G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands

Article abstract of DOI:10.1016/j.jinorgbio.2019.03.021

In this paper, three new Ruthenium(II) polypyridyl complexes containing ascididemin (ASC) as main ligand have been synthesized and characterized. Their interactions with different G-quadruplex (Htelo, c-myc and c-kit) (Htelo: human telomeric DNA, c-myc: cellular-myelocytomatosis viral oncogene, c-kit: oncogene c-kit promoter sequences) and duplex (ds26) DNA sequences were comparatively studied with the free ligand ASC by a series of spectroscopic techniques including UV–vis (ultraviolet-visible) spectroscopy, FID (fluorescent intercalator displacement) assay, and FRET (fluorescence resonance energy transfer) melting assay. Molecular docking studies were also performed to support the binding mode of the compounds with G-quadruplex DNA. Results indicated that [Ru(bpy)₂ASC]·(PF₆)₂ (1), [Ru(phen)₂ASC]·(PF₆)₂ (2), [Ru(tatp)₂ASC]·(PF₆)₂ (3) (bpy = 2,2′?bipyridine, phen = 1,10?phenanthroline, tatp = 1,4,8,9?tetra?aza?triphenylene) and ASC can effectively bind G-quadruplex and duplex DNA and stabilization ability lies in the order 3 > 2 > 1 > ASC. Complex 3 was determined to be the most promising candidate for further in vitro studies and potential anticancer drug.

Full text of DOI:10.1016/j.jinorgbio.2019.03.021

Accepted Manuscript
G-quadruplex and duplex DNA binding studies of novel
Ruthenium(II) complexes containing ascididemin ligands
Maierhaba Wumaier, Jing-Jing Shi, Tian-Ming Yao, Xiao-Chun
Hu, Ru-Ru Gao, Shuo Shi
PII:
S0162-0134(18)30685-8
DOI:
https://doi.org/10.1016/j.jinorgbio.2019.03.021
JIB 10681
Reference:
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
24 November 2018
22 March 2019
27 March 2019
Please cite this article as: M. Wumaier, J.-J. Shi, T.-M. Yao, et al., G-quadruplex and
duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin
ligands,
Journal
of
Inorganic
Biochemistry,
https://doi.org/10.1016/
j.jinorgbio.2019.03.021
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ACCEPTED MANUSCRIPT
G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes
containing ascididemin ligands
a
b
a
a
Maierhaba Wumaier, Jing-Jing Shi,* Tian-Ming Yao,* Xiao-Chun Hu, Ru-Ru
a
a
Gao, Shuo Shi*
a
Shanghai Key Laboratory of Chemical Assessment and Sustainability, School of
Chemical Science and Engineering, Tongji University, Shanghai, 200092 , P.R.
China
b
VARI/SIMM Center, Center for Structure and Function of Drug Targets, CAS-
Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, P.R. China
Corresponding author:
Shuo Shi, E-mail: shishuo@tongji.edu.cn.
Tian-Ming Yao, E-mail: tmyao@tongji.edu.cn.
Jing-Jing Shi, E-mail: shijingjing@simm.ac.cn
1

ACCEPTED MANUSCRIPT
G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
Abstract
In this paper, three new Ruthenium(II) polypyridyl complexes containing ascididemin
(ASC) as main ligand have been synthesized and characterized. Their interactions with
different G-quadruplex (Htelo, c-myc and c-kit) (Htelo: human telomeric DNA, c-myc:
cellular-myelocytomatosis viral oncogene, c-kit: oncogene c-kit promoter sequences)
and duplex (ds26) DNA sequences were comparatively studied with the free ligand
ASC by a series of spectroscopic techniques including UV-vis (ultraviolet-visible)
spectroscopy, FID (fluorescent intercalator displacement) assay, and FRET
(fluorescence resonance energy transfer) melting assay. Molecular docking studies were
also performed to support the binding mode of the compounds with G-quadruplex DNA.
Results indicated that [Ru(bpy) ASC]·(PF6)2 (1), [Ru(phen) ASC]·(PF ) (2),
2
2
6 2
[
Ru(tatp) ASC]·(PF ) (3) (bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline, tatp =
2 6 2
1
,4,8,9-tetra-aza-triphenylene) and ASC can effectively bind G-quadruplex and duplex
DNA and stabilization ability lies in the order 3 > 2 > 1 > ASC. Complex 3 was
determined to be the most promising candidate for further in vitro studies and potential
anticancer drug.
Keywords: Ruthenium complexes, G-quadruplex, duplex DNA, ascididemin
2

