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Experimental and quantum chemical study оn the DNA/protein binding and the biological activity of a rhodium(iii) complex with 1,2,4-triazole as an inert ligand.
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Živanovi, R. Puchta, D. Cocic, A. Scheurer, N. N. Milivojevi and J. Bogojeski, Dalton Trans., 2020, DOI:
10.1039/D0DT01343A.
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CDNan o- O dHuM eg tetlM ea llrs u U o r Wc N eyn .I cy CSllit A ce c Tp act hI ai O o erbn n N e s en ( teX a sH l.t +a, bXil i=z eSd, Spea,r Teen)t sulfenyl, selenenyl, or omissions in this Accepted Manuscript or any consequences arising
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Page 1 of 53 Dalton Transactions
Experimental and quantum chemical study оn the DNA/protein
binding, and the biological activity of rhodium(III) complex with
1,2,4-triazole as inert ligand
t
p
i
Angelina Petrović,a Marko Živanović,e Ralph Puchta,b,c,d Dušan Ćoćić,a Andreas Scheurer,b r
c
s
Nevena Milivojevic e and Jovana Bogojeski*a
u
n
a
M
a University of Kragujevac, Faculty of Science, Radoja Domanovića 12, 34000
d
Kragujevac, Serbia e
t
p
b Inorganic Chemistry, Department of Chemistry and Pharmacy, University of
e
c
Erlangen-Nürnberg, 91058 Erlangen, Germany c
A
c Computer Chemistry Center, Department of Chemistry and Pharmacy, University of
s
n
Erlangen-Nürnberg, 91058 Erlangen, Germany o
i
t
d ZISC (Zentralinstitut für Scientific Computing), Universität Erlangen-Nürnberg, c
a
s
Martensstrasse 5a, 91058 Erlangen, Germany
n
a
e University of Kragujevac, Institute of Information Technologies Kragujevac, Jovana
r
T
Cvijića bb, 34000 Kragujevac, Serbia
n
o
t
l
a
*Corresponding author:
D
Dr. Jovana Bogojeski
Department of Chemistry
Faculty of Science
University of Kragujevac
Radoja Domanovića 12
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Tel: +381(0)34336223
Fax: +381 (0)34 335040
e-mail: jovana.bogojeski@pmf.kg.ac.rs
t
p
i
r
c
Abstract s
u
n
a
Synthesis and structural characterization of a newly synthesized mononuclear rhodium(III) M
complex-Rhtrz, with a ligand (2,2,6-bis((4S,7R)-7,8,8-trimethyl-4,5,6,7-tetrahydro-1H-4,7- d
e
methanoindazol-3-yl)pyridine) and ligand of 1,2,4-triazole, are presented within this paper. t
p
e
Kinetic interactions of Rhtrz complex with essential biomolecules such as 5-GMP, L-Met,
c
c
and GSH were examined. The study of the biological interactions was focused on the binding
A
properties of the complex Rhtrz with CT-DNA and serum albumin. Interactions were
s
n
investigated using UV-vis spectrophotometry, fluorescence spectroscopy, viscosity
o
i
measurements, thermal denaturation studies, and electrophoresis. Fluorescence competition t
c
a
experiments with site-markers for BSA were used to locate the binding site of Rhtrz-complex
s
n
to the BSA. Molecular docking studies were carried out to obtain detailed binding information
a
r
of the complex with CT-DNA, BSA, and HSA. T
n
Furthermore, the apparent distance between the donor (HSA) and acceptor (Rhtrz) was
o
t
determined using fluorescence resonance energy transfer (FRET). Thermodynamic properties
l
a
and relative stabilities of the Rhtrz complex were examined, constructing the two model D
equation by DFT calculations (B3LYP(CPCM)/LANL2DZp). In vitro cytotoxicity and redox
status on human epithelial colorectal cancer cell line (HCT-116) and healthy human fibroblast
cell line (MRC-5) were also investigated.
