Anthracene-terpyridine metal complexes as new G-quadruplex DNA binders

Article abstract of DOI:10.1016/j.jinorgbio.2016.04.002

The formation of quadruple-stranded DNA induced by planar metal complexes has particular interest in the development of novel anticancer drugs. This is especially relevant for the inhibition of telomerase, which plays an essential role in cancer cell immortalization and is overexpressed in ca. 85-90% of cancer cells. Moreover, G-quadruplexes also exist in other locations in the human genome, namely oncogene promoter regions, and it has been hypothesized that they play a regulatory role in gene transcription. Herein we report a series of new anthracene-containing terpyridine ligands and the corresponding Cu(II) and Pt(II) complexes, with different linkers between the anthracenyl moiety and the terpyridine chelating unit. The interaction of these ligands and metal complexes with different topologies of DNA was studied by several biophysical techniques. The Pt(II) and Cu(II) complexes tested showed affinity for quadruplex-forming sequences with a good selectivity over duplex DNA. Importantly, the free ligands do not have significant affinity for any of the DNA sequences used, which shows that the presence of the metal is essential for high affinity (and selectivity). This effect is more evident in the case of the Pt(II) complexes. Moreover, the presence of a longer linker between the chelating terpyridine unit and the anthracene moiety enhances the interaction with G-quadruplex-forming sequences. We further evaluated the ability of the Cu(II) complexes to interact with, and stabilize G-quadruplex containing regions in oncogene promoters via a polymerase stop assay. These studies indicated that the metal complexes are able to induce G-quadruplex formation and stop polymerase activity.

Full text of DOI:10.1016/j.jinorgbio.2016.04.002

Journal of Inorganic Biochemistry 160 (2016) 275–286
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Anthracene-terpyridine metal complexes as new G-quadruplex DNA binders
Sofia Gama a, ,1, Inês Rodrigues a, Filipa Mendes a, Isabel C. Santos a, Elisabetta Gabano b, Beata Klejevskaja c,
⁎
Jorge Gonzalez-Garcia c, Mauro Ravera b, Ramon Vilar c, António Paulo a
a
Centro de Ciências e Tecnologias Nucleares (C2TN), Instituto Superior Técnico, Universidade de Lisboa, Portugal
Dipartimento di Scienze e Innovazione Tecnologica, Università del Piemonte Orientale “Amedeo Avogadro”, Alessandria, Italy
b
c
Department of Chemistry, Imperial College London, London SW7 2AZ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The formation of quadruple-stranded DNA induced by planar metal complexes has particular interest in the
development of novel anticancer drugs. This is especially relevant for the inhibition of telomerase, which plays
an essential role in cancer cell immortalization and is overexpressed in ca. 85–90% of cancer cells. Moreover,
G-quadruplexes also exist in other locations in the human genome, namely oncogene promoter regions, and it
has been hypothesized that they play a regulatory role in gene transcription. Herein we report a series of new
anthracene-containing terpyridine ligands and the corresponding Cu(II) and Pt(II) complexes, with different
linkers between the anthracenyl moiety and the terpyridine chelating unit. The interaction of these ligands
and metal complexes with different topologies of DNA was studied by several biophysical techniques. The
Pt(II) and Cu(II) complexes tested showed affinity for quadruplex-forming sequences with a good selectivity
over duplex DNA. Importantly, the free ligands do not have significant affinity for any of the DNA sequences
used, which shows that the presence of the metal is essential for high affinity (and selectivity). This effect is
more evident in the case of the Pt(II) complexes. Moreover, the presence of a longer linker between the chelating
terpyridine unit and the anthracene moiety enhances the interaction with G-quadruplex-forming sequences. We
further evaluated the ability of the Cu(II) complexes to interact with, and stabilize G-quadruplex containing re-
gions in oncogene promoters via a polymerase stop assay. These studies indicated that the metal complexes are
able to induce G-quadruplex formation and stop polymerase activity.
Received 2 November 2015
Received in revised form 15 March 2016
Accepted 3 April 2016
Available online 9 April 2016
Keywords:
G-quadruplex
Terpyridine derivatives
Platinum complexes
Copper complexes
DNA interaction
c-myc
© 2016 Elsevier Inc. All rights reserved.
1. Introduction
by the enzyme telomerase [4]. As telomerase is expressed in most
cancer cells at substantially higher levels than in normal somatic cells,
G-quadruplex DNA is a thermodynamically stable topology of DNA
which has been proposed to be functionally relevant to a number of
essential biological processes. In particular, it has been suggested that
quadruplexes play a role in regulation of gene expression, translation,
telomere-end maintenance, recombination and replication [1–5].
Therefore, G-quadruplexes have emerged as novel and distinctive tar-
gets for the development of anticancer drugs [6,7]. G-quadruplexes
are four-stranded DNA structures formed from stacked tetrads of
hydrogen-bonded guanine bases. These stable, non-classical, secondary
structures are formed in vitro at physiological salt concentration and
pH, from DNA sequences containing stretches of adjacent guanine
residues [8,9]. There is mounting experimental evidence indicating
that these non-canonical DNA structures are also present in vivo
[10–12]. Interestingly, stabilization of intramolecular telomeric DNA in
a G-quadruplex conformation has been shown to inhibit its elongation
the enzyme is a potentially attractive target for selective anti-cancer
therapy and drug development [13]. Telomerase itself is not an onco-
gene, but its repression and tight regulation in humans can be a tumour
suppressor, and its targeting has advantages such as relative universal-
ity and specificity for cancer cells [14–18].
In addition to telomeric DNA, an intramolecular-quadruplex forming
sequence has also been identified in the promoter region of the c-myc
oncogene, amongst other gene promoters [6], and stabilization of this
quadruplex structure has been implicated in the downregulation of
the transcription of the gene [19,20]. All these facts suggest that a
deeper understanding of quadruplex recognition by synthetic mole-
cules could provide the basis of a novel anticancer drug.
Thus, researchers have taken up the challenge of synthesizing
ligands that stabilize the quadruplex folded conformation focusing on
the fact that a desired molecule should bind a human G-quadruplex
structure with high affinity and with specificity over duplex DNA
[21–23]. The number of known G-quadruplex ligands has increased
steadily and several classes of quadruplex-targeting ligands have been
reported that selectively bind to and stabilize quadruplexes [24–30].
Many of these ligands present a large planar aromatic surface, cationic
⁎
Corresponding author.
E-mail address: sofia.gama@uni-jena.de (S. Gama).
Current adress: Institute für Anorganische und Analytische Chemie, Friedrich-Schiller-
1
Universität Jena, Germany.
http://dx.doi.org/10.1016/j.jinorgbio.2016.04.002
0162-0134/© 2016 Elsevier Inc. All rights reserved.

276
S. Gama et al. / Journal of Inorganic Biochemistry 160 (2016) 275–286
charges, and ability to adopt a terminal stacking mode [22,23]. In this
context, the use of metal complexes as G-quadruplex binders is becom-
ing very attractive due to the often easier synthetic access and their
promising DNA binding properties [22]. The cationic or highly polarized
nature of metal complexes is expected to promote the association with
the negatively charged G-quadruplex-DNA [22,23]. Therefore, this ap-
proach assumes that the central metal centre can be positioned over
the cationic channel of the quadruplex, optimizing the stacking interac-
tions of the surrounding chelating agent with the accessible G-quartet.