ACCEPTED MANUSCRIPT
G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
1
. Introduction
Metallodrugs plays an important role in cancer treatment [1]. The well-known metal
based chemotherapy drug is cisplatin. But its severe side effects and drug resistance
limited its application [2]. In order to overcome the drawbacks of currently used
chemotherapy and find more clinically efficient anticancer drugs, several classes of
alternative metal based compounds were developed [1]. Among these metal complexes,
ruthenium (Ru) complexes stood out, especially because of its physiologically
accessible oxidation states and lower toxicity toward healthy tissue probably due to the
ability of Ru to mimicking iron in binding to biomolecules [3-5]. A number of Ru
complexes showed promising anticancer activities and three of them, NAMI-A
(
[
[
[
imidazolium-trans-DMSO-imidazole-tetrachlororuthenate), KP1019 {indazolium trans-
tetrachlorobis(1H-indazole)ruthenate(III)]} and (N)KP1339 {sodium trans-
tetrachloridobis(1H-indazole)ruthenate(III)]} have successfully entered in clinical trials
6-8].
Binding of small molecules with DNA has been studied extensively since DNA is the
material of inherence and controls the structure and function of cells [9,10]. In this
respect Ru(II) complexes have attracted a great deal of attention due to their strong
DNA-binding and potential anticancer activities [11-14]. Furthermore, DNA is highly
polymorphic and can adopt a variety of different helical conformations [15,16] (B [17],
A [18], and Z [19]) as well as unusual structures (hairpins [20], cruciform [21], triplexes
[
22], i-motif [23] and G-quadruplexes [24]). It has been suggested that these secondary
DNA structures could be involved in the regulation of several key biological processes.
Among these different non-canonical DNA motifs, DNA G-quadruplex are probably the
3

ACCEPTED MANUSCRIPT
G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
most extensively studied [25,26]. Such structures are made up of G-quartet subunits,
where four coplanar guanines (G) are linked together by Hoogsteen hydrogen bonds
[
27-31]. G-quadruplex structures are highly dynamic and polymorphic too, which can
fold into different topologies such as, parallel, antiparallel and hybrid depending on the
base sequences, loop connectives and cations [32]. During the last decade, G-
quadruplex has emerged from being a structural curiosity observed in vitro, to being
recognized as a possible nucleic acid based mechanism for regulating multiple
biological processes in vivo [33,34]. There is great evidence that G-quadruplexes play
an overarching role in biology. G-quadruplexes are found throughout the human
genome [35] and in mRNA [36], especially in gene promoters and telomeres. For
example, human telomeric DNAs are made up of double stranded TTAGGG repeats and
ends 3' single stranded overhang [37-40]. The folding of the single stranded 3' overhang
into G-quadruplex makes the telomerase lose substrate, inhibits telomerase activity
(which is up regulated more than 85% cancer cells), and prevents telomere extension,
thus induces tumor cells senescence and apoptosis [40-44]. Owing to the strong
relations between telomerase and cancers, great efforts have been devoted to develop
the anticancer strategy based on small molecules that could target and stabilize G-
quadruplex structure, with the aim of inhibit telomerase and induce cancer cell death. In
addition, G-quadruplex formation in oncogene promoter regions such as c-kit (oncogene
c-kit promoter sequences), c-myc (cellular-myelocytomatosis viral oncogene), bcl-2 (b-
cell CLL/lymphoma 2) and K-ras (kirsten rat sarcoma viral oncogene) has been
proposed to function as a transcriptional controller of these genes [45], gene expression
can be suppressed by G-quadruplex stabilizing ligands [46].
4

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G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
Ru(II) complexes have been reported to be both duplex and G-quadruplex structure
stabilizers, and potent anticancer agents [11-14]. Barton, Norden, as well as Ji groups
have provided detailed information about recognition and reactions of classical duplex
DNA by Ru complexes [47-50]. Over the past 15 years, a rational approach to design
Ru complexes that can selectively interact with G-quadruplex DNA has emerged. For
example, a series of Ru(II) complexes synthesized by our lab, have shown remarkable
G-quadruplex formation and stabilization ability [51-54]. Rickling et al. studied the
4
+
interactions of the dinuclear [(tap) Ru(tpac)Ru-(tap) ] complex with the human
2
2
telomeric sequence of d(T AG3)4 (tap = 1,4,5,8-tetraazaphenanthrene, tpac =
2
tetrapyridoacridine). They found that the complex can damage these sequences by
intramolecular photocrosslinking [55]. Piraux et al. reported the new Ru(II) complexes
based on a novel dipyrazino [2,3-a:2',3'-h]phenazine ligand (dph), and these complexes
behave as selective probes for G-quadruplex [56]. Besides, a number of Ru based on
anticancer agents targeting G-quadruplex, such as, cis-[RuCl (S-(-)-FOA)(DMSO) ]
2
2
2
+
2+
2+
6
[
57], [Ru(bpy) (icip)] , [Ru-(bpy) (pdppz)]
and [Ru(bpy) (tactp)]
[58], (η -
2
2
2
C H )Ru(p-XPIP)Cl]Cl [59], [(bpy) Ru(bpibp)Ru(bpy) ](ClO ) and [(phen) Ru
6
6
2
2
4 4
2
(
bpibp)Ru(phen) ](ClO ) [60] have been reported too {FOA
=
4-(2,3-
2
4 4
dihydroxypropyl)-formamide oxoaporphine, bpy = 2,2'-bipyridine, icip = 2-(indeno[2,1-
b]chromen-6-yl)-1H-imidazo[4,5-f][1,10]phenanthroline, pdppz = phenanthro[4,5-
abc]dipyrido-[3,2-h:2',3'-j]phenazine, tactp
triphenylene, X = Cl, PIP = 2-phenylimidazole[4, 5f ][1,10]phenanthroline, bpibp = 4,
'-bis (1, 10-phenanthroline-[5, 6-d] imidazole-2-yl)-biphenyl, phen = 1,10-
phenanthroline}.
=
4,5,9,18-tetraazachryseno[9,10-b]-
4
5

