55.3 (CH3) 56.7 (C-β), 114.6 (C-9, C-11), 120.2 (C-
b), 123.7 (C-3, C-5), 127.4(C-7), 130.2 (C-8, C-12), 141.8 (C-a), 143.9
(C-2, C-6), 154.6 (C-4), 162.0 (C-10). Anal. calcd for C31H32Br2N2O2:
C, 59.63; H, 5.17; N, 4.49; found: C, 59.65; H, 5.12; N, 4.45. ESI-MS 1a in
H2O, m/z: calcd 232.3; found 232.9 [1a]2+
.
(pore size 0.45 μm) to remove any insoluble material. The concentra-
tions of DNA samples were determined by absorption measurements of
the diluted stock solution (1 : 20), using the molar absorption coefficient
ε260 = 12 824 cmꢀ 1Mꢀ 1(in base pairs, bp).
2.2.3. (E)-1,1’-(propane-1,3-diyl)bis(4-(4-(dimethylamino)styryl)
pyridinium) bromide (1b)
Red solid, yield 33 %, m.p. 176ꢀ 178 ◦C (dec.). 1H NMR (400 MHz,
CD3CN): 2.55 (m, 1H, H-
ω
); 3.04 (s, 6H, CH3); 4.44 (t, 2H, H-δ, J = 7.6,
2.4. Spectroscopic studies
7.3); 6.76 (d, 2H, H-9’, H-11’, J = 8.9); 7.02 (d, 1H, b’, Jtrans = 16.2);
7.57 (d, 2H, H-8’, H-12’, J = 8.9); 7.77 (d, 1H, a’, Jtrans = 15.9); 7.85 (d,
2H, H-3’, H-5’, J = 6.9); 8.33 (d, 2H, H-2’, H-6’, J = 6.7). 13C NMR (400
Electronic absorption spectra were recorded on a Varian-Cary 300
spectrophotometer. Fluorescence spectra were recorded on a Cary
Eclipse spectrofluorometer. The spectra of circular dichroism were
recorded on the automatic recording dichrograph SKD-2MUF. All mea-
surements were carried out in conventional quartz cells of 10 mm path
length at 20 ◦C. The preparation and handling of the solutions were
carried out under red light. The experiments were performed in the
phosphate buffer solution at pH = 7 or in deionized water. The fluo-
MHz, CD3CN): 31.2 (C-ω), 39.0 (CH3), 56.2 (C-δ), 111.6 (C-9’, C-11’),
116.2 (C-b’), 122.0 (C-7’), 122.4 (C-3’, C-5’), 130.2 (C-8’, C-12’), 142.7
(C-2’, C-6’),143.1 (C-a’), 152.4 (C-4’), 154.9 (C-10’). Anal. calcd for
C33H38Br2N4: C, 60.93; H, 5.89; N, 8.61; found: C, 60.98; H, 5.84; N,
8.58. ESI-MS 1b in H2O, m/z: calcd 245.3; found 245.3 [1b]2+
.
2.2.4. (E)-1-(3-bromopropyl)-4-(4-(dimethylamino)styryl)pyridinium
bromide (4)
rescence lifetime τ was measured in correlated photon counting mode
using a Horiba Jobin Yvon Fluorolog 3–221 spectrofluorimeter with 369
and 455 nm nanoleds.
A solution of compound 1b (0.1 g, 0.45 mmol) in DMF (10 mL) was
very slowly added dropwise to a solution of 1,3-dibromopropane (0.454
g, 2.25 mmol) in DMF (2 mL) under continuous stirring at 80 ◦C in the
argon atmosphere. After the addition was completed the obtained
The fluorescence quantum yield measurements were carried out in
buffer solutions at 20 ± 1 ◦C; the concentrations of the studied com-
pounds were 5 × 10ꢀ 6M. All measured fluorescence spectra were
2

M.A. Ustimova et al.
Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113378
corrected for the nonuniformity of the detector spectral sensitivity.
Coumarin 343 in ethanol (φfl = 0.63) and coumarin 6 (φfl = 0.78) in
ethanol was used as a reference for the fluorescence quantum yield
measurements. The fluorescence quantum yields were calculated using
Eq. (1):
simulation performed by using periodical boundary conditions and
rectangular box. The buffer between DNA-ligand complex and the pe-
riodic box wall was at least 15 Å. For neutralizing of the negative charge
of DNA backbone K+ ions and positive charge of 1a, 1b and 2 Clꢀ were
used. The parameters needed for interatomic energy calculation, were
taken from the force fields OL15 [44,45] for DNA and from general
amber force field (gaff2) for 1a, 1b and 2. At the beginning of the
calculation, the systems under study were minimized in two steps. At the
first stage, the location of solvent molecules was optimized using 1000
steps (500 steps of the steepest descent, followed by 500 steps of the
conjugate gradient), while the mobility of all solute atoms was
restrained with a force constant of 500 kcal*mol-1*Å-2. At the second
stage, the optimization was carried out without any restrictions using
2500 steps (1000 steps of the steepest decent, 1500 steps of the conju-
gate gradient). Then, gradual heating to 300 K in 20 ps was performed.
To avoid too large fluctuations for the systems under study, weak har-
monic constraints with a force constant of 10 kcal*mol-1*Å-2 were used
at this stage for all atoms except the solvent atoms. SHAKE algorithm
was applied to constrain bonds to hydrogen atoms, that allowed to use 2
fs step. Scaling of nonbonded van der Waals and electrostatic in-
teractions were performed by the standard Amber values. The cut off
distance for non-bonded interactions was equal to 10 Å and the
long-range electrostatics was calculated using the particle mesh Ewald
method. The MD simulations in production phase were carried out using
constant temperature (T =300 K) and constant pressure (p = 1 atm) over
80 ns. To control the temperature Langevin thermostat was used with
the collision frequency of 1 ps-1. Snapshot visualization was performed
using VMD. Energy was estimated using the MM-GBSA approach. The
polar contribution EGB was computed using the Generalized Born (GB)
method and the algorithm developed by Onufriev et al. for calculating
the effective Born radii [46]. The non-polar contribution to the solvation
energy (Esurf), which includes solute-solvent van der Waals interactions
and the free energy of cavity formation in solvent, was estimated from a
solvent-accessible surface area (SASA). Snapshot visualization was
performed using VMD with a donor-acceptor distance of 3.2 Å and angle
cut off of 20 degrees. To simulate the interaction of an ensemble of li-
gands with DNA, the distribution of ligands in the rectangular box was
modeled using the PACKMOL package [47] with tolerance distance 10
Å.
2
R
S
(1 ꢀ 10ꢀ A )n
φfl = φfl
∙
(1)
RSR (1 ꢀ 10ꢀ A)n2R
wherein φfl and φflR are the fluorescence quantum yields of the studied
solution and the standard compound respectively; A and AR are the
absorptions of the studied solution and the standard respectively; S and
SR are the areas underneath the curves of the fluorescence spectra of the
studied solution and the standard respectively; and n and nR are the
refraction indices of the solvents for the substance under study and the
standard compound.
2.5. Molecular modeling calculations
The 3D models of 1a, 1b, 2 and DNA were built using molecular
graphics software package Sybyl-X software (Certara, USA). Partial
charges on 1a, 1b and 2 atoms were calculated according to the
following scheme. First, to find the minimum energy conformation,
scanning of the conformational space of the 1a, 1b and 2 was performed
with the application of molecular-mechanical approach and Monte-
Carlo method by using Molsoft ICM-Pro 3.8.6 [31]. To calculate inter-
atomic interactions the force field MMFF [32] was used at this stage.