For instance, it has been reported that some Schiff base metal complexes
are highly effective stabilizers of the human telomeric DNA, in particular
square planar complexes of copper(II) and nickel(II) that are very prone
to stack with the G-quartet [31–33]. Other types of ligands, namely
derived from porphyrins and terpyridines, have been also tested and
the corresponding Cu(II) and Pt(II) complexes were shown to act as
G-quadruplex binders with high affinity and selectivity [34].
One of the features that quadruplex DNA binders should have if they
are to be leads in drug discovery, is high selectivity over duplex DNA to
minimise potential side effects. A successful strategy to improve such
selectivity relies on the use of structures containing more than one
interlinked planar polyaromatic moieties (e.g. anthracene, naphthalene,
bipyridine or pyrene). As reported by Teulade-Fichou et al. [35], this
type of strategy was successfully applied for macrocyclic organic
compounds of the bis-intercalator or tris-intercalator type. This success
inspired us to explore a similar approach to improve the quadruplex-
selectivity of M(II)-terpyridine (M = Cu, Pt) complexes, known to inter-
act with G-quadruplex sequences [34,36–40], by combining these
terpyridine metal cores with a planar intercalating anthracenyl group.
Herein we report on the synthesis of a series of new anthracene-
containing terpyridine ligands and the corresponding Cu(II) and Pt(II)
complexes (Scheme 1) and on the biophysical characterization of their
interaction with DNA of different topologies. In this work, the
terpyridine chelating unit and the anthracenyl moiety were assembled
using linker chains with different length and polarity, as we had antici-
pated that the different structural motifs (metal geometry, linker and
pendant DNA intercalator) could influence the selectivity of the com-
pounds' interaction with quadruplex DNA over duplex DNA. The inter-
actions between ligands and metal complexes with the different
topologies of DNA were studied by a variety of biophysical and spectro-
scopic techniques including FRET melting assays, fluorescence and CD
spectroscopy. A polymerase stop assay was also performed to assess
the ability of the compounds to interact and stabilize G-quadruplex
containing regions in an oncogene promoter.
2. Experimental section
All chemicals used were analytical grade and used as received. The
chemical reactions were followed by thin-layer chromatography
(TLC), which was performed on precoated silica plates 60 F254
(Merck). Visualization of the plates was carried out using Ultraviolet
(UV) light (254 nm). Gravity column chromatography was carried out
on silica gel (Merck, 70–230 mesh).The multinuclear NMR spectra
were measured on a Bruker 500 MHz nuclear magnetic resonance
(NMR) Spectrometer at 500 MHz (1H), 100.5 MHz (13C), and
85.9 MHz (195Pt), or in a Varian Unity 300 MHz spectrometer, and
chemical shifts are given in ppm, referenced with the residual solvent
resonances relative to SiMe4. 1H and 13C NMR chemical shifts were re-
ported in parts per million referenced to solvent resonances. 195Pt
NMR spectra were recorded using a solution of K2[PtCl4] in saturated
aqueous KCl as external reference. The shift for K2[PtCl4] was adjusted
to −1628 ppm relative to Na2[PtCl6] (δ = 0 ppm). For the Pt(II) com-
plexes, Reversed Phase-High-performance liquid chromatography
(RP-HPLC) and mass analysis were performed using a Waters LC-MS
(Liquid chromatography-mass spectrometry) instrument equipped
with Alliance 2695 separations module, 2487 dual lambda absorbance
detector, and 3100 mass detector. Electrospray ionisation mass
spectrometry (ESI-MS) was performed with a Micromass ZMD mass
spectrometer (Micromass, Manchester, UK). Typically, a diluted solu-
tion of the compound in methanol (with 2% of the suitable co-solvent)
was delivered directly to the spectrometer source at 0.01 mL min−1
by Hamilton microsyringe controlled by a single-syringe infusion
pump. The nebulizer tip operated at 3000–3500 V and 150 °C, with
nitrogen used both as a drying and a nebulizing gas. The cone voltage
was usually 30 V. For the chelators and Pt(II) complexes, ESI-MS was
performed on a QITMS (Quadrupole ion trap mass spectrometry) in-
strument in positive and negative ionisation mode. Peaks were assigned
on the basis of the m/z values and of the simulated isotope distribution
patterns. Elemental analysis (EA) was performed on an EA 110 CE
Instruments automatic analyzer.
2.1. Synthesis
2.1.1. Synthesis of 2-([2.2′:6′,2″-terpyridin]-4′-yloxy)-N-(anthracen-9-
ylmethyl)ethan-1-amine (AntTerp, L1)
To a solution of anthracene-9-carbaldehyde (2.062 g; 10 mmol) in a
25% CH3OH/CH2Cl2 solution (10 mL) 2-aminoethanol (0.72 mL,
12 mmol) was added. The reaction mixture was let to react overnight,
at room temperature and under N2. After vacuum removal of solvent,
the solid residue was redissolved in 50% CH3OH/CH2Cl2 solution
(20 mL) and NaBH4 (1.1349 g, 30 mmol) was added, at 0 °C. After
18 h reaction at room temperature, the solvent was removed and the
residue dissolved in CH2Cl2 (50 mL) and washed with a solution of
Na2CO3 (pH 10, 3 × 50 mL). The organic layer was dried over Na2SO4
and the solvent removed under vacuum to obtain 2-(anthracen-9-
ylmethylamino)ethanol (1, AntOH). Yield: 2.238 g (89%). 1H NMR
(CDCl3-d3) δ: 8.42 (1H, s, H28), 8.32 (2H, d, H23 + 33), 8.01 (2H, d,
H
26 + 30), 7.51 (4H, ddt, H25 + 24, H31 + 32), 4.75 (2H, d, H20), 3.70
(2H, td, H16), 3.2 (2H, m, H19), 2.17 (1H, s, NH) ppm.
Then, 2-(anthracen-9-ylmethylamino)ethanol (0.251 g, 1 mmol)
was let to react with 4′-chloro-2.2′:6′,2″-terpyridine (0.250 g,
0.9 mmol) and KOH (0.281 g, 5 mmol) in dimethyl sulfoxide (DMSO)
for 4 h at 60 °C. The reaction was extracted with CH2Cl2 (3 × 40 mL)
and the organic layer washed with water (3 × 30 mL). The organic
layer was dried over Na2SO4 and the solvent was removed under vacu-
um. The solid residue was purified by column chromatography (100%
CH2Cl2 to 95/5 CH2Cl2 /CH3OH) to afford 2-([2.2′:6′,2″-terpyridin]-4′-
yloxy)-N-(anthracen-9-ylmethyl)ethan-1-amine (AntTerp, L1), which
Scheme 1. Structural formulae of the new anthracene-containing terpyridine ligands
(L1 and L2) and the corresponding Cu(II) and Pt(II) complexes (CuL1, CuL2, PtL1 and PtL2).