ACCEPTED MANUSCRIPT
G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
From the previous studies, ideal G-quadruplex binder should show a high level of
selectivity between quadruplex and duplex DNA when attempting to develop a
powerful anticancer drug. However, anthracyclines daunomycin and doxorubicin that
are two widely used anticancer drugs [61], exhibit non-selective binding properties
between quadruplex and duplex DNA [62], which play an important role in their
chemotherapeutic effects [63]. Experimental evidence suggests that intercalation of an
anthracyclines into DNA duplex structure playing critical roles in suppressing tumor
growth [64]. Besides, these drugs can also interact with telomeric DNA quadruplexes
and induce cancer cell senescence and apoptosis [65]. These results hint some
anticancer drugs might bind both G-quadruplex and duplex DNA.
Ascididemin (ASC) is a pentacyclic marine pyridoacridine alkaloid, which extracts
from the marine animal tunicates and sponges [66-68]. Due to its significant cytotoxic
properties against a series of tumor cell lines, including multidrug resistant cancer cells,
ASC has received great interest [69]. It has been reported that, ASC intercalates into
DNA base pairs, especially at GC-rich sequences and inhibits topoisomerase II enzyme
[
66-69]. Bonnard and co-workers reported that, DNA binding is the likely reason of
ASC cytotoxic activity [69]. We conceive that Ru(II) complexes of ASC ligands would
be a potent G-quadruplex DNA binders, as they would possess a planar aromatic
ligands, which can stack on the flat surface of a terminal G-quartet in quadruplex
structures.
In this study, we have combined three Ru(II) polypyridyl complexes containing N-N-
chelating ligands, such as [Ru(L) Cl ] [L = bpy, phen and tatp (1,4,8,9-tetra-aza-
2
2
triphenylene) with ASC ligands to generate three novel complexes named
2
+
2+
2+
[
Ru(bpy) ASC] (1), [Ru(phen) ASC] (2) and [Ru(tatp) ASC] (3), the synthetic
2 2 2
6

ACCEPTED MANUSCRIPT
G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
route to complexes 1-3 are shown in Scheme 1. In order to evaluate the quadruplex
binding affinity, selectivity and structure-activity relationships of ruthenium complexes
1
-3 and ASC with different DNA structures, a series of spectroscopic analysis and
molecular docking studies have been carried out. In this experiment three different
quadruplex sequences, including Htelo (human telomeric DNA), c-myc and c-kit that all
+
can form G-quadruplex in the presence of K condition and duplex DNA (ds26) were
selected to study.
2
. Experimental section
Materials. All reagents and solvents were obtained from commercial sources and
used without further purification, and Ultrapure MilliQ water was used in all
experiments. DNA oligomers were purchased from Sangon (Shanghai, China).
Concentrations of these oligomers were determined by measuring the absorbance at 260
nm after melting. Single strand extinction coefficients were calculated from
mononucleotide data using a nearest-neighbor approximation [70,71]. The formation of
intramolecular G-quadruplexes were carried out as follows: the oligonucleotide samples
°
dissolved in different buffer solutions, heated to 90 C for 5 min, slowly cooled down to
°
room temperature and then incubated at 4 C overnight. The buffers were prepared as
follows: (A): 10 mM tris-HCl, 100 mM KCl, pH 7.0; (B):10 mM KH PO –K HPO ,
2
4
2
4
°
1
00 mM KCl, pH 7.0. Stock solutions were stored at 4 C and used after no more than 4
+
days. The structure of Htelo is the mixed parallel/antiparallel G-quadruplex in K buffer.
All of the tested compounds were dissolved in DMSO, and the concentration of DMSO
was 1% (v/v).
DNA sequences
Htelo: 5'-AGGGTTAGGGTTAGGGTTAGGG-3'
7

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G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
c-myc: 5'-TGGGGAGGGTGGGGAGGGTGGGGAAGG-3'
c-kit: 5'-AGGGAGGGCGCTGGGAGGAGGG-3'
ds26: 5'-GTTAGCCTAGCTTAAGCTAGGCTAAC-3'
LHtelo: 5'-FAM-AGGGTTAGGGTTAGGGTTAGGG-TAMRA-3'
Lc-myc: 5'-FAM-TGGGGAGGGTGGGGAGGGTGGGGAAGG-TAMRA-3'
Lc-kit: 5'-FAM-GGGAGGGCGCTGGGAGGAGGG-TAMRA-3'
Synthesis and characterization. The compounds quinoline-5,8-dione, 1,10-
phenanthroline-5,6-dione, cis-[Ru(bpy) Cl ]·2H O, cis-[Ru(phen) Cl ]·2H O and cis-
2
2
2
2
2
2
[
Ru(tatp) Cl ]·2H O were synthesized according to the literature procedures [72-75].
2 2 2
The ligand ASC used in this work was prepared according to the literature methods [68].
Ru(bpy) ASC]·(PF ) (1). Under an argon atmosphere, ASC (50.0 mg, 0.177 mmol,
[
2
6 2
1
eq) and cis-[Ru(bpy) Cl ]·2H O (92.0 mg, 0.177 mmol, 1eq) were mixed in
2 2 2
EtOH:H O (6:1, v/v).The reaction mixture was refluxed for 6-8 h. Upon cooling, the
2
reaction mixture was diluted with water and filtered to remove solid impurities. Then,
concentrated aqueous solution of NH PF was added to the filtrate until no more
4
6
precipitate formed. The crude product precipitate filtered, dried. The dried solid product
dissolved in MeCN, purified by silica chromatography eluting with MeCN: H O:NaNO3
2
1
(
40:4:1). This complex was obtained in 70% yield (122.0 mg) as a black solid. H NMR
(400 MHz, MeOD) δ 8.90 – 8.84 (m, 2H), 8.81 (d, J = 6.5 Hz, 1H), 8.74 (dd, J = 15.5,
8
.0 Hz, 4H), 8.53 (d, J = 7.7 Hz, 1H), 8.20 (t, J = 7.7 Hz, 3H), 8.16 – 8.10 (m, 4H), 8.06
t, J = 7.6 Hz, 1H), 7.96 (d, J = 5.6 Hz, 2H), 7.92 (d, J = 5.5 Hz, 1H), 7.88 (d, J = 5.2
Hz, 1H), 7.83 (dd, J = 7.7, 5.8 Hz, 1H), 7.57 (dd, J = 14.0, 7.4 Hz, 2H), 7.44 (dt, J =
(
2+
1
3
2.8, 6.4 Hz, 2H). HRMS (ESI) calcd for C H N ORu 348.5582 ([M] /2), found
38 25 7
2
+
48.5580 ([M] /2).
8