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Keywords
Rh(III) complex ∙ DNA interactions ∙ Albumin interactions ∙ Binding site markers ∙
t
p
Docking study ∙ Cytotoxic activity ∙ Redox status i
r
c
s
u
n
Introduction
a
M
d
Disease ranked second in the world by the number of deceased, based on the WHO report, is
e
t
cancer. For this reason, there is a constant need to find a cure that will successfully treat the p
e
disease without causing a large number of unwanted side effects. Transitional metals have taken c
c
a significant place in cancer therapy, after the successful use of cisplatin in the treatment for a A
substantial number of carcinoma.1 However, even though cisplatin was introduced in cancer s
n
therapy several decades ago, only a few other analogs have been used in treatment so far. In o
i
t
addition to the Pt(II) complexes, various ruthenium complexes have been intensively studied.2-5 c
a
Although they have proven to be significantly more efficient than the platinum complexes, no s
n
Ru complex has passed clinical testing and is entered into use as cytostatic. a
r
T
A large number of different metal complexes have been investigated worldwide, aiming to find
n
a drug that will effectively affect the carcinoma cells. For the last few years, researchers' interest o
t
has focused on metals like rhodium, osmium, and iridium.6-10 In the 1970s began the first l
a
D
interest in rhodium complexes as potential anticancer drugs with the attention focused on
binuclear rhodium(II) complexes.11 Rhodium complexes as potential anticancer agents were
not an attractive topic of research because of their kinetic inertness. However, some unreactive
compounds were known as active anticancer agents.12 Research has progressed over time to
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mononuclear species containing Rh(I) and Rh(III) ions.11 Recent studies with rhodium
complexes showed surprising anticancer activity, and they are seen as promising candidates for
the development of the anticancer drugs. Many studies have suggested that when talking about
anticancer drugs, the primary intracellular target is the DNA molecule.12,13 Metallo complexes
that can interact with the DNA molecule under physiological conditions attract increasing t
p
i
attention as possible metal-based anticancer drugs. Metallo complexes interact with the DNA r
c
s
through intercalation, groove binding, and electrostatic interactions.14 Except with the DNA,
u
n
metallo complexes can also strongly interact with proteins. At first, it was believed that for
a
M
ascertaining the anticancer effect of metallo complexes, it was only relevant to determine the
d
binding prosperities of the metallo complexes with DNA. However, later it was shown that the
e
t
binding of the metal complexes to the protein molecules represents an essential part of the p
e
pharmacokinetics and pharmacodynamics of possible anticancer drugs.15,16 The non-covalent
c
c
binding is the principal binding mode in the interactions between the proteins and the anticancer A
drugs. For the development of anticancer drugs, both DNA and protein interactions are equally s
n
important.17 o
i
t
The inertness of these metallo complexes that was first seen as a drawback, has now proven to c
a
be important for designing complexes with a specific target for DNA, alongside proteins and s
n
enzymes.6,7 Also, the combination of ligands and the complexes' coordination geometry have a a
r
T
substantial effect on the reactivities, binding preferences, and cellular uptake of these
n
complexes.6,7
o
t
The purpose of this study is the development of structure-activity relationships relevant to the l
a
D
design of promising medicinal agents. The primary goal is finding the correlation between
structural, thermodynamic, and kinetic properties of Rhtrz complex. Therefore, the synthesis,
characterization, mechanisms of reactions with biologically relevant molecules, as well as in
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vitro cytotoxicity and redox status on human epithelial colorectal cancer cell line (HCT-116)
and healthy human fibroblast cell line (MRC-5) were also investigated.
t
p
i
r
c
s
u
n
a
M
RESULTS AND DISCUSSION
d
e
t
Synthesis and characterization/Preparation and structure of [Rh(H 2 L*)(1,2,4- p
e
triazole) Cl]Cl (Rhtrz)
2 2 c
c
A
Complex [Rh(H L*)(1,2,4-triazole) Cl]Cl (Rhtrz) (Figure 1.) was synthesized by stirring one s
2 2 2
n
equivalent of RhCl •xH O, one equivalent H L* (2,2,6-bis((4S,7R)-7,8,8-trimethyl-4,5,6,7- o
3 2 2
i
t
tetrahydro-1H-4,7-methanoindazol-3yl)pyridine), and two equivalents of 1,2,4-triazole c
a
ligands in ethanol and refluxing overnight. The newly synthesized Rhtrz complex was s
n
characterized by 1H and 13C NMR spectroscopy, elemental analysis, IR, Uv-Vis and ESI-MS a
r
T
mass spectrometry (see Figures S1-S5).
n
o
t
l
a
D
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NH
X
N
N
N Rh Cl Cl 2 X N NH
N
X
NH 1,2,4-triazole
t
p
i
r
c
[Rh(H L*)(1,2,4-triazole) Cl]Cl s
2 2 2
u
n
Fig. 1. Structure of the investigated Rhtrz complex.
a
M
d
Elemental analyses on this complex was in very good agreement with a complex composition
e
t
of [Rh(H L*)(1,2,4-triazole) Cl]Cl . The 1H NMR, as well as the 13C NMR spectra of the Rhtrz
2 2 2 p
e
complex, indicated that only this distinct species is formed. The obtained spectra display a set
c
c
of signals for the pyrazole moieties, pyridine moiety, and camphor moiety, signals are A
significantly shifted, compared to the signals of the free ligand, as well as a signal of 1,2,4- s
n
triazole. Further, the complex was characterized by ESI-MS mass spectrometry, the m/z range o
i
t
of 200−700 included prominent peaks at m/z = 352.26 (2+), which represents fragments of the c
a
Rhtrz complex (see Supporting Information, Figure S3). s
n
a
r
T
Kinetic studies of the complex formation of Rhtrz
n
o
t
The UV-vis spectrophotometry was used to examine the kinetic reaction of substitution of the l
a
D
labile chloride ligand. Change in the absorbance (see SI Figure S6) was followed as a function
of time, and under physiological conditions: pH=7.2 (25 mM HEPES buffer/30 mM NaCl (to
suppress the solvolytic pathway) at 310 K. Biologically important nucleophiles (L-Met, GSH,
and 5′-GMP) were chosen based on their different binding prosperities, steric hindrance, and
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their nucleophilicity. With the nucleophile concentrations being at least 10-fold in excess, these
reactions were performed under pseudo-first-order conditions. Scheme 1 shows the proposed
reaction pathways for all observed substitution processes.
k t
2 p
[Rh(H L*)(1.2.4-triazole) Cl]+ + L [Rh(H L*)(1.2.4-triazole) L]+ + Cl-
2 2 2 2
k i
1 r
c
L=L-Met, GSH, 5'-GMP s
u
n
a
Scheme 1. Schematic representation of substitution reactions of complex Rhtrz with M
nucleophiles: L-Met, GSH, and 5'-GMP. d
e
t
p
e
c
The direct nucleophilic attack proceeds in a reversible manner, as shown in Scheme 1.
c
A
Substitution rate constants were determined under pseudo-first-order conditions by plotting the
s
n
linear dependence of k vs. total nucleophile concentration (see equation 1). All kinetic data
obsd
o
i
t
are summarized in Tables S1–S3 (see Supporting Information, SI).
c
a
s
n
k obsd = k 2 [Nucleophile] + k 1 [Cl-] (1) a
r
T
n
The direct nucleophilic attack is characterized by constant rate k , and the reverse reactions are
2 o
t
represented by constant rate k . The second-order constant rate k characterizes product l
1 2 a
D
formation and is evaluated from the slope of a plot k vs. nucleophile concentration.
obsd
Experimental results for the displacement of a chloride ion from the Rhtrz complex are shown
in Tables 1 and S4. Representative plots are shown in Figure 2.