S. Gama et al. / Journal of Inorganic Biochemistry 160 (2016) 275–286
277
was recovered as a pale brownish solid after removal of the solvent from
and washed with water, ethanol and diethyl ether. Yield: 703 mg
(80%). 1H NMR (CDCl3-d3) δ: 5.60 (4H, t, CH), 2.70 (2H, m, CH2\\CH),
2.30–2.16 (2H, m, CH2\\CH) ppm; 13C{1H} NMR (CDCl3-d3) δ: 100.0
(CH), 30.0 (CH2) ppm; 195Pt NMR (CDCl3-d3) δ: −3340 ppm.
the collected fractions. Yield: 205 mg (48%). ESI-MS (CH3OH): (m/z) =
483.3 [M
(dimethylformamide (DMF)-d7) δ: 8.75 (2H, d, H23 + 33), 8.72 (2H, d,
4 + 12), 8.58 (1H, s, H28), 8.60 (2H, d, H1 + 15), 8.12 (2H, d, H26 + 30),
+ ,
H]+ calcd. for C32H27N4O: 483.2 m/z. 1H NMR
H
2.1.4.2. Synthesis of chlorido(L1)platinum(II) chloride, [Pt(L [1])Cl]Cl
(PtL1). To a suspension of [Pt(cod)Cl2] (93 mg, 0.25 mmol) in
water (7 mL), a suspension of L1 (115 mg, 0.24 mmol) in DMF
(3 mL) was added with stirring, in the dark. After addition of the li-
gand, the color of the solution started to change from pale yellowish
to orange. The reaction mixture was stirred and heated on a sand bath at
70 °C for 50 min. Thereafter, the reaction mixture was cooled to room
temperature. The insoluble solids were removed by centrifugation and
the red-orange clear supernatant solution was dried under vacuum. The
resulting orange oil was stirred successively with diethyl ether to afford
a microcrystalline orange solid. After removal of diethyl ether, the solid
was dried under vacuum. Yield: 112 mg (62%). ESI-MS (CH3OH) m/z:
713.7 [M]+, calcd for C32H26ClN4OPt+: 713.14 m/z. 1H NMR (DMF-d7) δ:
9.053 (2H, d, H4 + 12), 8.773 (2H, d, H1 + 15), 8.563 (5H, m, H28 and
8.14 (2H, s, H7 + 9), 8.05 (2H, m, H3 + 13), 7.58 (2H, m, H2 + 14), 7.55
(4H, m, H24 + 32 + H25 + 31), 4.90 (2H, s, H20), 5.55 (2H, t, H16), 3.41
(2H, t, H19) ppm; 13C{1H} NMR (DMF-d7) δ: 158.59 (C6 + 10), 157.29
(C5
11), 150.19 (C1 + 15), 138.85 (C3
13), 136.42 (C26), 130.42
+
+
(C30), 128.83 (C27 + 29), 127.60 (C23 + 33), 126.22 (C24 + 32), 125.65
(C25 + 31), 125.19 (C4), 123.13 (C2 + 14, C12), 108.67 (C7 + 9), 68.39
(C16), 50.00 (C20), 45.63 (C19) ppm.
2.1.2. Synthesis of 2-(2-([2.2′:6′,2″-terpyridin]-4′-yloxy)ethoxy)-N-
(anthracen-9-ylmethyl)ethan-1-amine (AntPegTerp, L2)
2-(2-(Anthracene-9-ylmethylamino)ethoxy)ethanol (2, AntPegOH)
was synthesized as described above for compound 1, using 2-(2-
aminoethoxy)ethanol (1.2 mL, 12 mmol) instead of 2-aminoethanol.
Yield: 2.674 g (91%). 1H NMR (CDCl3-d3) δ: 8.42 (1H, s, H28), 8.34 (2H,
d, H23 + 33), 8.01 (2H, d, H26 + 30), 7.51 (4H, m, H24 + 32, H25 + 31), 4.79
(2H, s, H20), 3.66 (4H, dd, H17, H18), 3.54 (2H, m, H16), 3.04 (2H, t, H19),
2.28 (1H, s, NH) ppm.
H2 + 14 and H23 + 33), 8.401 (2H, s, H7 + 9), 8.080 (4H, m, H3 + 13 and
H26 + 30), 7.559 (2H, d, H24 + 32), 7.465 (2H, t, H25 + 31), 4.940 (2H, s,
H
20), 4.761 (2H, t, H16), 3.5 (2H, t, H19) ppm; 195Pt NMR (DMF-d7) δ:
AntPegTerp (L2) was synthesized as described above for AntTerp (L1)
but starting from 2-(2-(anthracene-9-ylmethylamino)ethoxy)ethanol
(0.295 g, 1 mmol). The final product was purified by column chromatog-
raphy (100% CH2Cl2 to 90/10 CH2Cl2/CH3OH) being recovered as a pale
brownish solid. Yield: 367 mg (78%). ESI-MS (CH3OH): (m/z) = 527.4
[M + H]+, calcd. for C34H31N4O2: 527.4 m/z. 1H NMR (DMF-d7) δ: 8.73
(2H, d, H23 + 33), 8.69 (2H, d, H4 + 12), 8.54 (1H, s, H28), 8.50 (2H, d,
−2720 ppm.
2.1.4.3. Synthesis of chlorido(L2)platinum(II) chloride, [Pt(L2)Cl]Cl (PtL2).
Compound PtL2 was synthesized using the same procedure as described
above for PtL1, by reacting [Pt(cod)Cl2] with L2 (200 mg, 0.38 mmol).
Yield: 182 mg (60%). ESI-MS (CH3OH) m/z: 756.6 [M]+, calcd for
C
34H30ClN4O2Pt+: 756.17 m/z. 1H NMR (DMF-d7) δ: 8.753 (2H, d,
H1
15), 8.09 (2H, d, H26
30), 8.08 (2H, s, H7
9), 8.04 (2H, m,
H
H
H
H
4+12), 8.625 (2H, d, H1
15), 8.460 (1H, s, H28), 8.432 (2H, t,
+
+
+
+
H3 + 13), 7.56 (2H, m, H2 + 14), 7.51 (4H, m, H24 + 32, H25 + 31), 4.78
2 + 14), 8.368 (2H, d, H23 + 33), 8.269 (2H, s, H7 + 9), 7.932 (2H, t,
3 + 13), 7.902 (2H, d, H26 + 30), 7.445 (2H, t, H24 + 32), 7.318 (2H, t,
25 + 31), 4.826 (2H, s, H20), 4.761 (2H, t, H16), 4.141 (2H, t, H17),
(2H, s, H20), 4.48 (2H, t, H16), 3.96 (2H, d, H17), 3.79 (2H, t, H18), 3.09
(2H, t, H19) ppm; 13C{1H} NMR (DMF-d7) δ: 167.40 (C8), 157.45
(C6 + 10), 155.75 (C5 + 11), 149.58 (C1 + 15), 137.48 (C3 + 13), 132.81
(C21), 131.86 (C22 + 34), 130.63 (C27 + 29), 129.14 (C26 + 30), 127.07
(C28), 126.11 (C23 + 33), 125.28 (C24 + 32), 125.05 (C25 + 31), 124.69
(C2 + 14), 121.26 (4 + 12), 107.28 (C7 + 9), 70.99 (C18), 69.29 (C17), 68.27
(C16), 49.63 (C20), 45.53 (C19) ppm.