ACCEPTED MANUSCRIPT
G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
[
Ru(phen) ASC]·(PF ) (2). With cis-[Ru(phen) Cl ]·2H O (100.0 mg, 0.177 mmol,
2 6 2 2 2 2
1
eq) in place of cis-[Ru(bpy) Cl ].2H O, this complex was obtained by a procedure
2 2 2
similar to that described for complex 1. This complex was obtained in 65% yield (119.0
1
mg) as a black solid. H NMR (400 MHz, MeOD) δ 8.84 (t, J = 7.7 Hz, 2H), 8.77 – 8.69
(m, 5H), 8.54 (d, J = 8.2 Hz, 1H), 8.45 (t, J = 4.5 Hz, 2H), 8.37 – 8.30 (m, 4H), 8.15 –
8
.03 (m, 6H), 7.86 (m, J = 13.5, 8.2, 5.3 Hz, 2H), 7.77 – 7.70 (m, 3H). HRMS (ESI)
2
+
2+
calcd for C H N ORu 372.5582 ([M] /2), found 372.5602 ([M] /2).
4
2
25
7
[
Ru(tatp) ASC]·(PF ) (3). It was prepared by the same method as above one but
2 6 2
starting from cis-[Ru(tatp) Cl ]·2H O (119.0 mg, 0.177 mmol, 1eq) and ASC (50.0 mg,
2
2
2
0
.177 mmol, 1eq). This complex was obtained in 52% yield (105.0 mg) as a black solid.
1
H NMR (400 MHz, DMSO) δ 9.64 – 9.52 (m, 1H), 9.40 (s, 1H), 8.97 (d, J = 8.2 Hz,
H), 8.83 – 8.75 (m, 1H), 8.58 (d, J = 8.3 Hz, 1H), 8.47 (d, J = 5.4 Hz, 1H), 8.32 (dd, J
1
=
5.3, 1.1 Hz, 1H), 8.26 (dd, J = 5.4, 1.1 Hz, 1H), 8.10 (m, J = 15.4, 13.7, 7.0, 3.3 Hz,
1
H), 7.93 (m, J = 8.2, 6.9, 5.4 Hz, 1H), 7.74 (dd, J = 8.0, 5.6 Hz, 1H). HRMS (ESI)
2
+
2+
calcd for C H N ORu 424.5644 ([M] /2), found 424.5644 ([M] /2).
4
8
25 11
1
Physical measurements. H NMR spectra were recorded on Varian Mercury-Plus
1
4
00 NMR instrument ( H 400 MHz). Abbreviations for data quoted are s, singlet; d,
doublet; t, triplet; dd, doublet of doublets; m, multiplet. Mass spectra were measured on
an agilent TOF-G6230B mass spectrometer. FID (fluorescent intercalator displacement)
measurements were recorded using an F-7000 fluorescence spectrophotometer (Hitachi
Ltd., Japan) in a 1.0 cm path length quartz cuvette. Absorption spectra titrations were
obtained by a UV-Vis (ultraviolet-visible) spectrophotometer (Hitachi U-3900). FRET
(fluorescence resonance energy transfer) melting measurements were carried out on
ABI-7500 real-time PCR (polymerase chain reaction) apparatus. UV-vis and FID
9