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0.0035
k /s-1
obsd GSH
0.003
0.0025 L-Met
0.002
t
p
0.0015
i
r
c
0.001
s
u
0.0005
n
5'-GMP
a
[nucleophile]/M
0 M
0 0.0005 0.001 0.0015 0.002 0.0025
d
e
Fig. 2. k vs. nucleophile concentration for the reaction of Rhtrz complex with t
obsd p
e
5’-GMP, L-Met, and GSH; pH = 7.2 and 310 K in 25 mM HEPES and 30 mM NaCl.
c
c
A
Table 1. The constant rates of the substitution reactions of the RhIII complexes with L-Met, s
n
5'-GMP, and GSH at pH = 7.2 (25 mM Hepes buffer) in the presence of 30 mM NaCl. o
i
t
c
a
5’-GMP L-Met GSH ref.
s
n
101k 101k 101k
2 2 2
a
r
M–1s–1 M–1s–1 M–1s–1
T
Rhtrz 0.3 ± 0.1 9.4 ± 0.1 13 ± 0.1 this work n
o
[RhIII(H L*)Cl ] (1a) 27 ± 0.1 2.4 ± 0.1 4.0 ± 0.1 18 t
2 3 l
a
[RhIII(Me L*)Cl ] (1b) 56 ± 0.1 3.8 ± 0.2 19 ± 0.1 18 D
2 3
Results in Table 1. indicate that the Rhtrz complex undergoes the process of substitution of
the coordinated chloride ligand with the selected 5'-GMP, L-Met, and GSH as nucleophiles.
The substitution reaction occurs most rapidly when the nucleophile is GSH, slightly slower with
L-Met and about ten times slower with 5'-GMP as a nucleophile. It can be noted that the
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investigated complex reacts much slower with 5'-GMP than the previously studied structurally
similar complexes [RhIII(H L*)Cl ] (1a) and [RhIII(Me L*)Cl ] (1b), while with GSH reacts at
2 3 2 3
a similar speed.18 It is unusual that the complex Rhtrz reacts faster with L-Met (2-3 times) than
complexes 1a and 1b, which indicates an influence of both steric and electronic effects on the
speed of substitution. t
p
i
The thermodynamic properties and relative stabilities of the RhIII complexes were examined r
c
s
constructing two model equation (Equation 2 and 3), based on the ligand
u
n
2,6-bis((4S,7R)-7,8,8-trimethyl-4,5,6,7-tetrahydro-1H-4,7-methomoindazol-3-yl)pyridine
a
M
(H L*). Equation 2 targets the relative stability of complexes based on H L* (see Fig. 3a)
2 2
d
compared to the analogue terpy complexes. In contrast, equation 3 is designed to evaluate the
e
t
preferred coordination of the monodentate ligands Gua, S(CH 3 ) 2 , HSCH 3 in comparison to p
e
monoanionic Cl- -anion. To avoid the spoiling influence of the Coulomb term, equation 2 and
c
c
3 is designed to compare isomeric systems. Thence the non-coordinated ligand was calculated A
in the second coordination sphere, do keep the charge and size of the system identical. s
n
(see. Fig. 3b). To get an indication of the influence of solvents, we applied for both equations o
i
t
the common and widely used implicit solvent model CPCM. c
a
s
n
2+ 3+ 3+ 2+ a
N NH N NH N NH N NH
r
NH NH T
N N N N N N N N
N Rh Cl + N Rh L N Rh L + N Rh Cl n
N N N N N N N N o
NH NH
t
N NH N NH N NH N NH l
a
L:Gua,S(CH ) ,HSCH
32 3 D
Equation 2. Model equation to evaluate the relative stability of [Rh(H L*)(1,2,4-
2
triazole) Cl]2+vs. [Rh(terpy)(1,2,4-triazole) Cl]2+.
2 2
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2+ 3+
N NH N NH
NH NH
N N L N N Cl-
N Rh Cl N Rh L
N N N N
NH NH
N NH N NH
t
p
L:Gua,S(CH ) ,HSCH
3 2 3 i
r
c
Equation 3. Model equation to evaluate the relative stability of the isomeric systems
s
u
{[Rh(H L*)(1,2,4-triazole) Cl]L}2+vs. {[Rh(H L*)(1,2,4-triazole) L]Cl}2+ .
2 2 2 2 n
a
M
d
e
t
p
e
c
c
A
s
n
o
i
t
c
a
s
n
a
r
T
n
o
t
l
a) a
D
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t
p
i
r
c
s
u
n
a
M
d
e
t
p
b) e
c
Fig. 3. Calculated structures (B3LYP/LANL2DZp) of a) [Rh(H L*)(1,2,4-triazole) Cl]2+and c
2 2
A
b) {[Rh(H L*)(1,2,4-triazole) (SCH ) ]Cl}2+.