Further optimization of the conformation found at the first step for the
purpose of searching geometry with the smallest energy and calculation
of electron density distribution were performed by using second-order
Møller–Plesset perturbation theory (MP2) [33] and implicit consider-
ation of the solvent effect with application of the conductor-like polar-
izable continuum model (CPCM) [34] and cc-pvdz basis sets. Then
Merz-Singh-Kollman scheme [35] was applied to obtained electron
density distribution for calculation of grid for the electrostatic potential
fitting with the following parameters: (6/41 = 10) - the number of
surfaces around the atoms and (6/42 = 17) - the density of test points on
these surfaces. The RESP (Restrained ElectroStatic Potential) method
[36] was applied to fitting of the grid obtained in the previous step for
calculation of partial atomic charges. All quantum mechanics simula-
tions were carried out using Gaussian 09 program [37]. To define the
most probable binding site of the ligands on the target surface, the
procedure of flexible ligand docking was performed using ICM-Pro
3.8.6. DNA conformation remained invariable during the docking pro-
cedure. Before starting a docking procedure the structures of DNA and
1a, 1b and 2 were converted into an ICM object according to the ICM
method, the molecular models were described using internal co-
ordinates as variables. The parameters needed for interatomic energy
calculation and the partial charges for atoms of the targets were taken
from the ECEPP/3 [38] and from force field MMFF for atoms of 1a, 1b
and 2. The biased probability Monte Carlo (BPMC) minimization pro-
cedure [39] was used for global energy optimization. Finally confor-
mational stack obtained from docking procedure was sorted by scoring
function. The selected complexes were used as targets during the second
docking procedure with subsequent optimization in modeling the pos-
sibility of aggregation of the ligands on the surface of the DNA. In the
second stage, the complexes, obtained by Molsoft ICM-Pro 3.8.6, were
minimized by using SYBYL X and Powell method [40]. The following
settings were used: partial charges on the DNA from Amber7 ff02 force
field, parameters for interatomic interactions and from Tripos force field
[41], a non-bonded cut-off distance equal to 8 Å, a distance-dependent
dielectric function, the number of iterations equal to 500, the simplex
method in an initial optimization, and an energy gradient convergence
criterion of 0.05 kcal/mol/Å.
3. Results and discussion
3.1. Synthesis of bisstyryl dyes 1a, 1b and 2
Symmetric bis(styryl) dyes 1a [48] and 1b [49] were obtained in a
two-step synthesis (Scheme 1). In the first step, aldol condensation of
4-picoline with benzaldehyde was performed according to published
protocols [28,29] resulting in the mono(styryl) dye 3a and 3b. In the
second step, mono(styryl) dyes reacted with 1,3-dibromopropane in
ratio 2:1 to give bis(styryl) dyes 1a and 1b. The asymmetric bis(styryl)
dye 2 was obtained in 51 % yield by quaternization of the intermediate 4
with 1,3-dibromopropane followed by alkylation of 3b with 1,3-dibro-
mopropane. All dyes 1a, 1b and 2 were identified and fully character-
ized by NMR spectroscopy, mass spectrometry and elemental analysis
(Fig. S1 in Electronic Supporting Information).
3.2. Optical properties of dyes 1a, 1b and 2
The absorption spectra in the phosphate buffer (BPE) solution at pH
= 7 of symmetric bis(styryl) dyes 1a and 1b exhibit long wavelength
absorption bands (LAB) centered at 380 and 468 nm since each of them
contains two identical chromophore fragments (Table 1). The positions
of LABs are similar to those of corresponding analogue mono(styryl)s 5a
and 5b (Table 1). The observed absorption spectra are attributed to an
intramolecular charge transfer from the donor OMe or NMe2 fragment to
the acceptor heterocyclic moiety. Absorption spectrum of
MD simulations were performed using Amber 18 software [42]. The
solvent effect was simulated using the OPC3 water model [43]. The
3

M.A. Ustimova et al.
Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113378
Scheme 1. Synthesis of the dyes 1a,b and 2 and structures of monostyryl dyes 5a,b [30]. *Reaction condition for obtaining of 3a. ** Reaction condition for obtaining
of 3b.
Table 1
Optical characteristics of dyes 1a,b, 2, 5a,b and their complexes with DNA (λabs, λem - absorption and emission spectral maximum wavelengths; IL – fluorescence
intensity of free or IL/ DNA - DNA-bound dye at λem
;
τ
- fluorescence lifetime at λem wavelength).
Stoke’s shift, cmꢀ 1
max
max
λ
, nm
λ
, nm
ϕ, %
τ
, ns (λem, nm) #
abs
em
ILmDaNxA/IL
free
dye
dye in the
presence of
DNA
free dye
dye in the
presence of
DNA
free
dye
dye in the
presence of
DNA
free
dye
dye in the
presence of
DNA
free dye
dye in the presence
Dye
(λem L·DNA)
of DNA (Cdye/CDNA
1:50)
=
< 0.1
(507) 100
%
0.32 (507) 84 %
1.74 (507) 16 %
1.26 (620) 43 %
2.78 (620) 57 %
0.33 (510) 96 %
5.47 (510) 4%
5a
5b
373
448
395
491
501
615
501
615
6850
6060
5360
4110
2.1
60
1.2
2.3
6.2
0.19
0.26
(620) 100
%
< 0.1
(510) 100
%
1a
1b
380
468
395
510,694
627
505
6710
5420
5520
4070
3.2
0.47
1.47
1.57
0.41 (560) 87 %
7.95 (560) 13 %
0.42 (620) 63 %
2.57 (620) 37 %
(660) 100
%
0.21
492
615
60
0.05
0.04
2.82
0.6
(520) 100
%
4940a
3830b
4310a
3460b
0.24
0.22 (490) 100 %
0.32 (620) 78 %
2.4 (620) 22 %
401,
503
500,
623
2
408, 507
495, 615
3.2, 23
(490) 100
%
#
- fluorescence decay curves are represented in ESI, Fig. S3.
- Stoke’s shift for O-chromophore in bisstyryl dye 2.
- Stoke’s shift for O-chromophore in bisstyryl dye 2.
a
b
unsymmetrical dye 2 contains two peaks in the long wavelength region
at 401 and 503 nm, which are characteristic of both mono(styryl) dyes
5a and 5b. Differences in intensity and some red shift (20–30 nm) in the
peak positions on going from equimolar mixture of corresponding mono
(styryl)s 5a (O-chromophore), 5b (N-chromophore) can be explained by
their intramolecular interaction and the increased polarization of the
chromophores and, hence, the more pronounced ICT interaction,
resulting from proximity of two positive charges in the structure of 2.
Dyes 1a, 1b and 2 demonstrate large Stoke’s shift (5420–6710 cm–1).
The fluorescence spectrum of dye 1a has a maximum at about 510 nm,
corresponding to the O-chromophore 5a, and a maximum at 694 nm.
Since the ratio of the intensity of fluorescence signals at 505 nm and 694
nm does not depend on the concentration of 1а in solution, it can be
concluded that the long-wavelength peak corresponds to the excimer
fluorescence. Thus, two styryl fragments of dye 1a can form intra-
molecular face-to-face sandwich H-dimer in aqueous solution (Fig. 1).
The fluorescence decay curves of dyes 5a,b and 1b in water are
monoexponential, which indicates the presence of only one type of
molecules in the excited state. Time-resolved measurements of 1a
showed the existence of short-lived and long-lived components, indi-
cating a relaxation process in the dye molecule with a linear structure
and in the form of an intramolecular excimer. When DNA is added to the
dyes, the fluorescence decay of the resulting solutions becomes multi-
exponential, which indicates that solutions contain two or more com-
ponents with different fluorescence lifetimes. The average fluorescence
lifetime of solutions also increases (Table 1). The large increase in the
4

M.A. Ustimova et al.
Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113378
Fig. 2. Mutual arrangement of 5a and 5b HOMO-LUMO obtained from elec-
trochemical data.
Fig. 1. Fluorescence of 1a at 20◦C in BPE buffer and proposed structure of
H-dimer.
two chromophores, and the distance between chromophores is 12.3 Å
(see Fig. S4 in ESI), which is quite far for Dexter electron transfer [52].
fluorescence lifetime is primarily due to the fact that interaction with
¨
At the same time, the fragments are close enough for FRET (Forster
DNA reduces the efficiency of torsional motion of dyes in an excited state
–
resonance energy transfer), and the HOMO-LUMO gap of 5b is less than
that of 5a, so FRET from O-chromophore to N-chromophore can occur.
The presence and efficiency of FRET was calculated by the optical
spectroscopy.
around the C C double bond, making it difficult to form the non-
–
fluorescent Z-isomer. In addition, due to the increased viscosity of
samples with a large mass percentage of DNA, the relaxation rate of the
excited state of the dye through the formation of the TICT state
decreases.
The equimolar mixture of mono(styryl) dyes 5a and 5b demonstrates
two absorption bands at 382 and 452 nm belonging to O- and N-chro-
mophores (Fig. 3a). Upon excitation the equimolar mixture of 5a and 5b
with 382 nm light, which is mostly absorbed by O-chromophore 5a, the
emission band at 505 nm is observed similar to that in the spectrum of
5a (Fig. 3b). The absorption spectrum of 2 recorded under the same
conditions is consisted of the same 382 nm and 495 nm bands (Fig. 3c).
But irradiation of 2 produces the red-shifted signal at 627 nm belonging
to the N-chromophore fluorescence (Fig. 3d). This result indicates
occurring of RET from O-chromophore unit to N-chromophore part of 2.
Noteworthy, comparison of the fluorescent spectra of 2 and 5b pre-
sented in Fig. 3b and d shows that the short wavelength part of the
emission band of 2 (Fig. 3d) is broadened to some extent by the residual
fluorescence of the donor unit. Using the emission intensities at 500 nm
where the acceptor chromophore does not emit light (see Fig. 3b) for
compound 2 (I) and equimolar mixture of 5a and 5b (I0), the energy
transfer efficiency was calculated to be as high as 0.97 (97 %) according
to the equation:
The fluorescence quantum yields of 1b, 1a and 2 are very low,
obviously due to the presence of efficient competitive nonradiative
relaxation processes involving the formation of twisted states and E–Z-
isomerization [50]. As shown earlier, the relaxation of the exited state of
ligands similar to 1b goes mainly through the formation of twisted states
with a subsequent non-radiative relaxation to the ground state via ‘loose
bolt’ mechanism [51]. To support the proposition, the measurements of
quantum yields of the fluorescence of dyes 1a, b and 2 in different
solvents have been done (Table S1 in ESI). In viscous glycerol and
ethylene glycol, an increase in fluorescence quantum yield was observed
by 5–6 times for 1a, 40 times for 1b and 32 times for 2 due to the
suppression of the possibility of rotation around single and double
bonds. Also in non-polar solvent the quantum yields of fluorescence are
higher than those in polar (Table S1 in ESI). This fact indicates the
possibility of the fluorescence enhancement for the dyes under study,
when, upon binding to DNA, the dyes pass from water to the hydro-
phobic environment of the nucleic acid.
The asymmetric dye 2 possesses two different chromophore units
between which the occurrence of energy transfer could be. To estimate
the possibility and mechanism of it, electrochemical study has been
prepared. The redox potentials of dyes 5a, 5b and 2 were investigated by
cyclic wave voltammetry in MeCN containing tetrabutylammonium
hexafluorophosphate as supporting electrolyte on Pt-electrode. The
resulting voltammograms, electrochemical data, its analysis and derived
HOMO-LUMO energy levels are summarized in ESI (Figs. S6–S8,
Table S3.) The mutual arrangement of HOMO-LUMO of 5a and 5b is
shown in Fig. 2.
I
ΦRET = 1 ꢀ
I0
A rather close value of ΦRET (99 %) was obtained using the theo-
¨
retical calculations by the Forster model. Theoretical calculation of ΦRET
is presented in part 3, Figs. S4, S5 and Table S2 in ESI.
3.3. Study of the interaction of 1a, b and 2 with ct-DNA by optical
methods
As shown in Fig. 2, the LUMOs of the O-chromophore and the N-
chromophore of the asymmetric dye 2 are close in energy. They are also
located closely on positively charged pyridinium fragments separated
only by a short alkyl bridge, which makes possible an electron transfer
from an excited chromophore to another one in the ground state.
However, the HOMOs of both fragments are localized at the far ends of
The interaction of bis(styryl) dyes 1a,b and 2 with calf thymus DNA
was investigated by spectrophotometric and spectrofluorimetric titra-
tions in BPE buffer at pH = 7. The changes in optical spectra were
analyzed by varying concentrations of DNA in the presence of a constant
concentration of each dye. For all dyes two processes have been
observed. Initially the addition of DNA to the solution of bis(styryl) dyes
5

M.A. Ustimova et al.
Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113378
Fig. 3. Absorption (a,c) and fluorescence (b,d) spectra of compounds 2 (c,d), 5a (a,b), 5b (a,b) and equimolar mixture of 5a and 5b (c,d) in BPE buffer. Concen-
trations of all compounds were 10 M. Excitation wavelengths λex were 450 nm for 5b and 382 nm for 2, 5a and mixture of 5a and 5b.
μ
1a,b and 2 causes a decrease in the intensity of the absorption band with
a small red shift of the absorption maximum (Fig. 4a–c). The further
increase in DNA concentration leads to a rise of the intensity of the
absorption band and its bathochromic shift up to 15 nm for 1a and 24
nm for 1b (Fig. 4a and b). The addition of DNA to the solution of bis
(styryl) dye 2 showed smaller changes in position of bands and intensity
of absorption. Here the DNA induced spectral changes may be explained
in terms of change in local polarity around the dye. The polarity of the
environment around the dye decreases with the addition of DNA, and
the energy gap between the highest occupied molecular orbital (HOMO)
and the lowest unoccupied molecular orbital (LUMO) of the dye de-
creases, which is reflected as bathochromic shift [53].
The interaction of monostyryl dyes 5a,b with DNA also demonstrates
the bathochromic shift of the long wavelength band in absorption
spectra (Figs S9 and S10 in ESI). The binding constants (Kb) and the
number of dsDNA base pairs occupied by one bound dye molecule (n) for
the dyes 5a,b were estimated by spectroscopic titrations. The approxi-
mation of the experimental results with the McGhee and von Hippel
equation shows that Kb values obtained for the dyes 5a and 5b were 4.9
× 103 Mꢀ 1 and 3.2 × 103 Mꢀ 1 respectively, the number of dsDNA base
pairs occupied by one bound dye molecule (n) were correspondingly
3.74 and 3.80 [54].
The fluorimetric response of the dyes to the association with DNA is
even more complicated (Fig. 5). For the bis(styryl) dye 1a, the position
Fig. 4. Photometric titration of ct-DNA to bis-styryl dyes 1a, cDNA/c1a = 0–49 (a); 1b, cDNA/c1b = 0–44 (b); 2, cDNA/c2 = 0–42 (c); clig = 10 μM; in BPE buffer. The
arrows show the change in the intensity of the absorption bands upon the addition of ct-DNA.
6

M.A. Ustimova et al.
Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113378
Fig. 5. Fluorimetric titration of ct-DNA to bis-styryl dyes 1a, cDNA/c1a = 0–29 (a); 1b, cDNA/c1b = 0–67 (b); 2, cDNA/c2 = 0–48 (c); clig = 10 μM; in BPE buffer. The
arrows show the change in the intensity of the emission bands upon the addition of ct-DNA.
of the fluorescence maximum change after the addition of the first ali-
quots of DNA (cDNA/c1a = 0.75 and 1.63), while the intensity increased
slightly. In this case, the destruction of the intramolecular excimer can
be observed, apparently when adding DNA to the dye, interaction with
ds-DNA becomes more beneficial. With the increasing concentrations of
DNA (cDNA/c1a = 29), the emission intensity of 1a significantly
increased with a 5 nm blue shift of the maximum (Fig. 5a). The first
additions of DNA to a solution of bis(styryl) 1b did not in any way affect
the change in the dye fluorescence signal, but with a further increase in
the DNA concentration (cDNA/c1b = 67), the intensity increased up to 60
times (Fig. 5b). For the dye 2, the first additions of DNA increased the
intensity of the far-wave fluorescence band, while the band at 500 nm
remained practically unchanged. Further, there was an intense increase
in fluorescence at 620 nm and a slight increase in the intensity of the
short-wavelength band (Fig. 5c). Such changes confirm the presence of
energy transfer in the molecule, therefore the binding of each of the
chromophores led to an increase in the fluorescence intensity of the N-
chromophore at 620 nm. Similar to what we saw when dyes 1a,b and 2
demonstrated the enhancement of fluorescence in viscous solvent, the
fluorescence of dyes increased when they placed in the DNA solution.
The restricted motional freedom for rotation around single and double
bond results in styryl energy dissipation predominantly throughout
emission via fluorescence. Analogue to glycerol solution, the most pro-
nounced enhancement was found for 1b and 2 (Table 1).
isoelliptic points coincide with the zero transition and are centered at
the wavelengths corresponding to the absorption maximum of the dye
(1a: 380 nm).This phenomenon is known as right-handed helical ag-
gregation in DNA template [62]. As the concentration of DNA increases,
the dimers begin to spread out along the groove. In CD spectra the rise in
DNA concentration leads to the splitting is lost (Fig. 6b–d).
The binding of a single molecule in the minor groove is favorable
based on the hydrophobicity of the dye. If a second dye enters the groove
to form a dimer, then the dyes will have more favorable van der Waals
interactions with each other. The assembly of one dimer widens the
minor groove, what do easer the assembly of additional dimers due to
the groove would already be pre-organized. The second level of coop-
erativity is the assembly of end-to-end aggregates in the minor groove at
high dye concentrations. While distorting the DNA should diminish the
face-to-face cooperativity, the van der Waals dye interactions are suffi-
ciently strong to compensate for this [2].
Since we did not detect the formation of dimers or aggregates in a
solution of the free dye 1b, and the cleavage of the CD signal indicates
the formation of aggregates in the DNA groove it can be assumed that
aggregates are formed already in the DNA body.
Upon the binding of 1b, the DNA helix preserves its right-handed
arrangement, whereas the exciton splitting of the dye ICD indicates
the formation of the left-handed helical assembly of the aggregating
chromophores [26]. For bisstyryl 1b the isoelliptic points appear at 476
and 537 nm, i.e. one is significantly red-shifted in comparison with the
absorption maximum of free 1b (Δλ = +69 nm) that may be indicative of
the formation of J-type dimers in the DNA groove.
3.4. Study of the interaction of 1a, 1b and 5 with ct-DNA by CD
spectroscopy
In the case of compound 2, the isoelliptic points appear at 439 nm
and 477 nm, i.e. it is shifted in comparison with the absorption
maximum of free 2 (Δλ1 = +38 nm, Δλ2 =ꢀ 26 nm). The extent of ag-
gregation is not very high because of the relatively low intensity of the
CD exciton coupled band that arises from side interactions when the
dimers in the groove have tight contact with each other. Such changes in
the CD spectra may indicate that bis(styryl) dye molecules interact with
DNA by folding into the groove.
For a better understanding the structure of dye-ds-DNA complexes,
we used CD spectroscopy as a highly sensitive method for detecting
conformational changes in the secondary structures of nucleic acid [55].
Dye molecules 1a, b and 2 are achiral and do not give a signal in the CD
spectra. Small achiral molecules can acquire an induced CD spectrum
when bound to nucleic acid (usually analyzed in the > 300 nm range),
which can be useful for determining binding modes (intercalation, as-
sociation, groove binding, etc.) [56]. Fig. 6a–e show the DNA-induced
CD spectra of dyes 5a,b, 1a,b and 2. Minor groove binding of 5a and
5b to ds-DNA gives a low-intensive positive ICD band (Fig. 6a, b). In the
ICD spectrum of dye 1а the appearance of a strong exciton CD signal is
observed (Fig. 6c). The exciton CD signal has a one positive and one
negative bands on either sides of the absorption maximum of the free
ligand [57,58]. Excitonic CD indicates the formation of dimers or higher
order complexes, either in the groove-binding mode or in the external
stacking-binding mode [56,59,60]. It is possible to propose the forma-
tion of a structure in which two dyes form a cofacial dimer within the
minor groove of DNA and adjacent dimers fill the minor groove along
the length of the DNA. The interaction between adjacent dimers in the
minor groove causes the splitting of the band into positive and negative
peaks [61]. The right-handed helical orientation of adjacent dimers re-
sults in order of the bands when positive is at long wavelength and
negative is at short wavelength. In the case of compounds 1a, the
3.5. Competitive displacement assay by bisstyryl dyes 1a, 1b and 2
The DNA-binding mode of bis(styryl) dyes 1a, 1b and 2 was further
investigated using a dye displacement assay which is simply based on
the principle that if a small molecule or bis (styryl) replaces the DNA-
bound dye, this indicates that the molecule binds to DNA in a similar
way. Substitution of a dye for a small molecule results in changes in
fluorescence intensity which can be easily identified by changes in the
fluorescence spectra.
To this end, we first studied the dye displacement assay using MeGr
(methylene green), ligand for DNA major groove [63]. As shown in
Fig. S11a-c in ESI, subsequent addition of bis(styryl) dyes 1a, 1b or 2 had
no effect on MeGr-ct-DNA complex, indicating that bis(styryl)s do not
follow major groove binding mode.
To further investigate the groove-binding mode of binding we
7

M.A. Ustimova et al.
Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113378
Fig. 6. Circular dichroism spectra of ct-DNA (cDNA = 0.1 mM b.p.) in the absence and presence of bis-styryl dyes 5a(a), 5b(b), 1a(c), 1b(d) and 2(e) at different LDRs
(ligand–DNA ratio): 0; 0.1; 0.3; 0.6. 5a, 5b, 1a, 1b, 2: in BPE buffer. The arrows show the changes in the bands with increasing dye concentration.
studied the displacement of the Hoechst 33258 dye, minor groove
binding dye [64]. The groove-binding molecules are able to displace
Hoechst 33258 from the minor groove of DNA helix, which leads to
decrease fluorescence intensity of the DNA-Hoechst system. As shown in
Figs. 7 and S12 in ESI, subsequent addition of dyes 1a, 1b or 2 to the
Hoechst-ct-DNA complex led to successive decrease in fluorescence
maxima intensity suggestive of minor groove binding mode of bis(styryl)
s.
3.6. Molecular modeling calculations of binding of bisstyryl dyes 1a, 1b
and 2 with DNA
In order to determine the probable conformation of compounds 1а,
1b, and 2 in solution, quantum-mechanical optimization was carried out
8

M.A. Ustimova et al.
Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113378
Fig. S15 in ESI shows the docking results. When interacting with DNA,
all compounds are located in a minor groove; the most probable
conformation is one in which the styryl fragments are in linear forms.
The docking procedure implicitly evaluates the effect of a solvent on
the mechanism of interaction with DNA; therefore, at the second stage of
calculations, to refine its results, we performed modeling using molec-
ular dynamics and adding interactions with solvent molecules to the
calculations. In the calculations, the starting conformations for all
compounds were taken in the form of both sandwich and open linear
structures. The starting conformations were localized at a distance that
almost excluded the interaction of ligands in the target. Analysis of the
simulation data showed that in the first close contact with the DNA
surface in all three cases occurred due to the convergence of the posi-
tively charged pyridine rings. During the interaction of 1а in the form of
a sandwich with DNA, it was observed to fold into a minor groove, as
well as the initial transition of the molecule into a linear form and
subsequent interaction with DNA. In cases 1b and 2, the conformation in
the form of a sandwich was retained when folded into the minor groove.
Fig. 9 shows the starting and obtained at the last step trajectories of the
conformation of DNA complexes with 1a, 1b, and 2, when the initial
conformation of the bisstyryl dye had a sandwich structure. Fig. 10
shows the process of interaction with DNA of linear dyes.
Fig. 7. Fluorimetric titration of complex Hoechst-ct-DNA by bis(styryl) dyes
1a, C1a/CHoechst = 0–12; CHoechst = 5 μM; CDNA = 0.15 mM in BPE buffer. The
arrows show the change in the intensity of the emission bands upon the addi-
tion of ct-DNA.
According to the data shown in Fig. S17 in ESI, the free energy of the
dye in the sandwich form is lower than those of linear form. This is
explained by that the mutual arrangement of styryl fragments in the
sandwich leads to a twofold increase in the interacting pairs and the
¨
using the second-order Moller-Plesset procedure and implicitly taking
into account the effect of the solvent in the framework of the conductor-
like polarizable continuum model. The results of quantum mechanical
simulation are shown in Fig. 8.
appearance of π-π interactions between pyridine and benzene rings. The
The results obtained in Fig. 8 indicate the possibility of the formation
of an intramolecular sandwich structure for all three molecules of bis-
styryl dyes in solution. The most stringent parallel arrangement of styryl
fragments is observed in the case of 1a; in the remaining molecules,
styryl fragments are displaced relative to each other. This is consistent
with the experimental fact that registration of excimer fluorescence is
observed only in the case of compound 1а. Molecular modeling also
showed the transition of dyes from a sandwich structure to a linear
structure as a result of thermal fluctuations. Future calculation analysis
of the bisstyryl dyes organization in solution is presented in ESI, part 7,
Figs S13, S14.
analysis of the change in the binding energy of the dye with DNA with
time obtained in the performed calculations is shown in Fig. S16 in ESI.
From the data obtained, it can be concluded that the interaction energy
of 1a, 1b and 2 in the linear form with DNA is 10ꢀ 15 kcal/mol lower,
even taking into account the fact that the transition from the sandwich
form to the linear one requires an energy of 8ꢀ 10 kcal/mol. This is due
to the fact that, in the case of a sandwich, the area of contact with the
DNA surface is two times less, which leads to a significant decrease in the
Coulomb and Van der Waals interactions and the nonpolar component of
the solvation energy.
To analyze the formation of aggregates in the minor groove of DNA,
calculations of the DNA complex formed in the presence of 50 molecules
1a and 1b were performed. Modeling for dye 2 was not performed, since,
according to experimental data, the aggregation process for this dye is
insignificant. The structure of aggregates in the groove of DNA formed
from dyes in a sandwich form is shown in Fig. 9. For compound 1a, the
most probable arrangement of molecules is along a narrow groove in
such a way that the pyridine site of one sandwich is adjacent to the aryl
fragment of the other. Aggregates formed from 1b fit into a minor groove
perpendicular to the DNA surface (Fig. 9). This type of molecular
packing provides a tighter packing and, consequently, a stronger hy-
drophobic interaction of molecules with the DNA groove.
Modeling of compounds 1a, 1b and 2 with DNA was carried out in
two stages. The sequence 5’-AACCGGTTACGTACGT-3 ’was used to
build a DNA model. First, a docking procedure was carried out in order
to establish the probable geometry of the dye on the DNA surface.
Fig. 10 shows the calculated structures of dimeric aggregates formed
from dyes 1a and 1b in the minor groove of DNA. Fig. S18 in ESI presents
graphs characterizing the change in the mutual arrangement of styryl
dyes 1a and 1b in dimeric aggregates. As parameters, we used the dis-
tance between the centers of mass of the pyridine and benzene frag-
ments, and the angle between the vectors connecting the centers of mass
of the pyridine and benzene fragments related to the molecules located
one above the other. An analysis of the parameter values shows that the
arrangement of the styryl fragments of two molecules one above the
other is stable. The overlap occurs at an acute angle, the value of which
in case 1b is less than in case 1a. The shift of the styryl fragment relative
to the other along the minor groove is more pronounced in case 1a.
4. Conclusion
Fig. 8. Conformations of 1a, 1b and 2 obtained as a result of quantum me-
chanical optimization. Atoms are colored as follows: carbons - yellow, nitrogen
- blue, oxygen - red. Hydrogen is not shown.
The investigation of mechanism of the bis(styryl) dye – ct-DNA
interaction has been done applying of three different bis(styryl)
9

M.A. Ustimova et al.
Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113378
Fig. 9. Conformations, starting and obtained at the last step of MD calculations, of complexes DNA with 1a, 1b and 2 (initial dyes in sandwich form).
Fig. 10. Conformations, starting and obtained at the last step of MD calculations, of complexes DNA with 1a, 1b and 2 (initial dyes 1b and 2 in linear form, dye 1a as
H-dimer).
10

M.A. Ustimova et al.
Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113378
pyridinium dyes possessing OMe or/and NMe2 substituents in phenyl
ring by using of UV–vis and fluorescence spectroscopic techniques, cir-
cular dichroism (CD), Hoechst 33258 displacement experiments and
quantum-chemical calculations. According to MD calculations the initial
dyes can exist in water solution in linear and intramolecular sandwich
forms. The interaction with DNA can occur with both forms of dyes. For
all studied dyes the two step interaction with DNA has been found. At
high dye concentration the binding of a pair of dyes in the minor groove
forces the groove to widen, making it energetically favorable for a sec-
ond pair of dyes to bind adjacent to the first dimer along the long axis of
the minor groove. According to the MD calculations the formation of
aggregates can occurs from the dyes as in sandwich H-dimer as well as
linear forms. The formation of dye aggregates was confirmed by
experimental methods, but the obtained experimental data do not allow
making an unambiguous conclusion about what type of aggregation
(“folded” or “extended”) is observed for the dyes under study. Cooper-
ative assembly of multiple dyes in DNA minor groove results in the
formation of helical dye aggregates that exhibit large circular dichroism
(CD) signals. In case of bis(styryl) dye 1a containing OMe substituent in
phenyl ring, the obtained results indicate the formation of aggregates of
right-handed chirality. For bis(styryl) dye 1b the left-handed helical
assembly of the aggregating chromophores was revealed. We assumed
that the difference in aggregate chirality could be due to the fact that
formation of 1a aggregates occurs from “extended” dye form, whereas,
dye 1b forms the aggregates in “sandwich” form. Dye 2 composed from
OMe- and NMe2-containing chromophores is able to form the aggregates
in less extend if compare with dyes 1a and 1b. The formation of ag-
gregates in solution or in the body of DNA has not been found for
monostyryl dyes 5a, 5b. This is consistent with the lack of literature data
on the formation of aggregates of monostyryl dyes in the presence of
DNA.