3.976 (2H, t, H18), 3.286 (2H, t, H19) ppm; 13C{1H} NMR (DMF-d7) δ:
169.68 (C8), 158.51 (C6
142.55 (C3
(C4 + 12,C26 + 30), 128.38 (C28), 126.50 (C23 + 33), 126.06 (C24 + 32),
1125.37 (C25 + 31), 124.70 (C2 + 14), 111.26 (C7 + 9), 70.57 (C18), 69.39
(C17), 65.54 (C16), 49.11 (C20) ppm; 195Pt NMR (DMF-d7) δ: −2700 ppm.
10), 155.50 (C5
11), 151.37 (C1
15),
+
+
+
13), 131.50 (C22
34), 130.59 (C27
29), 129.32
+
+
+
2.1.3. Synthesis of anthracene-terpyridine-copper(II) complexes
To a solution of L1 or L2 (100 mg) in methanol (10 mL) was added
CuCl2·5H2O (1 equivalent). The mixture was stirred at room tempera-
ture for 3 h. The resulting green precipitates were recovered by filtra-
tion, washed with methanol and diethyl ether, and dried under vacuum.
Table 1
Crystallographic data and refinement parameters for compound L1.
Empirical formula
C32H26 N4O
Formula weight
Temperature/K
Crystal system
Space group
a/Å
b/Å
482.57
150(2)
Triclinic
P1
5.0226(2)
10.5037(5)
2.1.3.1. AntTerpCu(II) (CuL1). Yield: 63 mg (49%). ESI-MS (CH3OH):
(m/z) = 652.4 [M + Cl− −; CHN (%): experimental 47.99 C, 4.37
]
H, 6.40 N; calculated for C32H26Cl2CuN4O·3HCl·5.5H2O 47.60 C,
4.74 H, 6.94 N.
c/Å
α/°
β/°
γ/°
13.6134(6)
102.588(3)
97.886(2)
102.783(2)
670.45(5)
1
1.195
0.074
2.1.3.2. AntPegTerpCu(II) (CuL2). Yield: 52 mg (41%). ESI-MS (CH3OH):
(m/z) = 696.3 [M + Cl− −
] ; CHN (%): experimental 51.91 C, 5.18 H,
Volume/Å3
Z
6.90 N; calculated for C34H30Cl2CuN4O2·HCl·5H2O 51.85 C, 5.25 H,
7.11 N.
ρ
calcg/cm3
μ/mm−1
2.1.4. Synthesis of anthracene-terpyridine-platinum(II) complexes
F(000)
254.0
0.2 × 0.16 × 0.12
Crystal size/mm3
2.1.4.1. Synthesis of cis-dichlorido(1.5-cyclooctadiene)platinum(II),
[Pt(cod)Cl2]. K2PtCl4 (1.000 g, 2.4 mmol) was dissolved in water
(16 mL) and the resulting solution was filtered. To the filtered deep red
solution, acetic acid (24 mL) and 1,5-cyclooctadiene (cod) (1.00 mL,
8.1 mmol) were added, in the dark. The reaction mixture was stirred
and heated at ca. 90 °C in a water bath. Over 30 min the deep red solution
slowly became pale yellow and some precipitation was observed. After
ca. 2 h, the volume of the solution was reduced to 10 mL by evaporation
under reduced pressure. The obtained precipitate was then centrifuged
Θ range/°
Index ranges
2.89 to 25.68
−6 ≤ h ≤ 6, −12 ≤ k ≤ 11, −16 ≤ l ≤ 15
6388
3799 [Rint = 0.0199]
3532/4/338
1.059
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
Final R indexes [I N =2σ (I)]
Final R indexes [all data]
Largest diff. peak/hole/e Å−3
Flack parameter
R
R
1 = 0.0743, wR2 = 0.1369
1 = 0.0756, wR2 = 0.1460
0.202/−0.199
0(2)

278
S. Gama et al. / Journal of Inorganic Biochemistry 160 (2016) 275–286
2.2. X-ray diffraction analysis
2.3. DNA interaction
Crystals of L1 suitable for X-ray diffraction studies were obtained
from a concentrated dichloromethane solution of the compound, after
standing for two days. The crystals were mounted on a loop with pro-
tective oil. X-ray data were collected at 150 K in the ω and φ scans
mode on a Bruker APEX II CCD diffractometer using graphite
monochromated Mo Kα radiation (0.71073 Å) and operating at 50 kV
and 30 mA. Cell parameters were retrieved using Bruker SMART [41]
software and refined using Bruker SAINT [41] on all observed reflec-
tions. Semi-empirical absorption corrections were applied using
SADABS [42]. Structure solution and refinement were performed using
direct methods with program SIR97 [43] and SHELXL97 [44], both in-
cluded in the package of programs WINGX-Version 2013.3 [45]. A full-
matrix least-squares refinement was used for the non-hydrogen
atoms with anisotropic thermal parameters, except for disordered
atoms that were refined isotropically. All hydrogen atoms were inserted
in idealized positions and allowed to refine riding in the parent carbon
atom. Molecular graphics were prepared using ORTEP3 [46]. A summary
of the crystal data, structure solution and refinement parameters are
given in Table 1. Crystallographic data for the structure of L1 have
been deposited at the Cambridge Crystallographic Data Centre as sup-
plementary publication CCDC 1,431,619. Copies of the structures can
be obtained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
2.3.1. Photophysical experiments
The labelled oligonucleotides HTelo DNA, (5′-FAM-AGGGTTAGGGTTAG
GGTTAGGG-TAMRA-3′), c-myc (5′-FAM-GGGGAGGGTGGGGAGGGTGGG-
TAMRA-3′) and ds26 (5′-FAM-CAATCGGATCGAATTCGATCCGATTG-
TAMRA-3′) (FAM – fluorescein amidite, TAMRA – tetramethylrhodamine)
were purchased from Eurogentec. The unlabelled oligos were also acquired
from Eurogentec and Calf thymus DNA (ct-DNA) was purchased from
Sigma Aldrich. Tested compounds were dissolved in DMSO to give
10 mM stock solutions. All solutions were stored in 100 μL aliquots at
−20 °C, thawed, and diluted immediately before use. Oligonucleotide
concentrations were calculated using the following extinction coeffi-
cients (L cm−1 mol−1) at 260 nm given by the supplier: 220,590
(HTelo), 240,699 (c-myc), 258,900 (ds26), for quadruplex forming
oligos per strand. The extinction coefficient of ct-DNA at 260 nm was
taken to be 13,200 L cm−1 per base pair. [47].
2.3.1.1. Fluorescence resonance energy transfer (FRET) assays. FAM -
TAMRA labelled DNA oligos were prepared as 20–30 μM stock solutions
in MilliQ water. Oligo DNA samples diluted to 0.40 μM in lithium
cacodylate (LiCad) buffer (pH 7.4). LiCad buffer was prepared from a
stock solution of 10 mM LiOH, titrated with cacodylic acid until 7.4 pH
was reached. To this solution, 1 mM KCl and 99 mM LiCl or 10 mM
KCl and 90 mM LiCl were added to obtain the final buffer solution
Scheme 2. Synthesis of ligands L1 and L2. The numbering schemes are those used to identify the 1H and 13C NMR signals.

S. Gama et al. / Journal of Inorganic Biochemistry 160 (2016) 275–286
279
Table 2
Selected bond lengths (Å) and angles (°) for compound L1.