2 2 3 2
s
n
o
Table 2. Calculated relative stabilities for the model Equation 2 and 3. i
t
c
[kcal/mol] equation 2 equation 3 a
s
n
B3LYP B3LYP
a
ligand (L) B3LYP B3LYP
r
(CPCM) (CPCM)
T
HSCH -14.83 -5.09 48.47 24.57 n
3
o
S(CH ) -14.12 -5.49 45.87 22.02 t
3 2
l
a
Gua -15.51 -4.87 40.72 22.72 D
aB3LYP: RB3LYP/LANL2DZp + ZPE(B3LYP/LANL2DZp) B3LYP(CPCM):
RB3LYP(CPCM)/LANL2DZp // RB3LYP/ LANL2DZp + ZPE(B3LYP/LANL2DZp).
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As demonstrated by our equation 2 and 3 (see Table 2. and Figure 3), independent if we compare
the gas phase or the CPCM model is applied, generally the terpy based RhIII-complex is favored
if the monoanionic chloride ligand is coordinated and the neutral ligand L binds to the
[Rh(H L*)(1,2,4-triazole) ]3+-fragment. As the model reaction’s energy for all three ligands L
2 2
(Gua, S(CH ) , HSCH ) is significantly lowered to 30 % by applying the CPCM solvent model, t
3 2 3 p
i
we tend to attribute this results mainly to the one-third bigger ligand 2,6-bis((4S,7R)-7,8,8- r
c
s
trimethyl-4,5,6,7-tetrahydro-1H-4,7-methomoindazol-3-yl)pyridine (H L*) compared to
2
u
n
traditional terpy ligand. The larger quantity of non-hydrogen atoms in H L* allows a better
2
a
M
stabilization of the overall positive charges in [Rh(H L*)(1,2,4-triazole) Cl]2+ (see Fig. 3a).
2 2
d
Equation 3 allows a more detailed investigation of the preferred coordination mode of the RhIII-
e
t
center. This equation (with and without CPCM) (see Table 2) stresses stabilization by charge p
e
diminution at the metal center, because here the systems with the coordinated monoanionic
c
c
chlorido ligand {[Rh(H2L*)(1,2,4-triazole) 2 Cl]L}2+ and the neutral ligand L in the second A
coordination sphere is always much more stable than the isomeric complex {[Rh(H2L*)(1,2,4- s
n
triazole) L]Cl}2+ with the coordinated neutral ligand L and the Cl- ion the second coordination o
2
i
t
sphere (see Fig. 3b). c
a
On a first glance, these thermodynamic findings seem to contradict the kinetic experiment, but s
n
one has to keep in mind that the concentrations of the nucleophile were least 10-fold in excess, a
r
T
and the solvent was water, that can add giant stabilization to the leaving Chloride ion by explicit
n
hydrogen bonds, we had to discount in our model equation.
o
t
l
a
D
DNA-binding studies
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To further examine the reactivity of the Rhtrz complex towards potential biological targets,
interactions with CT-DNA were performed using the UV-vis spectrophotometry, fluorescence
quenching, viscosity measurements, and thermal denaturation studies.
Electronic absorption method t
p
i
r
c
s
One of the most commonly used methods for monitoring the way metallo complexes bond to
u
n
the DNA is electronic absorption spectroscopy. It is known that transition metal-complexes
a
M
bind to the DNA fragments via covalent and/or non-covalent interactions.19 Potential
d
interactions between the examined metallo complex and the DNA molecule, can be revealed
e
t
by following the changes in the absorption spectra of the complex after the addition of DNA, p
e
when applying different [complex]/[DNA] molar ratios. The absorption intensity of the
c
c
examined complex can either decrease (hypohromatic shift) or grow (hyperchromic shift) with A
a slight change in absorption wavelength maximum after the DNA addition. Spectral changes s
n
shown in Figure 4, were obtained by recording the UV-vis spectra of the Rhtrz complex o
i
t
(constant concentrations, 8 μM; PBS) in the absence and presence of different concentrations c
a
of the CT-DNA solution (0-40 μM). On the spectrum shown in Figure 4, s
n
a hyperchromic effect and a displacement of the absorption maximum at a wavelength of 258 a
r
T
nm, i.e., red shift, can be observed.
n
o
t
l
a
D
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0.3
0.2
0.1
0
220 270 320 370 420 470
14
ecnabrosbA
6
5
4
3
2
1
0 0 0.5 1 1.5 2 2.5 3 3.5 4
λ (nm)
mc2M
)fε-aε(/]AND[9-01
Dalton Transactions Page 14 of 53
t
p
105[DNA] i
r
c
s
u
n
a
M
d
e
t
Fig. 4. Absorption spectra of the Rhtrz complex in 10 mM PBS after the addition of DNA. p
e
[complex] = 8 μM, [DNK] = 0-40 μM. The arrow indicates a change in the absorbance with c
c
increasing DNA concentration. A
s
n
o
The binding constant, K , is determined by monitoring the change in the absorbance at the
b
i
t
corresponding wavelength after the addition of growing concentration of the DNA solution, c
a
s
based on the following equation (4):
n
a
r
T
[DNA]/(ε – ε) = [DNA]/(ε – ε) + 1/[K (ε – ε)] (4)
A f b f b b f
n
o
t
l
K is calculated from the slope and the y-intercept ratio [DNA] / (εA - εf) = f ([DNA]) a
b
D
(Figure 4), where [DNA] is DNA concentration, ε = A /[complex], ε is the extensional
A obsd f
coefficient of the uncoordinated complex, and ε is the extinction coefficient of the coordinated
b
complex. The values obtained for the constant K are shown in Table 3.
b
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Typical behavior of a classical electrostatic interaction is the hyperchromism and blue shift of
the absorption bands of the complexes and DNA (in many cases, they may be located at similar
wavelengths).20 However, in our examined system, we recognize hyperchromism and red shift.