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G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
spectrophotometric measurements were carried out in buffer A and FRET melting
measurements were performed in buffer B.
UV-Vis absorption titration. Initially, 3000 μL solutions of the blank buffer and the
Ru complex sample (10 μM) were placed in the reference and sample cuvettes (1.0 cm
path length), respectively, and then the first spectrum was recorded in the range of 200-
6
00 nm. During the titration, an aliquot of buffered DNA solution (100 μM) was added
to each cuvette to eliminate the absorbance of DNA itself, and the solutions were mixed
by repeated inversion. Complex-DNA solutions were incubated for 5 min before
absorption spectra were recorded. The titration processes were repeated until there was
no change in the spectra for four titrations at least, indicating binding saturation had
been achieved. The changes in the metal complex concentration due to dilution at the
end of each titration were negligible.
The intrinsic binding constants K to DNA were determined based on the following
b
equations (1) and (2) [52,76]:
2
2
1/2
(
ε -ε )/(ε -ε )=(b-(b -2K C [DNA]/s) )/2KC (1)
a f b f t t
b=1+KC +K[DNA]/2s
(2)
t
where [DNA] is the concentration of DNA in base pairs, ε , ε , and ε are the apparent
a
f
b
extinction coefficient A /[M], the extinction coefficient for the free metal (M) complex,
abs
and the extinction coefficient for the metal (M) complex in the fully bound form,
−1
respectively; K is the equilibrium binding constant in M , C is the total metal complex
t
concentration, and s is the binding size.
FID assay. To a mixture of the pre-folded G-quadruplex or duplex DNA (1 μM) and
thiazole orange (TO) (2 μM), in a buffer A (100 mM), 10.0 μL corresponding
compounds (200 μM) was added each time. After an equilibration time of 3 min the
1
0

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G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
emission spectrum was recorded between 500 and 650 nm with an excitation
wavelength of 480 nm. The titration processes were repeated until there was no change
in the spectra for at least four titrations, indicating that binding saturation had been
achieved. The percentage of TO displacement is calculated using the following equation:
percentage of displacement = 100 - [(F /F ) × 100], where F is the fluorescence
t
0
t
intensity at each titration point and F is the fluorescence of TO bound to DNA without
0
any added complex as described earlier [70]. FID curves were obtained by plotting
percentage of displacement versus concentration of complex. DC50 represents the
amount of complex required to displace 50% of bound TO from a G-quadruplex.
FRET melting assay. The fluorescent double labeled oligonucleotides (5'-FAM-
[
DNA]-TAMRA-3'), (FAM: carboxyfluorescein, donor fluorophore, TAMRA: 6-
carboxytetramethylrhodamine, acceptor fluorophore), used as the FRET probe was
°
diluted in buffer B and then annealed by heating to 90 C for 5 min, followed by slow
°
cooling to room temperature, kept at 4 C overnight. Double labeled DNAs were
incubated with different concentrations of compounds for 1 h in the dark. The samples
were prepared by aliquoting 10 μL of the annealed DNA (0.25 μM) into each strip,
followed by 10 μL of the compound solutions. Then, the strips were put into a PCR
apparatus, and denaturation of oligonucleotides was performed using the following
°
°
protocol: incubation at 25 C for 2 min, and then increasing temperature to 95 C in
°
0
.5 C increments, and the recording was performed after a 30 s stabilization to ensure a
stable value. Measurements were made in triplicate with an excitation wavelength of
92 nm and emission wavelength of 516 nm, in a volume of 20 μL for each sample.
4
Final analysis of the data was carried out by using Origin Pro 8.5 data analysis. A DNA
competition FRET melting assay was carried out to investigate the selectivity of the
1
1

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G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
compound to the quadruplex. All of the conditions of the experiment were similar to
those used in the FRET melting assay, except that, added various concentrations of ds26
as competitors.
Molecular docking. Electronic structures of the compound were obtained in water
using density functional theory (DFT) calculations. DFT calculations were carried out
using Gaussian 09 by Becke's three parameter hybrid functional with the Lee-Yang-Parr
correlation functional (B3LYP) methods. The geometries of compounds were optimized
with the standard 6-311G** basis set for carbon, hydrogen, nitrogen, oxygen, while
SDD basis set was used for Ru atom. The crystallographic structure of G-quadruplex
DNA was down loaded from the Protein Data Bank (PDB) (http://www.rcsb.org./pdb).
+
The 26-mer mixed parallel/antiparallel G-quadruplex structure in a K solution (PDB ID:
2
HY9) was used as the initial template for the docking studies. Before docking,
necessary modifications were carried out according to a literature method [76].
Visualization of the docked molecules has been made with Accelrys Discovery Studio
2
.5 software.
The molecular dynamic (MD) simulations were performed using GROMACS
program. GROMOS force field GROMOS96 53A6 was introduced to the G-quadruplex
before the energy minimization [77]. After a 1000 steps of energy minimization in
vacuum, the G-quadruplex were placed in cubic box and the distance between the G-
quadruplex and the box edge was set to be 1.0 nm, which could turn off the boundary
effect of the system. The box was then filled with single point charge (SPC) water
molecules, and 150 mM of sodium chloride was added to modify the ion condition and
to neutralize the system. An energy-minimization in water with a steepest descent
method for 3000 steps was carried out. The results of these minimizations produced the
1
2