Length/Å
Angle/°
Atom
Atom
Length
Atom
Atom
Length
Atom
Atom
Atom
Angle
Atom
Atom
Atom
Angle
O(1)
N(1)
N(2)
N(3)
N(4)
C(5)
C(16)
C(8)
C(4)
C(6)
C(11)
C(17)
C(6)
1.367(5)
1.339(5)
1.339(4)
1.364(5)
1.466(5)
1.489(5)
1.514(6)
O(1)
N(1)
N(2)
N(3)
N(4)
C(10)
C(18)
C(16)
C(5)
1.433(4)
1.365(5)
1.340(5)
1.348(5)
1.473(5)
1.490(5)
1.512(6)
C(8)
C(4)
C(6)
N(2)
N(2)
C(11)
C(17)
O(1)
O(1)
O(1)
N(1)
N(2)
C(6)
C(10)
N(3)
N(4)
C(8)
C(8)
C(16)
C(5)
C(10)
C(5)
C(11)
C(15)
C(18)
C(7)
117.3(3)
116.6(4)
117.8(3)
115.9(3)
115.2(3)
116.0(4)
112.5(3)
124.1(3)
116.9(3)
O(1)
N(1)
C(1)
N(3)
C(12)
C(20)
C(32)
N(4)
N(4)
C(16)
C(5)
C(5)
C(11)
C(11)
C(19)
C(19)
C(17)
C(18)
C(17)
C(6)
C(6)
C(10)
C(10)
C(18)
C(18)
C(16)
C(19)
106.6(3)
115.4(3)
122.8(3)
115.7(3)
122.1(3)
118.9(3)
120.5(3)
111.0(3)
109.8(3)
C(10)
C(15)
C(18)
C(11)
C(19)
C(17)
C(9)
used for c-myc oligo or HTelo and ds26, correspondingly. To anneal,
samples were heated to 95 °C for 5 min, and allowed to cool slowly to
room temperature overnight. c-myc was annealed in 1 mM KCl,
99 mM LiCl, 10 mM Lithium cacodylate and HTelo and ds26 in 10 mM
KCl, 90 mM LiCl, 10 mM Lithium cacodylate buffer. For FRET melting ex-
periments 8-well optical tube strips were used. The final volume of each
sample was 40 μL, with a final DNA concentration of 0.20 μM and in-
creasing concentration of tested compound (0–8 μM). In the case of
the competition assays, each well was prepared with a final oligo con-
centration of 0.20 μM, 1 μM compound, and the ct-DNA concentration
to test (0 to 120 μM). Measurements were performed on a PCR
Stratagene Mx3005P (Agilent Technologies) with FAM excitation at
450–495 nm and detection at 515–545 nm. Readings were taken from
25 °C to 95 °C every 1 °C and at 1 °C/min melting rate. To obtain melting
curves, normalised FAM fluorescence signal was plotted against
temperature. From the non-linear fitting of the plot ΔTm [Tm (with
ligand) − Tm (without ligand) obtained from at least three independent
measurements] vs. ligand concentration, the ΔTm value for 1 μM
concentration of ligand was obtained.
GTGGGGAAGATTCCCGACTTCGTATTAAGTACTCTAGCCTT-3′) and the
corresponding partially complementary FAM label-sequence (5′-FAM-
AAGGCTAGAGTACTTAATACGA-3′) were used. A mixture of each
oligonucleotide (1 μM each) and increasing concentrations of the
compounds (0, 0.3, 1, 2.5, 5, 10 and 20 μM) were annealed in TRIS
(Tris(hydroxymethyl)aminomethane)-HCl buffer (50 mM, pH 7.4).
After cooling to room temperature, the PSA reactions were performed
in 1× Taq (Thermus aquaticus) buffer, containing the previously
annealed solution (0.2 μM), dNTPs (deoxyribose nucleotide triphos-
phates (0.2 mM), MgCl2 (1.25 mM) and Taq DNA polymerase (2.5 U)
at 37 °C during 30 min. The reactions were quenched by adding an
equal volume of stop buffer (90% formamide, 10 mM NaOH). PSA
products were then analysed on a denaturing polyacrylamide gel
(20%) in 1 x TBE (Tris/Borate/EDTA) and visualized with FAM dye. As
a control experiment, a non-forming quadruplex sequence was used
(5′-GCGGCTCCTGTGAGGGTGAAGAGGGTGGGGA AGATTCCCGACTTCGT
ATTAAGTACTCTAGCCTT-3′).
3. Results and discussion
2.3.1.2. Circular dichroism (CD). CD titrations were done by adding a
metal complex to test (0–50 μM) to the annealed c-myc or HTelo at
5 μM. To anneal, as previously described (Section 2.3.1.1) samples
were heated to 95 °C for 5 min, and allowed to cool slowly to room tem-
perature overnight. c-myc was annealed in 1 mM KCl, 99 mM LiCl,
10 mM Lithium cacodylate and HTelo in 10 mM KCl, 90 mM LiCl,
10 mM Lithium cacodylate buffer. CD experiments were performed on
a Jasco J 810 spectropolarimeter using a 0.2 cm path length quartz cu-
vette and the spectra were recorded from 200 to 320 nm at 1 nm
bandwidth.
3.1. Synthesis of anthracene-terpyridine chelators and the corresponding
M(II) (M = Cu, Pt) complexes
The anthracene-terpyridine chelators (L1 and L2) were obtained
using 4′-chloro-2.2′:6′,2″-terpyridine as starting material, which was
O-alkylated using the appropriate alcohol derivative of 2-
aminomethylanthracene via a Williamson reaction (Scheme 2). The al-
cohol derivatives of anthracene, compounds 1 and 2 containing an eth-
ylenic or pegylated linker, respectively, were synthesized by reduction
2.3.1.3. Spectroscopic titrations. The electronic absorption spectra were
obtained on a Perkin-Elmer UV/Vis spectrometer and fluorescence spec-
tra on a Varian Cary-Eclipse, all in quartz cuvettes (1 cm). Spectroscopic
studies were performed in 50 mM Tris HCl buffer (pH 7.4) with 10 mM
KCl at 20
0.5 °C. Titrations were performed keeping a constant
concentration of the compound (2.5 and 15 μM for emission and
absorption, respectively) and concentrated DNA was then added to
the solution containing the compound until changes in the absor-
bance/emission became noticeably indifferent. Samples were excited
at 368nm, where upon titration of DNA, absorbance changes were
negligible. Emission spectra were recorded from 380 to 600nm. Two
independent repeats were performed. Binding data obtained from spec-
trophotometric and spectrofluorimetric titrations were cast into
Scatchard plot of r/Cf versus r. Non-linear binding isotherms observed
were fitted to a theoretical curve drawn according to the excluded site
model developed by McGhee and von Hippel [48].
2.3.2. Polymerase stop assay (PSA)
The oligonucleotide containing a quadruplex-forming sequence
derived from c-myc oncogene (5′-GCGGCTCCTGTGAGGGTGGGGAGG
Fig. 1. ORTEP diagram of L1. Ellipsoids were drawn at the 50% probability level.

280
S. Gama et al. / Journal of Inorganic Biochemistry 160 (2016) 275–286
Fig. 2. Pack diagram of L1.
of the imine function of the corresponding Schiff base precursors, as
shown in Scheme 2.