It is noted that the investigated complex interacts moderately with the molecule of DNA with a
constant order of 104, which may be indicative of interaction with DNA through a mode that t
p
i
involves a stacking interaction of the aromatic chromophore and the base pairs of the DNA. r
c
s
Investigated complex shows less affinity for the interaction with the DNA molecule compared
u
n
to structurally similar RhIII complexes, Table 3.
a
M
d
Table 3. DNA binding constants (K ) and Stern-Volmer constants (K ) for the examined
b sv
e
t
complex and structurally similar complexes Rh(III). p
e
c
c
CT-DNA Ref. A
K [M–1] K [M–1] s
b sv
n
o
Rhtrz (2.5 ± 0.1) × 104 (1.3 ± 0.1) × 104 this work
i
t
c
[RhIII(H L*)Cl ] (1a) (5.0 ± 0.1) × 104 (5.0 ± 0.1) × 104 18
2 3 a
s
[RhIII(Me L*)Cl ] (1b) (8.3 ± 0.1) × 104 (5.5 ± 0.1) × 104 18 n
2 3
a
[RhIII(terpy)Cl ] (1c) (7.0 ± 0.1) × 104 (3.8 ± 0.1) × 104 18 r
3
T
[RhIII(H 2 LtBu)Cl 3 ] (9.7 ± 0.1) × 104 (1.9 ± 0.1) × 104 21 n
o
t
l
a
D
Fluorescence spectroscopic methods
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Ethidium bromide - EB (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide) intensely
fluoresces in the presence of a DNA molecule, and if excited at 527 nm shows an intense
fluorescence emission at 612 nm. This intense fluorescence emission is a result of the
intercalation of the planar EB ring between the pairs of the DNA bases. However, a decrease in
the fluorescence intensity of the EB-DNA par will happen in the presence of a molecule t
p
i
(metallo complex) that has the ability to replace EB from the EB-DNA pair.22,23 The resulting r
c
s
reduction in fluorescence of the DNA-EB pair can be used to determine the extent and method
u
n
of the interaction for that molecule to DNA. The relative binding of complexes to CT-DNA
a
M
was determined by calculating the quenching constant (K ) from the slopes of straight lines
sv
d
obtained from the Stern-Volmer equation (equation 5).
e
t
p
e
I /I = 1+ K [Q] (5)
0 sv c
c
A
I and I represent emission intensities in the absence and presence of quencher (complex s
0
n
Rhtrz), [Q] is the total concentration of the quencher, K is the Stern-Volmer quenching o
sv
i
t
constant, which was obtained from the slope of the plot of I /I vs. [Q]. c
0
a
Based on the absorption spectroscopic measurements, it has been established that there is a s
n
certain degree of interaction between the examined complex and the DNA molecule. a
r
T
Fluorescence measurements were subsequently made to confirm the type of interaction between
n
the DNA molecule and the examined complex. In Figure S7, a dependence of the intensity of
o
t
EB-DNA emission in the absence and presence of the Rhtrz complex in the function of the l
a
D
wavelength is presented. Adding the solution of the Rhtrz complex in rising concentrations
leads to a significant reduction in the emission intensity to about 610 nm, indicating the
competition between the complex and the EB in binding to the DNA (Figure S7).24,25 This
decrease in the intensity of fluorescence of the DNA-EB complex indicates that the investigated
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complex can suppress EB from the EB-DNA complex, i.e., that the Rhtrz complex shows an
affinity for intercalation with the DNA molecule.
The data obtained from the fluorescence measurement can be used to determine the number of
the binding sites for the complex to the DNA molecule, n, as well as the equilibrium binding
constant, K , based on the Scatchard25 equation, t
bin p
i
r
c
s
log(I -I)/I = logK + nlog[Q] (6)
0 bin
u
n
a
M
The number of binding sites (n), and the binding constant (K ) were calculated from the plot
bin
d
of log(I -I)/I vs. log[Q], and the obtained values are shown in Table 3 and Figure S8. For the
0
e
t
investigated complex, a value of n = 1.03 was obtained, indicating that there was one binding p
e
site for the CT-DNA complex. Constant K has a value of 1.3x104 M-1, which indicates that
bin c
c
the complex A
moderately intercalates between the CT-DNA strands. When comparing the obtained values s
n
for the investigated complex with structurally similar complexes (Table 3), it can be observed o
i
t
that Rhtrz interacts with lower intensity, which may be due to steric disturbances. In Table 3, c
a
it can be noted that there is a good agreement between the constants obtained by the absorption s
n
and fluorimetric method. a
r
T
n
Determination of viscosity
o
t
l
In order to define the method of interaction of the examined Rhtrz complex with the DNA a
D
molecule as accurately as possible, the viscosity of the DNA solution was measured.
Measurements were performed in the presence and absence of rising concentrations of the
Rhtrz complex (Figure 5).