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G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
starting structures for the MD simulations. The MD simulations were then carried out
with a constant number of particles (N), pressure (P), and temperature (T), that is, NPT
ensemble. The long-range electrostatic interactions were calculated by the Particle-
Mesh Ewald (PME) method.
A constant pressure of 1 bar was applied with a coupling constant of 1.0 ps; G-
quadruplex, water molecules, and ions were coupled separately to a bath at 310 K with a
coupling constant of 0.1 ps. The equation of motion was integrated at each 2 fs time
steps.
3
. Results and discussion
Synthesis and characterization. Initially, the synthesis of the main ligand ASC
requires the generation of the non-commercially available quinolone-5,8-dione in two
steps. Sodium hydrogen sulfite reduction of available 5-nitro-8-hydroxy-quinoline
afforded 5-amino-8-hydroxyquinoline in moderate yield. 5-amino-8-hydroxyquinoline
was converted into quinolone-5,8-dione in the presence of potassium dichromate under
acidic conditions [72]. Then the synthesis of ASC was achieved mainly in three steps
[
68]. First of all, the alkyne building block was prepared from N-Boc-protected
propargylamine and 2-iodoanilines by a Sonogashira coupling reaction, in good yield.
Secondly, the oxidative amination between alkyne building block and quinoline-5,8-
dione gives aminoquinone. And then, under an oxygen atmosphere, aminoquinone
underwent the Brønsted Acid-Promoted Domino Cyclization to afford the ASC. Lastly,
the coordination between the main ligand ASC and precursor complexes cis-
[
Ru(bpy) Cl ]·2H O, cis-[Ru(phen) Cl ]·2H O and cis-[Ru(tatp) Cl ]·2H O afforded
2 2 2 2 2 2 2 2 2
the product 1, 2 and 3, respectively. After further purification, the complexes were
1
obtained with satisfactory purity and characterized by H NMR and HRMS, details are
1
3

ACCEPTED MANUSCRIPT
G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
described in Fig. S1-S8. These compounds have a weak solubility in water, but have a
high solubility in DMSO. Importantly, all complexes display remarkable stability in the
buffered solution (10 mM Tris-HCl, 100 mM KCl buffer solution with pH value of 7.0,
containing 1% DMSO) at 298 K for at least 3 days, as verified by UV-Vis spectroscopy
(Fig. S9).
Absorption spectra titrations. UV absorption spectroscopy studies were carried out
in order to investigate the binding affinity and binding mode of the compounds to G-
quadruplex. As shown in Fig. 1, upon incremental additions of Htelo quadruplex to the
corresponding compound solutions resulted in considerable hypochromicity of the intra-
ligand charge transfer (ILCT) (λ = 250-260 nm) band and metal-to-ligand charge
transfer (MLCT) (λ = 400-420 nm) band. A slight red shift was also observed. These
features indicated strong interactions between complex 3 and ASC with DNA base pairs
and π-π stacking interactions between the aromatic regions of compounds and the G-
quartets of the quadruplex. The spectra for complexes 1-2 are provided in Fig. S10. In
order to compare the DNA binding affinities of these compounds to quadruplex, the
intrinsic binding constants (K ) were calculated by monitoring the changes of the
b
MLCT absorbance. The intrinsic binding constants for complexes 1-3 and ASC were
6
-1
calculated to be K = 2.78, 3.00, 3.12 and 0.77 × 10 M , respectively. Such
b
comparisons suggest that the trend in G-quadruplex DNA binding affinity is 3 > 2 > 1 >
ASC, which indicates that complex 3 binds G-quadruplex more tightly than other
compounds. The results are due to the different ancillary ligands. On going from bpy,
phen to tatp, the plane area increase, leading to a greater DNA-binding affinity for
complex 3. Furthermore, the positive charges of Ru complexes are in favour of G-
quadruplexes DNA binding, thus, all Ru complexes binds to DNA more strongly than
1
4

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G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
ASC does. Control experiments with ancillary ligands [Ru(L) Cl ] (L = bpy, phen and
2
2
tatp) were also performed (Fig. S11), as expected that these ligands have lower binding
affinity than corresponding complexes 1-3.
FID assay. We also utilized a FID assay to study interactions of these compounds
with different sequences of DNA [78]. Four different DNA sequences were studied:
three quadruplex forming sequences, namely, HTelo, c-myc and c-kit DNA and one
duplex DNA (ds26) sequence. In our experiments, DNA was first treated with TO,
yielding a fluorescent increase upon binding. Addition of the compounds results in a
decrease of fluorescence intensity and finally quenched due to the displacement of TO
from the DNA. The binding affinity of a small molecule for quadruplex and duplex
DNA is determined by the DC50 value, which corresponds to the compound’s
concentration at which TO fluorescence decreases by 50%. Graphical representation of
the TO displacement from quadruplex and duplex DNA for complexes 1-3 and ASC are
shown in Fig. 2. Table 1 summarizes the DC values obtained from all FID assay. The
5
0
DC value follows the order 3 < 2 < 1 < ASC for both quadruplex and duplex DNA.
5
0
For all DNA sequences, low concentration of 3 was required to displace 50% TO,
indicating that 3 is the strongest displacer and better G-quadruplex and duplex DNA
binder than complexes 1-2 and ASC. FID results indicated that Ru complexes interact
with both G-quadruplex and duplex DNA structures, showed lack of selectivity for
different DNA structures. We also carried out the control experiments with ligands
[
Ru(L) Cl ] (L = bpy, phen and tatp) (Fig. S12 and Table S1), results indicated that
2 2
complexes 1-3 have more stronger binding ability for G-quadruplex and duplex DNA
than corresponding ligands.
1
5