The synthesized new anthracene-terpyridine (Ant-tpy) chelators, L1
and L2, were used to obtain complexes of the type [Cu(Ant-tpy)Cl2] and
[Pt(Ant-tpy)Cl]+. As mentioned in the introductory section, we have
considered that the presence of metal centres with different geometry
would influence the affinity and selectivity of metallated anthracene-
terpyridine derivatives towards DNA structures of different topology.
For example, Teulade-Fichou et al. have shown previously that the com-
plex [Cu(ttpy)(NO3)2] (ttpy = tolyl-terpyridine) has a higher selectivity
for G-quadruplex DNA over duplex DNA if compared with the Pt(II)
counterpart [Pt(ttpy)Cl]+. This difference was accounted by the square
pyramidal coordination geometry of the Cu(II) complex that might
hinder intercalation between two pairs of duplex DNA, unlike the
square planar Pt(II) congener that has the propensity to act as a DNA
intercalator [34]. Similar results were obtained by Vilar et al. with
a different set of copper(II) and platinum(II) terpyridine complexes
[36,39,40].
The chemical identity of compounds L1 and L2 was ascertained based
on their characterization by 1H and 13C NMR spectroscopy and by ESI-
MS. For L1, X-ray diffraction analysis confirmed the proposed
formulation. X-ray quality crystals of L1 were obtained by recrystalliza-
tion from a saturated solution of the compound in dichloromethane. L1
crystallized in a triclinic system, space group P1 with one independent
molecule in the asymmetric unit. The crystal data and final
refinement details are given in Table 1. A selection of bond lengths
and angles are given in Table 2. The respective ORTEP diagram is
presented in Fig. 1.
The molecular structure of L1 is defined by two almost planar frag-
ments, terpyridine and anthracene, connected by a spacer (Fig. 1).
These two planar units are almost perpendicular to each other, with a
dihedral angle between the respective l.s. planes of 77.70(4)° as illus-
trated in Fig. 2. The three pyridine rings present a trans-trans configura-
tion and are not coplanar. The dihedral angles between the l.s. planes of
the rings containing atoms N1 and N2, and atoms N2 and N3 are
The copper complexes (CuL1 and CuL2) were obtained by reacting
stoichiometric amounts of the desired anthracene-terpyridine chelator
and copper(II) chloride in methanol at room temperature (Scheme 3),
using a similar approach to the one described originally to synthesize
the simplest member of this family of coordination complexes,
[Cu(tpy)Cl2] [49]. The formation of CuL1 and CuL2 was almost
4.25(2)° and 8.91(2)°, respectively. Several short C\\H…O or C\\H…
C
contacts between adjacent molecules give rise to a three-dimensional
network as shown in Fig. 3.
Fig. 3. Pack diagram of L1 showing short C\\H…C and C\\H…O contacts (C3\\H3…O1 = 2.635, C13\\H13…C26 = 2.683, C15\\H15…C29 = 2.791, C21\\H21…C4 = 2.761).

S. Gama et al. / Journal of Inorganic Biochemistry 160 (2016) 275–286
281
with chemical shifts that are well in agreement with the values reported
in the literature for [Pt(tpy)Cl] (−2694 ppm [52]).
3.2. Biophysical studies: evaluation of G-quadruplex DNA binding
We have investigated the DNA binding of PtL1,2 and CuL1,2 in com-
parison with the respective metal-free ligands, L1 and L2, using a variety
of techniques (FRET melting studies, UV/Vis, CD and fluorescence titra-
tions, and polymerase stop assay). We also compared these results with
previously reported DNA binding studies with other metal-terpyridine
complexes.
Scheme 3. Synthesis of Cu(II) complexes.
immediate as indicated by the sudden appearance of a green color upon
addition of ligands. The complexes precipitated from each reaction mix-
ture as a green solid, and were characterized by ESI-MS and elemental
analysis. The negative ion mode ESI-MS spectra of CuL1 and CuL2 have
shown major peaks corresponding to the [M + Cl]− ions with the ex-
pected isotopic distribution pattern. This finding corroborated the for-
mation of the desired Cu(II) complexes having a single anthracene-
terpyridine coordinated ligand.
The Pt(II) compounds with the Ant-tpy chelators were synthesized
according to a procedure based on a method described by Annibale
et al. [50] for the synthesis of the congener [Pt(tpy)Cl]Cl. In this method,
[Pt(cod)Cl2] [51] was used as the starting precursor; the precursor rap-
idly reacted with L1 and L2 to afford PtL1 and PtL2, which were recov-
ered in moderate yield (ca. 60%) as orange solids (Scheme 4). We
have found that the use of a small excess of platinum (ca. 3%) and a lon-
ger reaction time (50 min vs. 15 min) increased the yield of our synthe-
sis (from 25% to ca. 60%) if compared with the synthesis carried out
using the conditions originally reported to obtain the parental complex
[Pt(tpy)Cl]Cl.
The chemical identity of the newly synthesized Pt(II) complexes was
ascertained by LC-MS and by multinuclear NMR spectroscopy. For both
complexes the positive ESI-MS spectra in methanol have shown the
presence of intense peaks corresponding to the cationic species
[Pt(L)Cl]+ (L = L1 and L2). By comparing the 1H and 13C NMR spectra
of PtL1 and PtL2, obtained using freshly prepared solutions of the
complexes, with the spectra of the corresponding ligand in the same
solvent, we observed a downfield shift in the peaks correspondent to
the terpyridine protons and carbons, namely for the 1/15 (1H: Δδ =
0.25 ppm, 13C: Δδ = 1.80 ppm), 2/14 (1H: Δδ = 0.37), and 3/13 (1H:
Δδ = 0.39 ppm; 13C: Δδ = 5.06) carbon/hydrogen atoms (see
Scheme 2 for atom numbering), due to the influence of the metal-
ligand bond. The 195Pt{1H} NMR spectrum exhibited only one signal
for each compound (−2720 ppm for PtL1 and −2700 ppm for PtL2)
3.2.1. Fluorescence resonance energy transfer (FRET)
Fluorescence resonance energy transfer (FRET) melting studies were
conducted to investigate the effect of the four metal complexes (PtL1,2
and CuL1,2) on the stability of two G-quadruplex DNA structures, name-
ly human telomeric (HTelo) and c-myc promoter (c-myc) sequences
(see Experimental Details for exact sequences). In addition, the affinity
of these compounds towards a duplex DNA sequence (ds26) was also
assessed. FRET melting curves of HTelo, c-myc and ds26 were obtained
(Figs. S1–S3) and melting temperature differences (ΔTm) (see Table 3)
were determined, with increasing concentrations (from 0 to 10 μM) of
the tested compounds. As shown in Table 3, the metal complexes induce
some stabilization of G-quadruplex (HTelo and c-myc); in contrast, they
presented very low values of ΔTm with the duplex DNA sequence.
To evaluate the effect of the metal on quadruplex DNA stabilization,
the corresponding metal-free ligands were also analysed under the
same experimental conditions (Table 3). The ΔTm values at 1 μM obtain-
ed for L1 and L2 are lower than the ΔTm values obtained for the corre-
sponding metal complexes; particularly, in case of the Pt(II)
complexes and with the c-myc sequence where the ΔTm values are ca.