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2.8
(η/η )1/3
0
Rhtrz
2.6
1a
2.4 1b
2.2
t
2
p
1.8 i
r
c
1.6
s
u
1.4
n
1.2 a
r = [Complex]/[DNA]
M
1
0 0.2 0.4 0.6 0.8 1
d
e
Fig. 5. Relative viscosity (η/η )1/3 of the DNA solution in 10 mM PBS in the presence of the t
0
p
e
complex Rhtrz of different concentrations (r). (1a = [RhIII(H L*)Cl ]; 1b = [RhIII(Me L*)Cl ]-
2 3 2 3
c
c
ref. 8)
A
s
n
With the change in the length of the DNA chain, the viscosity of the DNA solution changes. In
o
i
the absence of crystalline structure data, viscosity is considered to be an important data in the t
c
a
identification of binding patterns for the DNA in a solution.27,28 Adding rising concentrations
s
n
(up to r = 1.0) of the Rhtrz complex in a DNA solution (0.01 mM) led to a moderate increase
a
r
in the relative viscosity of the DNA. In the case of classical intercalation, T
n
the compound is inserted between the pairs of DNA bases, which leads to an increase in the
o
t
length of the DNA chain and, therefore, the viscosity of the DNA solution. The intercalation
l
a
strength is usually proportional to the increase in the viscosity of the DNA. Based on the data D
shown in Figure 5, it can be noted that the investigated Rhtrz complex intercalates weakly
between DNA strands compared to the structurally similar complexes 1a = [RhIII(H L*)Cl ];
2 3
1b = [RhIII(Me L*)Cl ],28 which agrees with the results obtained by fluorescence measurements.
2 3
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Thermal denaturation studies
Thermal DNA denaturation studies were performed to better understand the nature of the
complex–DNA interaction. These studies show if the examined complex intercalates in the
DNA-helix, using the change in the melting temperature value. If the melting temperature value
t
of the DNA solution increases in the presents of the examined complex compared to the melting
p
i
temperature of DNA solution alone, it indicates that the type of interaction between the r
c
s
examined complex and the DNA is intercalation. No change in the value means that
u
n
intercalation is not the type of interaction between the examined complex and the DNA
a
M
molecule.26 The values of DNA melting temperature (Tm) were calculated from the derivative
d
plot (dA /dT vs. T) of the melting profile, Figure S9. The melting temperature of CT-DNA
260 e
t
(Tm) alone was lower (348.12 K) than in the presence of Rhtrz (359.15 K). An increase in the p
e
melting temperature of CT-DNA is due to the intercalation of the Rhtrz in the DNA-helix. c
c
Results obtained using thermal denaturation studies are in agreement with the results obtained A
s
using the fluorescents spectroscopy and the viscosity measurements.
n
o
i
t
Protein binding studies c
a
s
n
Fluorescence spectroscopy of BSA a
r
T
Albumin is the most abundant protein in the bloodstream. The most important role of albumin
n
in the human organism is the transport of various matters through the blood plasma to the organs o
t
l
and tissues. Albumin transports hormones, fatty acids, ions, drugs, etc. It is, therefore, important
a
D
to examine the interaction between serum albumin proteins (BSA) and transitional metal
complexes as potential drugs. The interaction of the newly synthesized Rhtrz complex and
serum albumin (BSA and HSA) was examined using fluorescence spectroscopy.
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The BSA solution was excited at 295 nm, and at this wavelength, fluorescence comes from
tryptophan residues.29 By adding the Rhtrz complex to the BSA solution, an increase in
fluorescence appears at about λ = 367 nm, as shown in Figure S10b. Fluorescence increase after
the addition of the complex to the BSA solution can indicate that the Rhtrz complex fluoresces
in the selected wavelength bandwidth (Figure S11), so a series of the growing complex t
p
i
concentration without the presence of BSA were recorded, Figure S10a. After correcting the r
c
s
obtained values of the fluorescence of the BSA solution for the fluorescence of the complex,
u
n
the graph shown in Figure 6 was obtained. The values of the Stern-Volmer constant (K ) for
sv
a
M
the interactions of the Rhtrz complex with serum albumin were determined using the Stern-
d
Volmer equation (5), where I is the initial fluorescence intensity of tryptophan in serum
0
e
t
albumin, I is the intensity of fluorescence of tryptophan in serum albumin after the addition of p
e
Rhtrz complex, and [Q] is the concentration of the complex. K can be calculated from the
sv c
c
linear dependence of I 0 /I in relation to [Q] (Figure 6). The values of the K sv constant for the A
interaction of the studied complex with serum albumin are given in Table 4. s
n
o
i
t
c
a
s
n
a
r
T
n
o
t
l
a
D
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1.14
I /I
0
1.12
1.1
200 I (a.u.)
1.08
180
160
1.06 140
120
t
100 p
1.04
80
60 i
r
1.02 40
c
20
1 0 313 340 367 394 421 448λ (nm) s
105[Q]M u
0.98 n
0 2 4 6 8 10 a
M
d
e
Fig. 6. The dependence of I /I on the concentration [Q] (Q = complex), where the experimental
0 t
p
points are denoted by (■), and the full lines represent a linear dependence. Embedded graph: e
c
Emission spectra of serum albumin in the presence of the Rhtrz complex [serum albumin] = 2 c
A
μM, [complex] = 0-10 μM, λex = 295 nm. Arrows show changes in intensity after adding
s
n
solutions of the growing concentration complex
o
i
t
c
The resulting decrease in fluorescence can be attributed to the changes in the tertiary structure a
s
of the protein, as a result of changes in the environment of tryptophan in serum albumin, due to n
a
the binding of the complex to protein.30 r
T
Based on the obtained K values shown in Table 4, it can be observed that there is an interaction n
sv
o
between the Rhtrz complex and the BSA. By comparing the value of the K constant for the
sv t
l
a
examined complex, with the values for structurally similar complexes ([RhIII(H L*)Cl ] = (3.5
2 3 D
± 0.1) × 104 M-1; [RhIII(Me L*)Cl ](1б) = (3.9 ± 0.1) × 104 M-1)18 it can be noted that the
2 3
investigated Rhtrz complex interacts with BSA with about two times lower K constant. This
sv
may be due to steric disturbances of the additional 1,2,4-triazole molecules.