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G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
FRET melting assay. The stabilization ability of the compounds to G-quadruplex
structures was investigated using FRET melting assay [52]. When the melting
temperature increases, the quadruplexes structure opens up and the fluorophore and
quencher move apart and fluorescence is restored. The emission of fluorescence was
normalized between 0 and 1, and the T was defined as the melting temperature for
m
which the normalized emission is 0.5 [52]. Compounds ability to stabilize G-quadruplex
was determined by quantifying the change in melting temperature (ΔT ) values. As
m
summarized in Table 2, the melting temperature increase notably with different
concentrations of compounds added into each quadruplex solution (except c-myc). As
shown Fig. 3, upon treatment of the Htelo G-quadruplex (0.25 μM) with 3.5 μM
complex 3 and ASC, ΔT values were 11.5 ˚C and 3 ˚C, respectively. And for c-kit G-
m
quadruplex, ΔT values were 11.0 ˚C and 4 ˚C, respectively. The melting curves for
m
complexes 1 and 2 are shown in Fig. S13. These results indicating that complex 3 is a
more effective G-quadruplex stabilizer than complexes 1-2 and ASC, which is
agreement with the above experimental results. The difference may originate from the
different DNA-binding affinity.
The interaction of complexes 1-3 and ASC with the duplex DNA was also
investigated. As depicted in Table 2, Fig. 3 and Fig. S13, complexes 1-2 and ASC were
found to be no obvious stabilization effect for the ds26, ΔT values were 1 ˚C, 0 ˚C and
m
2
˚C induced by complexes 1-2 and ASC, respectively. Meanwhile, upon treatment of
the ds26 with the same concentration of complex 3, the melting temperature increased 7
˚C, indicating that complex 3 exhibits the strongest stabilization effects for duplex DNA.
A DNA competition FRET melting assay was also applied to explore the quadruplex
versus duplex selectivity. More specifically, DNA competition FRET melting assay was
1
6

ACCEPTED MANUSCRIPT
G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
performed to show ΔT changes for a fixed concentration (3.5 μM) of added compound
m
with G-quadruplex (Htelo, 0.25 μM) through the addition of increasing excess of duplex
DNA (ds26). As shown in Fig. 4, using classical 5 and 15 μM competitor concentrations
(equivalent to 20 and 60 times that of G-quadruplex), G-quadruplex melting
temperature in the presence of complexes 1-3 and ASC is decreased dramatically, in
comparison to the absence of this group of compounds, G-quadruplex stability
significantly affected by the addition of excess amounts of ds26. These results indicate
that complexes 1-3 and ASC are not selective for quadruplex versus duplex DNA and
they can interact both quadruplex and duplex DNA. Control experiments with ligands
[
Ru(L) Cl ] (L = bpy, phen and tatp) were also carried out, as summarized in Fig. S14-
2 2
S15 and Table S2, these ligands have lower stabilizing ability to G-quadruplex and
duplex DNA than corresponding complexes 1-3.
Molecular docking. To elucidate the most favourable binding mode between the
compounds and G-quadruplex DNA, molecular docking studies were carried out, which
could corroborate the experimental results [70]. It has been reported that, small
molecules potentially bind to a G-quadruplex by external stacking, intercalating
between the quartets, or nonspecifically binding to the DNA strand [79]. And external
stacking was the most potential binding mode for the many metal complexes and
quadruplex. Our docking results revealed that, complexes 1-3 and ASC interact with
mixed parallel/antiparallel G-quadruplex through an “external end-stacking” binding
mode (results for all compounds are provided in Fig. S16-S17).
To further investigate these possible binding modes and get more reliable information,
MD simulations and binding free energy calculations were carried out. MD simulation
results suggested that similar external binding modes were achieved for complexes 1-3
1
7

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G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
and ASC. It was also found that the binding modes between the complexes 1-3 and G-
quadruplex DNA is a combination manner: π-π end stacking interactions (between the
ligand and G-quartet), electrostatic interactions (between the metal and DNA’s
backbone) and hydrophobic effects. It is noteworthy that because of the combination
binding mode of complex, it can bind well and tightly than the free ligand ASC. As
shown in Fig. 5, complex 3 and ASC could stack on the both surfaces of the terminal G-
quartet planes (5'- or 3' terminus) and the ligand preferred to stack on the center of a
terminal G-quartet end with π-π stacking interactions, results for complexes 1-2 and
ligands [Ru(L) Cl ] (L = bpy, phen and tatp) are shown in Fig. S18-S19.
2
2
As summarized in Table. S3, the energetic differences between different poses were
-1
about 2-3 kcal·mol . Due to the relatively larger π–aromatic contact surfaces and
-
stronger ligand/DNA interactions, the lower binding free energy values (-12.8 kcal·mol
1
-1
for site-1 and -9.4 kcal·mol for site-2) were observed for complex 3 (Table. S4).
These results were consistent with the spectral studies and confirmed again that
complex 3 was the strongest binder.
Conclusions
To summarize, the act of conjugating a ASC ligands to a Ru(II) polypyridyl subunits
resulted in a class of excellent DNA binders. A comparison of the DNA binding
abilities of the free ASC ligand and the complexes, has revealed that the octahedral
Ru(II) center played a crucial role in yielding good G-quadruplex and duplex DNA
binders. Among three new complexes, complex 3 exhibited the strongest G-quadruplex
and duplex DNA stabilization ability. Complex 3 was determined to be the most
promising candidate for further in vitro studies and to be a potent anticancer drug.
Though these complexes showed lack of selectivity for G-quadruplex over duplex DNA,
1
8