3-fold (L1 vs PtL1) or 7-fold (L2 vs PtL2) higher for the complexes.
These results show that the presence of the metal is essential for high af-
finity (and selectivity) to quadruplex. This effect is more evident in the
case of the platinum(II) complexes, which display selectivity for c-myc
quadruplex DNA not only over duplex DNA but also over HTelo
quadruplex DNA. Moreover, the presence of a longer linker between
the chelating terpyridine unit and the anthracene moiety enhances
the interaction with G-quadruplex-forming sequences.
The selectivity G-quadruplex vs. duplex DNA was also evaluated and
confirmed by competition FRET melting assays where increasing
amounts of ct-DNA were added to a fixed mixture of FRET-labelled
G-quadruplex oligo and metal complex (at 1 μM concentration). No
significant change to the ΔTm values were observed upon addition of
300 equivalents of ct-DNA and in some cases (mainly with c-myc
Scheme 4. Synthesis of Pt(II) complexes.

282
S. Gama et al. / Journal of Inorganic Biochemistry 160 (2016) 275–286
DNA) even after addition of 600 equivalents of ct-DNA. This confirms
the selectivity of the tested Pt(II) and Cu(II) compounds to the
G-quadruplexes (Fig. 4).
The results obtained suggest that the inclusion of the anthracenyl
moiety in the terpyridine-metal complex system seems to increase the
interaction with G-quadruplexes when compared with simple
platinum(II) or copper(II) terpyridine complexes, i.e. without any
substituents in the aromatic ring. Some of our complexes display ΔTm
values that are similar to those exhibited by toluene-terpyridine Pt(II)
and Cu(II) derivatives (see Table S1 in the SI). For the later complexes,
their augmented G-quadruplex interaction was associated with the ex-
tension of the interlinked aromatic system with consequent increase of
π-π interactions. This is not likely to occur with the Pt(II) and Cu(II)
complexes described herein as the anthracenyl ring is attached to the
tpy unit using flexible linkers of different length. One possibility is that
the anthracenyl moiety interacts via π-π stacking with bases present
in the loops of the G-quadruplex structure providing higher affinity
and distinct selectivity to the metal-terpyridine complexes. However,
further studies will be required to clarify the exact binding mode of
the new complexes.
3.2.2. CD measurements
To study the effect of metal complexes on the HTelo and c-myc
quadruplex structures, CD titrations were carried out. It is known that
G-quadruplexes can exist in solution as a mixture of different topologies
(e.g. parallel and antiparallel G4s). The spectra of parallel quadruplexes
have a dominant positive band at 260 nm, whereas the spectra of anti-
parallel quadruplexes have a negative band at 260 nm and positive band
at 290 nm. In presence of K+, HTelo DNA adopts an antiparallel/parallel
hybrid conformation and exhibits a maximum at 290 nm and a
“shoulder” at around 260 nm. With the addition of the metal complexes
(1–10 eq) to HTelo quadruplex, the negative band at 260 nm appeared
and a decrease of the 290 nm band was observed, suggesting a change in
the conformation of HTelo quadruplex induced by the binding of the
metal complexes. C-myc quadruplex in the presence of K+ adopts a
predominantly parallel conformation. Addition of metal complexes
resulted in the decrease of the positive band intensity at 260 nm,
suggesting interaction with the structure (see Fig. 5 for a representative
example). Overall CD titration experiments suggest that the metal
complexes interact with HTtelo and c-myc quadruplex. It should be
noted that at higher concentrations, compound precipitation was
observed.
Fig. 4. FRET competition experiments of selected complexes at 1 μM concentration
showing the variation (ΔTm) of a) FAM-c-myc-TAMRA and b) FAM-HTelo-TAMRA DNA
melting temperature with increased concentration (0–120 μM) of ct-DNA
(corresponding to 0 to 600 equivalents).
transfer processes between the fluorophore and the terpyridine/metal-
terpyridine. A similar behaviour has been reported by Bonnet et al. for a
metal-terpyridine system attached to a fluorophore where some energy
absorbed by the fluorophore is donated to the metal centre whilst in
the metal-free terpyridine system, this process is hampered [53]. Further
studies will have to be carried out to characterise fully the photophysical
behaviour of these compounds upon DNA binding.
An analogous behaviour was observed upon addition of ct-DNA: a
decrease in the emission of the metal complexes (CuL1, PtL1, CuL2 and
PtL2) was observed upon increasing amounts of ct-DNA (Fig. S7 in SI)
while a very large increase in emission was observed for the metal-
free ligands (L1 and L2). Absorption titrations for these compounds
were conducted to obtain the affinity constants (Fig. S8).
3.2.3. Spectrofluorimetric titrations
In order to quantify the affinity of the compounds towards quadruplex
and duplex structures, we performed fluorimetric titrations with duplex
DNA (ct-DNA) and G-quadruplex DNA sequence c-myc. Upon addition
of c-myc DNA to solutions of the corresponding metal complex, a decrease
in the emission intensity was observed. In contrast, an increase in fluores-
cence emission was observed upon addition of G4 to L1 or L2 solutions
(Fig. 6 and S6 in SI). The different emission behaviours between the free
ligands and the metal complexes could be related to differences in energy
Table 3
ΔTm for 1 μM concentration of compound. Values determined from fitting average ΔTm
pointsa obtained from FRET melting curves of labelled DNA oligomers in the presence of
the different compounds.
ΔTm/°C
ds26
HTelo
c-myc
L1
0.68 0.76
0.00 0.65
0.00 0.84
0.53 0.38
0.39 0.81
0.58 0.79
2.91 0.54
9.42 0.56
5.31 0.45
5.00 0.70
9.57 0.49
5.92 0.65
2.98 0.68
10.22 0.56
2.07 0.65
2.5 1.0
15.60 0.70
7.26 0.27
PtL1
CuL1
L2
PtL2
CuL2
Fig. 5. CD spectra of 5 μM HTelo DNA recorded in the presence of different concentrations
of PtL2 in LiCac pH 7.2 buffer (KCl 10 mM, LiCl 90 mM, lithium cacodylate 10 mM).
a
std. dev. on a minimum of three replicates.

S. Gama et al. / Journal of Inorganic Biochemistry 160 (2016) 275–286
283
Fig. 6. Spectrofluorimetric titrations of c-myc quadruplex DNA with (a) L2, (b) CuL2 and (c) PtL2 in Tris HCl buffer (Tris HCl 50 mM, pH 7.4, KCl 10 mM). Insets show the plotting of emission
fluorescence at 412 nm vs. molar ratio ([DNA]/[Compound]).

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S. Gama et al. / Journal of Inorganic Biochemistry 160 (2016) 275–286
Table 4
(polyacrylamide gel electrophoresis) gels obtained after running the
polymerase stop assay in the presence of CuL2 (see SI for L1, L2 and
CuL1). The relative intensities of the band associated to the shorter
oligonucleotide formed (i.e. the one formed up to the point that the
quadruplex forms) were plotted and the values corresponding to the
50% of ‘G4 band’ formation in relation to the control (i.e. the system in
the absence of ligand or K+) were calculated. It should be noted that
even at the highest concentration of CuL2, we did not see a signifi-
cant decreases (ca. 30%) of the upper band associated to the oligo
formed if G-quadruplex is not formed. This suggests that CuL2 is
not a particularly strong G-quadruplex binder, which is consistent
with the other biophysical data (see above). It should be noted that
when we carried out polymerases stop assays with the platinum
complexes PtL1 and PtL2, precipitation was observed and hence
these two complexes could not be studied using this assay (Figs.