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Data obtained from the fluorescence measurements was used to determine the number of
binding sites, n, as well as the equilibrium binding constant, K , based on the Scatchard25,
bin
equation 6, and the obtained values, are shown in Table 4, and Figure S12. For n, the value of
1.28 is obtained, indicating that there is one binding site for the examined complex on the BSA
molecule. t
p
i
r
c
s
Competitive experiments with BSA and site markers
u
n
Serum albumins are heart-shaped and comprise of three (I-III) domains, each comprising two
a
M
subdomains (A and B).31 Binding sites for metallo complexes are located at subdomain IIA and
d
subdomain IIIA of the albumin molecule.32 To locate if the newly synthesized Rhtrz complex
e
t
binds at the site I and/or II of the BSA molecule, competitive experiments with site markers p
e
were performed. The fluorescence titration methods were used to identify the binding location
c
c
on the BSA molecule, with eosin Y as a marker for the site I of the subdomain IIA, and A
ibuprofen as a marker for site II of the subdomain IIIA.33 The excitation wavelength for the s
n
fluorescence titration was 295 nm, with the emission range of 300-500 nm. BSA and the o
i
t
markers were added in equimolar concentrations (2x106 M), and the Rhtrz complex was then c
a
gradually added in rising concertation. The changes in the emission of the Rhtrz complex with s
n
BSA in the absence and presence of the markers separately, were monitored. After correcting a
r
T
the obtained values from the fluorescence of the BSA- site marker solution in the presence of
n
the increasing complex concentration, for the fluorescence of the complex, Figure S13 was
o
t
obtained. l
a
D
The decrease in fluorescence intensity of the BSA solution, after the addition of the site marker,
indicates that the site marker molecule has bounded to BSA. If the complex binds to the same
site as the corresponding marker, when added to the site marker-BSA system, the complex must
compete with the marker in order to bind to BSA, which would lead to a significant change in
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the constant value. The emission quenching data were analyzed according to the Scatchard
equation25, equation 6. In Table 4. it can be seen that the binding constant is mostly affected in
the presence of the site marker - eosin Y.
Table 4. Values of K for the complex-BSA system in the presence and absence of the site t
bin p
i
markers (eosin Y and ibuprofen) with values for R and number of the binding sites-n r
c
s
u
n
K [M–1] N R
bin
a
M
Complex-BSA (1.67 ± 0.1) x106 1.28 0.95
d
Complex-BSA-Ibuprofen
e
t
(5.08 ± 0.1) x104 0.97 0.90 p
e
Complex-BSA-Eosin Y c
c
(1.98 ± 0.1) x103 0.67 0.97 A
s
n
o
Results obtained in the BSA study showed that the Rhtrz complex has one binding site on the
i
t
c
BSA molecule, meaning that the complex can bind either to site I or site II, but not both. As
a
s
shown in Table 4, the binding constant is remarkably affected in the presence of eosin Y, while
n
a
a slighter change is noted in the presence of ibuprofen. Results indicate that the binding of the
r
T
examined complex should be mainly located within the site I of BSA, and that the complex has
n
to compete with eosin Y in order to bind to BSA. However, the change in the constant value in o
t
l
the presence of ibuprofen, could not be excluded, and for this reason, the docking measurements a
D
were performed. Docking measurements confirmed that the complex has binding preferences
for the site I of the BSA molecule. Taking all of this into consideration, it is believed that the
Rhtrz complex binds to the site I of the BSA molecule driven by the hydrophobic interaction.
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However, the role of other possible interactions such as hydrogen bonds, electrostatic, van der
Waals interactions, and steric contacts cannot be entirely ruled out.