Acknowledgments
Financial support from the Russian Foundation for Basic Research
◦
project N 19-03-00535 (synthesis and optical studies), Russian Scien-
◦
tific Foundation N 19-73-20187 (CD spectroscopy studies and MD
calculations) and equipment facilities from Center of collective facilities
of A.N. Nesmeyanov Institute of Organoelement compounds of Russian
Ministry of Sciences and High Education are gratefully acknowledged.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jphotochem.2021.
113378.
References
[1] S. Mukamel, D.S. Chemla, Preface, Chem. Phys. 210 (1996) vii–viii, https://doi.
org/10.1016/s0301-0104(96)90047-6.
[2] A. Pawlik, S. Kirstein, U. De Rossi, S. Daehne, Structural conditions for spontaneous
generation of optical activity in J-aggregates, J. Phys. Chem. B 101 (1997)
5646–5651, https://doi.org/10.1021/jp9708308.
[3] A. Kurutos, O. Ryzhova, V. Trusova, U. Tarabara, G. Gorbenko, N. Gadjev,
T. Deligeorgiev, Novel asymmetric monomethine cyanine dyes derived from
sulfobetaine benzothiazolium moiety as potential fluorescent dyes for non-covalent
labeling of DNA, Dyes Pigm. 130 (2016) 122–128, https://doi.org/10.1016/j.
dyepig.2016.03.021.
[4] H. Hilal, J.A. Taylor, Cyanine dyes for the detection of double stranded DNA,
J. Biochem. Biophys. Methods 70 (2008) 1104–1108, https://doi.org/10.1016/j.
jprot.2007.12.008.
[5] T.Y. Ogul’chansky, M.Y. Losytskyy, V.B. Kovalska, V.M. Yashchuk, S.M. Yarmoluk,
Interactions of cyanine dyes with nucleic acids. XXIV. Aggregation of monomethine
cyanine dyes in presence of DNA and its manifestation in absorption and
fluorescence spectra, Spectrochim. Acta - Part A Mol. Biomol. Spectrosc. 57 (2001)
1525–1532, https://doi.org/10.1016/S1386-1425(01)00437-1.
[6] B.A. Armitage, Cyanine dye-DNA interactions: intercalation, groove binding, and
aggregation, Top. Curr. Chem. 253 (2005) 55–76, https://doi.org/10.1007/
b100442.
The fluorescence response upon binding of dyes to DNA strongly
depends on the nature of the substituents in the dye molecules. Thus,
dyes 1b, 2 and 5b containing NMe2 substituents demonstrate an increase
in fluorescence quantum yield up to 56 times upon binding to DNA.
Whereas for dyes 1a and 5a containing OMe groups, the increase in the
fluorescence quantum yield upon binding to DNA does not exceed 3.1
times.
[7] A.L. Mikheikin, A.L. Zhuze, A.S. Zasedatelev, Binding of symmetrical cyanine dyes
into the DNA minor groove, J. Biomol. Struct. Dyn. 18 (2000) 59–72, https://doi.
org/10.1080/07391102.2000.10506647.
[8] R. Cao, C.F. Venezia, B.A. Armitage, Investigation of DNA binding modes for a
symmetrical cyanine dye trication: effect of DNA sequence and structure,
J. Biomol. Struct. Dyn. 18 (2001) 844–856, https://doi.org/10.1080/
07391102.2001.10506712.
In free and bound with DNA dye 2 the effective resonance energy
transfer was proved between OMe- and NMe2-containing chromophores.
Such chromophoric pair provides the system with large Stoke’s shift. It
absorbs at 408 nm and emits at 615 nm.
[9] H.J. Karlsson, P. Lincoln, G. Westman, Synthesis and DNA binding studies of a new
asymmetric cyanine dye binding in the minor groove of [poly(dA-dT)]2, Bioorg.
Med. Chem. Lett. 11 (2003) 1035–1040, https://doi.org/10.1016/S0968-0896(02)
00463-7.
[10] H.J. Karlsson, M. Eriksson, E. Perzon, B. Åkerman, P. Lincoln, G. Westman, Groove-
binding unsymmetrical cyanine dyes for staining of DNA: syntheses and
characterization of the DNA-binding, Nucleic Acids Res. 31 (2003) 6227–6234,
https://doi.org/10.1093/nar/gkg821.
The study of bis(styryl) dye-DNA interaction reveals what structural
fragments in composition of dye provides the desirable mode of com-
plex, the investigation could be very important for designing an
improved imaging and therapeutic agents.
[11] J.G. Pelton, D.E. Wemmer, Structural modeling of the distamycin A-d
(CGCGAATTCGCG)2Complex using 2D NMR and molecular mechanics,
Biochemistry 27 (1988) 8088–8096, https://doi.org/10.1021/bi00421a018.
[12] J.G. Pelton, D.E. Wemmer, Structural characterization of a 2:1 distamycin A ⋅ d
(CGCAAATTGGC) complex by two-dimensional NMR, Proc. Natl. Acad. Sci. U. S. A.
86 (1989) 5723–5727, https://doi.org/10.1073/pnas.86.15.5723.
[13] X. Chen, B. Ramakrishnan, S.T. Rao, M. Sundaralingam, Binding of two distamycin
a molecules in the minor groove of an alternating b-DNA duplex, Nat. Struct. Biol.
1 (1994) 169–175, https://doi.org/10.1038/nsb0394-169.
Statement
Maria A. Ustimova: Investigation, Validate, Visualization, Writing-
Original draft preparation. Yury V. Fedorov: Methodology, Formal
analysis, Writing- Reviewing and Editing. Vladimir B. Tsvetkov:
Investigation, Methodology, Formal analysis, Validate, Writing- Orig-
inal draft, Visualization. Sergey D. Tokarev: Investigation, Validate.
Nikolai A. Shepel: Investigation. Olga A. Fedorova: Methodology,
Formal analysis, Conceptualization, Writing- Original draft preparation,
Writing- Reviewing and Editing.
[14] X. Chen, B. Ramakrishnan, M. Sundaralingam, Crystal structures of the side-by-side
binding of distamycin to AT-containing DNA octamers d(ICITACIC) and d
(ICATATIC), J. Mol. Biol. 267 (1997) 1157–1170, https://doi.org/10.1006/
jmbi.1997.0941.
[15] D. Suh, J.B. Chaires, Criteria for the mode of binding of DNA binding agents,
Bioorg. Med. Chem. Lett. 3 (1995) 723–728, https://doi.org/10.1016/0968-0896
(95)00053-J.
[16] M.Q. Wang, S. Liu, C.P. Tang, A. Raza, S. Li, L.X. Gao, J. Sun, S.P. Guo, Flexible
amine-functionalized triphenylamine derivative as a fluorescent “light-up” probe
for G-quadruplex DNA, Dyes Pigm. 136 (2017) 78–84, https://doi.org/10.1016/j.
dyepig.2016.08.041.
Declaration of Competing Interest
[17] A.O. Balanda, K.D. Volkova, V.B. Kovalska, M.Y. Losytskyy, V.P. Tokar, V.
M. Prokopets, S.M. Yarmoluk, Synthesis and spectral-luminescent studies of novel
4-oxo-4,6,7,8-tetrahydropyrrolo[1,2-a]thieno[2,3-d]pyrimidinium styryls as
fluorescent dyes for biomolecules detection, Dyes Pigm. 75 (2007) 25–31, https://
doi.org/10.1016/j.dyepig.2006.05.010.