ACCEPTED MANUSCRIPT
G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
the present work provides the basis for the rational development of ASC derivatives as
promising telomerase inhibitors and potential anticancer drugs.
Abbreviations
Ru
ruthenium
ASC
ascididemin
Htelo
c-myc
c-kit
human telomeric DNA
cellular-myelocytomatosis viral oncogene
oncogene c-kit promoter sequences
b-cell CLL/lymphoma 2
bcl-2
K-ras
UV-vis
FID
kirsten rat sarcoma viral oncogene
ultraviolet-visible
fluorescent intercalator displacement
fluorescence resonance energy transfer
molecular dynamic
FRET
MD
bpy
2,2'-bipyridine
phen
1,10-phenanthroline
tatp
1,4,8,9-tetra-aza-triphenylene
imidazolium-trans-DMSO-imidazole-tetrachlororuthenate
indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]
sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]
1,4,5,8-tetraazaphenanthrene
NAMI-A
KP1019
(N)KP1339
tap
tpac
dph
FOA
tetrapyridoacridine
dipyrazino [2,3-a:2',3'-h]phenazine
4-(2,3- dihydroxypropyl)-formamide oxoaporphine
1
9

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G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
icip
2-(indeno[2,1-b]chromen-6-yl)-1H-imidazo[4,5-f][1,10]phenanthroline
phenanthro[4,5-abc]dipyrido- [3,2-h:2',3'-j]phenazine
4,5,9,18-tetraazachryseno[9,10-b]-triphenylene
X : Cl, PIP : 2-phenylimidazole[4, 5f][1,10]phenanthroline
4, 4'-bis (1, 10-phenanthroline-[5, 6-d] imidazole-2-yl)-biphenyl
dimethyl sulfoxide
pdppz
tactp
XPIP
bpibp
DMSO
EtOH
MeCN
MeOH
NMR
ethanol
acetonitrile
methanol
nuclear magnetic resonance spectroscopy
HRMS (ESI) high resolution mass spectrometry (electrospray ionization)
PCR
polymerase chain reaction
thiazole orange
TO
DC50
half maximal displacement concentration
carboxyfluorescein
FAM
TAMRA
DFT
6-carboxytetramethylrhodamine
density functional theory
B3LYP
Becke's three parameter hybrid functional with the Lee-Yang-Parr
correlation functional
PDB
protein data bank
SPC
single point charge
PME
ILCT
MLCT
Particle-Mesh Ewald
intra-ligand charge transfer
metal-to-ligand charge transfer
2
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G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
Acknowledgements
This work was supported by the National Natural Science Foundation of China (No.
2
1671150, 21877084, 81871730 and 21472139), Science and Technology Commission
of Shanghai Municipality (No. 14DZ2261100) and the Fundamental Research Funds for
the Central Universities.
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2
641-2648.
2
6

ACCEPTED MANUSCRIPT
G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
Figure Captions
2
+
2+
Scheme 1 Synthetic route to [Ru(bpy) ASC]
(1), [Ru(phen) ASC]
(2),
2
2
2
+
[
Ru(tatp) ASC] (3).
2
2
+
Fig. 1 Absorption spectra of [Ru(tatp) ASC] (3) (A) and ASC (B) (10 μM) titration by
2
G-quadruplex DNA in presence of Htelo in 10 mM Tris-HCl, 100 mM KCl buffer,
pH=7.0, [DNA]=(0-15 μM). Inset: plots of (ε -ε )/(ε -ε ) vs [DNA].
a
f
b
f
Fig. 2 Graphical representation of the TO (2 μM) displacement from Htelo (A), c-kit (B),
c-myc (C) G-quadruplexes and ds26 (D) duplex DNA (1 μM) upon increasing
concentration of complexes 1-3 and ASC.
Fig. 3 Normalized FRET melting curves of G-quadruplex and duplex (ds26) DNA (0.25
+
2+
μM) in K buffer, with increasing concentrations (0-3.5 μM) of [Ru(tatp) ASC] (3)
2
(A-D) and ASC (E-H) measured by real-time PCR system.
Fig. 4 Competitive FRET-melting curves of Htelo (0.25 μM) with 3.5 μM of
2
+
2+
2+
[
Ru(bpy) ASC] (1) (A), [Ru(phen) ASC] (2) (B), [Ru(tatp) ASC] (3) (C), ASC (D)
2 2 2
and duplex competitor ds26 (5-15 μM) in buffer containing 100 mM KCl.
Fig. 5 The calculated model of the compounds bind to the mixed hybrid type G-
2+
quadruplex DNA at site-1 and site-2 (A) [Ru(tatp) ASC] (3), (B) ASC.
2
2
7

ACCEPTED MANUSCRIPT
G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
2
+
2+
Scheme 1 Synthetic route to [Ru(bpy) ASC]
(1), [Ru(phen) ASC]
(2),
2
2
2
+
[
Ru(tatp) ASC] (3).
2
2
8

ACCEPTED MANUSCRIPT
G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands
2+
Fig. 1. Absorption spectra of [Ru(tatp) ASC] (3) (A) and ASC (B) (10 μM) titration
2
by G-quadruplex DNA in presence of Htelo in 10 mM Tris-HCl, 100 mM KCl buffer,
pH=7.0, [DNA]=(0-15 μM). Inset: plots of (ε -ε )/(ε -ε ) vs [DNA].
a
f
b
f
Fig. 2. Graphical representation of the TO (2 μM) displacement from Htelo (A), c-kit
B), c-myc (C) G-quadruplexes and ds26 (D) duplex DNA (1 μM) upon increasing
concentration of complexes 1-3 and ASC.
(
2
9