Fluorescence spectroscopy of HSA
Fluorescence spectroscopy measurements for the interaction of the newly synthesized Rhtrz t
p
i
complex and human serum albumin (HSA) were also made. The HSA solution was excited at r
c
s
295 nm, and at this wavelength, fluorescence is caused only by tryptophan.31 An increase in
u
n
fluorescence after the addition of the Rhtrz complex solution to the HSA solution appears
a
M
because Rhtrz complex fluoresces in the selected wavelength. Solutions of the growing
d
concentration of the complex without the presence of HSA were recorded for this reason. After
e
t
correcting the obtained values from the fluorescence of the HSA solution in the presence of the p
e
increasing complex concentration, for the fluorescence of the complex (similar to the BSA
c
c
corrections), the graph shown in Figure S14 was obtained. The resulting decrease in A
fluorescence can be attributed to the changes in the tertiary structure of the protein, and it s
n
indicates that there is an interaction between the Rhtrz complex and the HSA. In Figure 7, a o
i
t
graphical representation of the obtained values of K constants for the tested complex for c
sv
a
interaction with DNA and serum albumin is given. s
n
a
r
T
n
o
t
l
a
D
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t
p
i
r
c
s
u
n
a
M
Fig. 7. Graphical representation of the obtained values of the K constants for the interaction of
sv
d
the examined complex Rhtrz with CT-DNA, BSA, and HSA. e
t
p
e
c
Based on all of the values obtained in this paper, it can be noticed that the investigated Rhtrz
c
A
complex interacts moderately with both the DNA molecule and serum albumin. Showing a
s
higher affinity for the interaction with the serum albumin, Figure 7, Tables 3, and 4. n
o
i
t
c
Energy transfer efficiency between HSA and Rhtrz complex
a
s
Fluorescence resonance energy transfer (FRET) represents a non-destructive spectroscopic n
a
method that can give us information about relative orientation and the distance of a donor (Trp
r
T
214, D) and an acceptor fluorophore (Rhtrz complex, A). According to the Förster theory,
n
o
efficiency of energy transfer between a donor and acceptor E, the average distance between
t
l
a
them r and the critical distance for a 50% energy transfer R were calculated using following
0
D
equations34 (7,8):
E = R 6/( R 6+r6) = 1-I/I (7)
0 0 0
R 6 = 8.8 x 1025K2n-4Ф J (8)
0 D
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Where I and I are fluorescence intensities of the donor in a presence and absence of acceptor,
0
respectively. Biochemically, orientation space factor K2=2/3, refracted index of the medium n
= 1.33, and the fluorescence quantum yield of a donor Ф =0.118. The resonance transfer is
D
more efficient when the spectral overlap of the donor emission and acceptor absorption is large, t
p
i
Figure 8. r
c
s
u
n
a
M
d
e
t
p
e
c
c
A
s
n
o
i
t
c
a
s
n
a
r
Fig. 8. The overlap of the absorption spectrum of a complex Rhtrz and fluorescence spectrum T
n
of HSA. The ration of Rhtrz complex and HSA is 1:1.
o
t
l
a
D
The effect of the spectral overlap between the emission of the donor and the absorption
spectrum of the acceptor J was calculated by the following equation35 (9):
𝐽=∫ ∞ 𝐼(𝜆)𝜀(𝜆)𝜆4 ∫𝐼(𝜆)𝑑(𝜆) (9)
0
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Where I(λ) is a corrected fluorescence intensity of a donor in the wavelength range from λ to
λ+ Δλ, and ε(λ) is an extinction coefficient of an acceptor at λ.
We have calculated the overlap integral (J = 8.71 x 109 nm4/Mcm), the energy transfer
efficiency (E = 0.153), the Förster’s distance at which energy transfer is 50% efficient t
p
i
(R = 5.16 Å), and the D-A distance (r = 6.86 Å). Since the distances between D and A are r
0
c
s
usually in the range 2–8 Å and the obtained r value obeys the condition 0.5R < r < 1.5R ,36 we
0 0
u
n
may conclude that the energy transfer between HSA and Rhtrz complex is a highly feasible
a
M
process.
d
e
t
Molecular docking p
e
Molecular docking simulations were used to test how the investigated complex interacts with
c
c
the DNA and serum albumins (BSA and HSA). In the case of the DNA docking, two possible A
interactions were studied. The predicted top-ranking pose for complex with the lowest energy s
n
was applied for suggesting the best possible geometry of compounds inside the DNA double o
i
t
helix as well as the binding inside for bovine serum albumin cavity. MolDock, Docking, c
a
Rerank, and Hbond scoring functions were used for the assessment of complex-DNA/SA- s
n
binding affinity.37 a
r
T
Our complex was docked into the DNA fragments representing either (i) canonical
n
B-DNA (PDB 1BNA) or (ii) DNA with an intercalation gap (PDB 1Z3F). 1BNA is the crystal
o
t
structure of a synthetic DNA dodecamer, while 1Z3F is the crystal structure of a 6 bp DNA l
a
D
fragment in complex with an intercalating anticancer drug, ellipticine. The best-docked poses
of complexes with DNA are displayed in Figure 9 and Figure 10, and top-ranked poses
according to used scoring functions are presented in Table 5.
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t
p
i
r
c
s
u
n
a
M
Fig. 9. Computational docking model illustrating interactions between complex Rhtrz and
d
DNA with the intercalation gap. e
t
p
e
c
c
A
s
n
o
i
t
c
a
s
n
a
r
T
n
o
t
l
a
D
Fig. 10. Computational docking model illustrating interactions between complex Rhtrz and
canonical B-DNA.
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DOI: 10.1039/D0DT01343A
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Table 5. Score values of DNA docking with complex Rhtrz.
PDB ID of DNA Poses: MolDock Rerank Docking
A -180.363 -77.828 -177.239
1Z3F – intercalation gap
B -175.648 -74.556 -173.240
t
p
1BNA – canonical gap C -184.274 -80.091 -181.604
i
r
c
s
Investigated complex exhibits similar affinities to interact with DNA via intercalation and/or
u
n
by minor groove binding, which can be seen from the values presented in Table 5. Intercalation
a
M
into the gap created by elliptic can be performed either by H L* or 1,2,4-triazole moiety of
2
d
Rhtrz complex, in which according to the used scoring functions, the intercalation via H L*
2
e
t
ligand is slightly more preferabale.
p
e
The docking results from the MVD program revealed that the complex Rhtrz binds to the
c
c
subdomain IIA (the site I) of SA proteins, which is consistent with the experimental data by
A
which with the increasing amount of complex, a fluorescence quenching was observed due to s
n
the interaction between the complexes and Trp residue. Interaction results between Rhtrz and o
i
t
SA proteins are illustrated in Figure 11, while top-ranked poses according to the used scoring c
a
functions are presented in Table 6. s
n
a
r
T
n
o
t
l
a
D
29
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DOI: 10.1039/D0DT01343A