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.
[18] V.B. Kovalska, M.Y. Losytskyy, D.V. Kryvorotenko, A.O. Balanda, V.P. Tokar, S.
M. Yarmoluk, Synthesis of novel fluorescent styryl dyes based on the imidazo[1,2-
a]pyridinium chromophore and their spectral-fluorescent properties in the
11

M.A. Ustimova et al.
Journal of Photochemistry & Photobiology, A: Chemistry 418 (2021) 113378
presence of nucleic acids and proteins, Dyes Pigm. 68 (2006) 39–45, https://doi.
[39] R. Abagyan, M. Totrov, Biased probability Monte Carlo conformational searches
and electrostatic calculations for peptides and proteins, J. Mol. Biol. 235 (1994)
983–1002, https://doi.org/10.1006/jmbi.1994.1052.
org/10.1016/j.dyepig.2004.12.010.
[19] S. Udayan, V.K. Ramachandran, M. Sebastian, P. Chandran, V.P.N. Nampoori,
S. Thomas, Effect of DNA-CTMA complex on optical properties of LDS 821 dye,
Opt. Mater. (Amst.) 69 (2017) 49–53, https://doi.org/10.1016/j.
optmat.2017.04.016.
[40] M.J.D. Powell, Restart procedures for the conjugate gradient method, Math.
Program. 12 (1977) 241–254, https://doi.org/10.1007/BF01593790.
[41] M. Clark, R.D. Cramer, N. Van Opdenbosch, Validation of the general purpose
tripos 5.2 force field, J. Comput. Chem. 10 (1989) 982–1012, https://doi.org/
10.1002/jcc.540100804.
[20] N. Nizomov, E.N. Kurtaliev, S.N. Nizamov, G. Khodjayev, Spectral-luminescent
study of the interaction of some styrylcyanine dyes with bovine serum albumin and
DNA in aqueous solutions, J. Mol. Struct. 936 (2009) 199–205, https://doi.org/
10.1016/j.molstruc.2009.07.040.
[42] D.A. Case, I.Y. Ben-Shalom, S.R. Brozell, D.S. Cerutti, T.E. Cheatham III, V.W.
D. Cruzeiro, T.A. Darden, R.E. Duke, D. Ghoreishi, M.K. Gilson, H. Gohlke, A.
W. Goetz, D. Greene, R. Harris, N. Homeyer, Y. Huang, S. Izadi, A. Kovalenko,
T. Kurtzman, T.S. Lee, S. LeGrand, P. Li, C. Lin, J. Liu, T. Luchko, R. Luo, D.
J. Mermelstein, K.M. Merz, Y. Miao, G. Monard, C. Nguyen, H. Nguyen, I. Omelyan,
A. Onufriev, F. Pan, R. Qi, D.R. Roe, A. Roitberg, C. Sagui, S. Schott-Verdugo,
J. Shen, C.L. Simmerling, J. Smith, R. Salomon-Ferrer, J. Swails, R.C. Walker,
J. Wang, H. Wei, R.M. Wolf, X. Wu, L. Xiao, D.M. York, P.A. Kollman, Amber 2018,
University of California, San Francisco, 2018.
[21] V.B. Kovalska, I.O. Kocheshev, D.V. Kryvorotenko, A. Balanda, S.M. Yarmoluk,
Studies on the spectral-luminescent properties of the novel homodimer styryl dyes
in complexes with DNA, J. Fluoresc. 15 (2005) 215–219, https://doi.org/10.1007/
s10895-005-2620-5.
[22] C. Gao, S.Y. Liu, X. Zhang, Y.K. Liu, C. De Qiao, Z.E. Liu, Two-photon fluorescence
and fluorescence imaging of two styryl heterocyclic dyes combined with DNA,
Spectrochim. Acta - Part A Mol. Biomol. Spectrosc. 156 (2016) 1–8, https://doi.
org/10.1016/j.saa.2015.11.014.
[43] S. Izadi, A.V. Onufriev, Accuracy limit of rigid 3-point water models, J. Chem.
Phys. 145 (2016) 74501, https://doi.org/10.1063/1.4960175.
[23] V.P. Tokar, M.Y. Losytskyy, T.Y. Ohulchanskyy, D.V. Kryvorotenko, V.B. Kovalska,
A.O. Balanda, I.M. Dmytruk, V.M. Prokopets, S.M. Yarmoluk, V.M. Yashchuk,
Styryl dyes as two-photon excited fluorescent probes for DNA detection and two-
photon laser scanning fluorescence microscopy of living cells, J. Fluoresc. 20
(2010) 865–872, https://doi.org/10.1007/s10895-010-0630-4.
ˇ
´
ˇ
[44] M. Zgarbova, F.J. Luque, J. Sponer, T.E. Cheatham, M. Otyepka, P. Jurecka,
Toward improved description of DNA backbone: revisiting epsilon and zeta torsion
force field parameters, J. Chem. Theory Comput. 9 (2013) 2339–2354, https://doi.
org/10.1021/ct400154j.
ˇ
´
[24] R. Tropcheva, N. Lesev, S. Danova, S. Stoitsova, S. Kaloyanova, Novel cyanine dyes
and homodimeric styryl dyes as fluorescent probes for assessment of lactic acid
bacteria cell viability, J. Photochem. Photobiol. B Biol. 143 (2015) 120–129,
https://doi.org/10.1016/j.jphotobiol.2015.01.002.
[45] M. Zgarbova, J. Sponer, M. Otyepka, T.E. Cheatham, R. Galindo-Murillo,
ˇ
P. Jurecka, Refinement of the sugar-phosphate backbone torsion Beta for AMBER
force fields improves the description of z- and B-DNA, J. Chem. Theory Comput. 11
(2015) 5723–5736, https://doi.org/10.1021/acs.jctc.5b00716.
[25] N. Akbay, M.Y. Losytskyy, V.B. Kovalska, A.O. Balanda, S.M. Yarmoluk, The
mechanism of benzothiazole styrylcyanine dyes binding with dsDNA: studies by
spectral-luminescent methods, J. Fluoresc. 18 (2008) 139–147, https://doi.org/
10.1007/s10895-007-0252-7.
[46] A. Onufriev, D.A. Case, D. Bashford, Effective born radii in the generalized Born
approximation: the importance of being perfect, J. Comput. Chem. 23 (2002)
1297–1304, https://doi.org/10.1002/jcc.10126.
[47] L. Martinez, R. Andrade, E.G. Birgin, J.M. Martínez, PACKMOL: A package for
building initial configurations for molecular dynamics simulations, J. Comput.
Chem. 30 (2009) 2157–2164, https://doi.org/10.1002/jcc.21224.
[26] D.V. Berdnikova, N.I. Sosnin, O.A. Fedorova, H. Ihmels, Governing the DNA-
binding mode of styryl dyes by the length of their alkyl substituents-from
intercalation to major groove binding, Org. Biomol. Chem. 16 (2018) 545–554,
https://doi.org/10.1039/c7ob02736b.
[48] S.H. Li, X. Xu, Y. Zhou, Q. Zhao, Y. Liu, Reversibly tunable white-light emissions of
styrylpyridiniums with cucurbiturils in aqueous solution, Org. Lett. 19 (2017)
6650–6653, https://doi.org/10.1021/acs.orglett.7b03377.
[27] A.Y. Lebedeva, O.A. Fedorova, V.B. Tsvetkov, V.Y. Grinberg, N.V. Grinberg, T.