S9–10 in SI).
The polymerase stop assays show that the copper complexes have
the ability to halt polymerase – upon stabilization of the G-quadruplex
– at lower concentrations (EC50 3.18 μM and 1.34 μM for CuL1 and
CuL2 respectively) that the metal-free ligands (EC50 for both L1 and
L2 N 20 μM). These results also show that CuL2 is more efficient at stop-
ping the activity of polymerase than CuL1. These results are consistent
with the biophysical studies presented in the previous sections.
To confirm that the halting of the polymerase activity was due to
quadruplex formation, a control experiment was carried out where
the polymerase stop assay was performed with a template oligonucleo-
tide which does not containing a quadruplex-forming sequence
(Fig. S10). This experiment didn't show any polymerase pausing prod-
uct in the presence of L1, L2, CuL1 or CuL2 indicating that the EC50 values
obtained in the presence of the copper(II) complexes (see above) are
due to the formation of quadruplexes.
Binding constantsa (log Ks) calculated from the spectroscopic titrations of compounds
with c-myc quadruplex DNA and calf thymus DNA.
Compound
c-myc
Calf thymus DNA
L1
3.11b
5.62
5.82
3.71b
6.13
6.32
5.17
4.89
5.22
4.99
5.17
5.16
CuL1
PtL1
L2
CuL2
PtL2
a
Processing of titration data by means of Scatchard equation.
Data obtained from UV–Vis titrations.
b
The affinity binding constants were calculated through Scatchard plot
analysis and are shown in Table 4. The log K values ranged from 3.1 to 6.1
depending on the compound and DNA structure used. The log K values
confirm the ΔTm obtained by FRET: the metal complexes have signifi-
cantly higher affinities (log K ≈ 5.6–6.3) than the metal-free ligands
(log K ≈ 3.1–3.7), highlighting the important role the metal centre
plays. We hypothesise that the metal centre ‘forces’ the terpyridine
moiety into a planar arrangement thus making available for efficient π-
stacking. Compounds with a longer linker (L2, CuL2 and PtL2) display
higher affinities than those with the shorter linker (L1, CuL1 and PtL1).
Furthermore, the Pt(II) complexes also display higher log K values to-
wards c-myc quadruplex DNA than the Cu(II) complexes [54,55].
3.2.4. Polymerase stop assay
In order to evaluate the ability of the compounds to form a G-
quadruplex structure as part of a longer sequence, we performed poly-
merase stop assays. As has been discussed elsewhere [56], in this assay a
polymerase enzyme is added to a mixture of a labelled primer and a
DNA template which contains a quadruplex forming sequence. The po-
lymerase is able to extend the full length of the template if no
quadruplex forms. However, if a stable quadruplex is formed (e.g. by
adding mM concentrations of K+-lane 8 in Fig. 7 – or a quadruplex-
stabilising ligand – lanes 3–7 in Fig. 7) in the template sequence, the po-
lymerase stops at the point where the quadruplex forms. Therefore, the
length of the oligonucleotides formed in this assay is an indication of
whether a stable quadruplex forms or not. Fig. 7 shows the PAGE
4. Conclusions
The new Pt(II) and Cu(II) complexes herein reported showed affinity
for quadruplex-forming sequences (HTelo and c-myc) with a good
selectivity over duplex DNA (ds26 and ct-DNA), as shown by the FRET
assays. Importantly, the metal-free ligands do not have significant affin-
ity to any of the DNA sequences investigated, which shows that the
Fig. 7. Polymerase Stop Assay of the c-myc sequence in the presence of CuL2 highlighting the bands (enclosed in rectangle) corresponding to the shorter oligonucleotide obtained when
quadruplexes form. The 1st lane shows the primer, the 2nd lane shows the mixture of primer and template, lanes 3 to 7 show the result of the polymerase assay upon mixing the primer,
template and increasing concentrations of compound; the 8th lane shows the result of the assay with the primer, template and K+. The plot of the relative intensities vs. concentration of
compound is on the left.

S. Gama et al. / Journal of Inorganic Biochemistry 160 (2016) 275–286
285
presence of the metal is essential for high affinity (and selectivity). This
effect is more evident in the case of the platinum(II) complexes. More-
over, the presence of a longer linker between the chelating terpyridine
unit and the anthracene moiety enhances the interaction with G-
quadruplex-forming sequences. These trends were further
corroborated and quantified by the determination of the affinity binding
constants for the interaction of the different compounds with G-
quadruplex and duplex DNA structures, which were measured based
on fluorescence and spectrophotometric titrations.
Tpy
TRIS
UV
terpyridine
tris(hydroxymethyl)aminomethane
ultraviolet
UV/Vis ultraviolet-visible
Acknowledgements
This work was supported by COST action CM1105 (Functional metal
complexes that bind to biomolecules), Fundação para a Ciência e a
Tecnologia – FCT (projects PTDC/QUI-QUI/114139/2009, RECI/QEQ-
QIN/0189/2012 and EXCL/QEQ-MED/0233/2012; grants SFRH/BPD/
29564/2006 to SGama and FCT Investigator Grant to FMendes), EPSRC
(EP/H005285/1), Collaborative Research Centre ChemBioSys (CRC
1127) funded by the Deutsche Forschungsgemeinschaft (DFG) and
Newton Fellow of Royal Society. The authors would also like to thank
Célia Fernandes for the Mass Spectrometry analysis, which was carried
out on a QITMS instrument, acquired with the support of the Programa
Nacional de Reequipamento Científico (Contract REDE/1503/REM/
2005-ITN) of FCT and is part of RNEM-Rede Nacional de Espectrometria
de Massa.
Interestingly, the [Pt(Ant-tpy)Cl]+ complexes described herein tar-
gets the G-quadruplex DNA structures more selectively than the copper
analogue, [Cu(Ant-tpy)Cl2], despite the expected better ability of the
planar [Pt(tpy)Cl]+ unit to intercalate between adjacent base pairs of
duplex DNA if compared with the [Cu(Ant-tpy)Cl2] unit. Altogether,
these results point out that the affinity and selectivity of these
anthracene-containing M(II)-tpy (M = Cu, Pt) complexes towards
G-quadruplex DNA structures are certainly justified by an intricate in-
terplay of effects such as π-stacking with the accessible G-tetrad or
with loops and grooves of the quadruplex DNA elements, as well as elec-
trostatic interactions involving the more polar pendant arm.
Our results indicate that the combination of the terpyridine scaffold
linked to a ‘classical’ DNA intercalator can afford metal(II) complexes
with affinity and selectivity for G-quadruplex DNA. This effect needs
to be better explored, in particular in order to clarify whether the
anthracenyl and metal-terpyridine moieties are acting in a synergistic
way when the complexes bind to G-quadruplexes. Nevertheless, these
encouraging results give impetus to pursue with these studies using
different pendant intercalators, namely those having a more extended
aromatic structure and/or a cationic nature that might improve the
binding affinity and water solubility of the final compounds. It should
be noted that when.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jinorgbio.2016.04.002.
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