V. Burova, A.S. Dubovik, K.K. Babievsky, Y.V. Fedorov, Novel 18-crown-6-ether
containing mono- and bisstyryl dyes derived from pyridine moiety as fluorescent
dyes for non-covalent interaction with DNA, Dyes Pigm. 157 (2018) 80–92,
https://doi.org/10.1016/j.dyepig.2018.04.038.
[49] Y. Huang, T. Cheng, F. Li, C.H. Huang, T. Hou, A. Yu, X. Zhao, X. Xu, Photophysical
studies on the mono- and dichromophoric hemicyanine dyes I. Photoelectric
conversion from the dye modified ITO electrodes, J. Phys. Chem. B 106 (2002)
10020–10030, https://doi.org/10.1021/jp020876n.
[28] S. Brasselet, F. Cherioux, P. Audebert, J. Zyss, New octupolar star-shaped strucures
for quadratic nonlinear optics, Chem. Mater. 11 (1999) 1915–1920, https://doi.
org/10.1021/cm990093n.
[50] E.V. Tulyakova, O.A. Fedorova, Y.V. Fedorov, G. Jonusauskas, A.V. Anisimov,
Effect of arrangement of the styryl fragment on the optical properties and
complexation of mono-and bis(styryl)-substituted N-methylpyridinium
perchlorates containing benzo-15-crown-5 ether moieties, Russ. Chem. Bull. 56
(2007) 2166–2174, https://doi.org/10.1007/s11172-007-0341-4.
[29] M.F. Pepitone, G.G. Jernigan, J.S. Melinger, O.K. Kim, Synthesis and
characterization of donor-acceptor chromophores for unidirectional electron
transfer, Org. Lett. 9 (2007) 801–804, https://doi.org/10.1021/ol063000y.
[30] A.S. Brown, L.M. Bernal, T.L. Micotto, E.L. Smith, J.N. Wilson, Fluorescent
neuroactive probes based on stilbazolium dyes, Org. Biomol. Chem. 9 (2011)
2142–2148, https://doi.org/10.1039/c0ob00849d.
[51] E.V. Tulyakova, O.A. Fedorova, Y.V. Fedorov, G. Jonusauskas, A.V. Anisimov,
Spectroscopic study of mono- and bis(styryl) dyes of the pyridinium series
containing azathiacrown ether residue, J. Phys. Org. Chem. 21 (2008) 372–380,
https://doi.org/10.1002/poc.1349.
¨
[31] R. Abagyan, M. Totrov, D. Kuznetsov, ICM—A new method for protein modeling
and design: applications to docking and structure prediction from the distorted
native conformation, J. Comput. Chem. 15 (1994) 488–506, https://doi.org/
10.1002/jcc.540150503.
[52] A. Olaya-Castro, G.D. Scholes, Energy transfer from Forster-Dexter theory to
quantum coherent light-harvesting, Int. Rev. Phys. Chem. 30 (2011) 49–77,
https://doi.org/10.1080/0144235X.2010.537060.
[53] D. Sahoo, P. Bhattacharya, S. Chakravorti, Quest for mode of binding of 2-(4-
(dimethylamino)styryl)-1-methylpyridinium iodide with calf thymus DNA, J. Phys.
Chem. B 114 (2010) 2044–2050, https://doi.org/10.1021/jp910766q.
[54] M.A. Ustimova, P.A. Chernikova, N.E. Shepel, Y.V. Fedorov, O.A. Fedorova, Effect
of N-substituent in 4-styrylpyridinium dyes on their binding to DNA, Mendeleev
Commun. 30 (2020) 217–219, https://doi.org/10.1016/j.mencom.2020.03.029.
[32] T.A. Halgren, Merck molecular force field. I. Basis, form, scope, parameterization,
and performance of MMFF94, J. Comput. Chem. 17 (1996) 490–519, https://doi.
org/10.1002/(SICI)1096-987X(199604)17:5/6<490::AID-JCC1>3.0.CO;2-P.
[33] C. Møller, M.S. Plesset, Note on an approximation treatment for many-electron
systems, Phys. Rev. 46 (1934) 618–622, https://doi.org/10.1103/PhysRev.46.618.
[34] V. Barone, M. Cossi, Quantum calculation of molecular energies and energy
gradients in solution by a conductor solvent model, J. Phys. Chem. A 102 (1998)
1995–2001, https://doi.org/10.1021/jp9716997.
´
[55] Alison Rodger, Bengt Norden Circular Dichroism and Linear Dichroism, Oxford
University Press, New York, 1997 ch. 2.
´
[56] M. Eriksson, B. Norden, Linear and circular dichroism of drug-nucleic acid
[35] U.C. Singh, P.A. Kollman, An approach to computing electrostatic charges for
molecules, J. Comput. Chem. 5 (1984) 129–145, https://doi.org/10.1002/
jcc.540050204.
complexes, Methods Enzymol. 340 (2001) 68–98, https://doi.org/10.1016/S0076-
6879(01)40418-6.
´
[57] B. Norden, F. Tjerneld, Structure of methylene blue–DNA complexes studied by
[36] C.I. Bayly, P. Cieplak, W.D. Cornell, P.A. Kollman, A well-behaved electrostatic
potential based method using charge restraints for deriving atomic charges: the
RESP model, J. Phys. Chem. 97 (1993) 10269–10280, https://doi.org/10.1021/
j100142a004.
linear and circular dichroism spectroscopy, Biopolymers 21 (1982) 1713–1734,
https://doi.org/10.1002/bip.360210904.
´
[58] H.K. Kim, J.M. Kim, S.K. Kim, A. Rodger, B. Norden, Interactions of intercalative
and minor groove binding ligands with triplex poly(dA)⋅[poly(dT)]2 and with
duplex poly(dA)⋅poly(dT) and poly[d(A-T)]2 studied by CD, LD, and normal
absorption, Biochemistry 35 (1996) 1187–1194, https://doi.org/10.1021/
bi951913m.
[37] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman,
G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato,
X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada,
M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda,
O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro,
M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi,
J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi,
M. Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken,
C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin,
R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski,
´
[59] B. Norden, T. Kurucsev, Analysing DNA complexes by circular and linear
dichroism, J. Mol. Recognit. 7 (1994) 141–155, https://doi.org/10.1002/
jmr.300070211.
[60] A. Rodger, S. Taylor, G. Adlam, I.S. Blagbrough, I.S. Haworth, Multiple DNA
binding modes of anthracene-9-carbonyl-N1-spermine, Bioorg. Med. Chem. Lett. 3
(1995) 861–872, https://doi.org/10.1016/0968-0896(95)00086-V.
[61] J.L. Seifert, R.E. Connor, S.A. Kushon, M. Wang, B.A. Armitage, Spontaneous
assembly of helical cyanine dye aggregates on DNA nanotemplates, J. Am. Chem.
Soc. 121 (1999) 2987–2995, https://doi.org/10.1021/ja984279j.
[62] K. Nakanishi, N. Berova, R.W. Woody, Circular Dichroism: Principles and
Applications, VCH Publishers, New York, 1994.
¨
G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas, J.
B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian 09, Revision A.1,
Gaussian, Inc., Allingford CT, 2009.
[38] Y.A. Arnautova, A. Jagielska, H.A. Scheraga, A new force field (ECEPP-05) for
peptides, proteins, and organic molecules, J. Phys. Chem. B 110 (2006)
5025–5044, https://doi.org/10.1021/jp054994x.
´
[63] S.K. Kim, B. Norden, Methyl green, FEBS Lett. 315 (1993) 61–64, https://doi.org/
10.1016/0014-5793(93)81133-K.
[64] S. Neidle, DNA minor-groove recognition by small molecules, Nat. Prod. Rep. 18
(2001) 291–309, https://doi.org/10.1039/a705982e.
12