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Bipyrimidine ruthenium(II) arene complexes: structure, reactivity and cytotoxicity.
Original citation:
Betanzos-Lara, Soledad, Novakova, Olga, Deeth, Robert J., Pizarro, Ana M., Clarkson,
Guy J., Liskova, Barbora, Brabec, Viktor, Sadler, Peter J. and Habtemariam, Abraha.
(2012) Bipyrimidine ruthenium(II) arene complexes : structure, reactivity and cytotoxicity.
JBIC Journal of Biological Inorganic Chemistry, Vol.17 (No.7). pp. 1033-1051. ISSN
0949-8257
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1
For submission to JBIC.
2
Bipyrimidine Ruthenium(II) Arene Complexes:
3
Structure, Reactivity and Cytotoxicity
4
Soledad Betanzos-Lara,a,b Olga Novakova,c Robert J. Deeth,a Ana M. Pizarro,a Guy J.
5
Clarkson,a Barbora Liskova,c Viktor Brabec,c Peter J. Sadlera and Abraha
6
Habtemariama
a
7
Department of Chemistry, University of Warwick, Coventry, UK CV4 7AL; bCurrent
8
address: Departamento de Química Inorgánica, Facultad de Química, Universidad Nacional
9
Autónoma de México (UNAM), Ciudad Universitaria, Coyoacán, México, D.F. 04510;
10
11
12
c
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska
135, CZ61265 Brno, Czech Republic
E-mail: A.Habtemariam@warwick.ac.uk
13
1
1
ABSTRACT. The synthesis and characterization of complexes [(η6-arene)Ru(N,N')X][PF6]
2
where
3
hexamethylbenzene (hmb), indane (ind), or 1,2,3,4-tetrahydronaphthalene (thn); N,N' is
4
2,2'-bipyrimidine (bpm), and X is Cl, Br, or I are reported, including the X-ray crystal
5
structures of [(η6-p-cym)Ru(bpm)I][PF6] (3), [(η6-bip)Ru(bpm)Cl][PF6] (4), [(η6-
6
bip)Ru(bpm)I][PF6] (6), and [(η6-etb)Ru(bpm)Cl][PF6] (7). Complexes in which N,N' is
7
1,10-phenanthroline (phen), 1,10-phenanthroline-5,6-dione (phendio), or 4,7-diphenyl-
8
1,10-phenanthroline (bathophen) were studied for comparison. The RuII arene complexes
9
undergo ligand exchange reactions in aqueous solution at 310 K; their half-lives for
10
hydrolysis vary from 14 to 715 min. Density functional theory (DFT) calculations on [(η6-
11
p-cym)Ru(bpm)Cl][PF6] (1), [(η6-p-cym)Ru(bpm)Br][PF6] (2) and 3–6 suggest that
12
aquation occurs via an associative pathway and that the reaction is thermodynamically
13
favorable when the leaving ligand is I > Br ≈ Cl. pKa* values for the aqua adducts of the
14
complexes range from 6.9 to 7.32. A binding preference for 9-ethylguanine (9-EtG)
15
compared to 9-ethyladenine (9-EtA) was observed for 1, [(6-hmb)Ru(bpm)Cl]+ (8), [(6-
16
ind)Ru(bpm)Cl]+ (9), [(6-thn)Ru(bpm)Cl]+ (10), [(6-p-cym)Ru(phen)Cl]+ (11) and [(6-
17
p-cym)Ru(bathophen)Cl]+ (13) in aqueous solution at 310 K. The X-ray crystal structure of
18
the guanine complex [(6-p-cym)Ru(bpm)(9-EtG-N7)][PF6]2 (14) shows multiple H-
19
bonding. DFT calculations show that the 9-EtG adducts of all complexes are
20
thermodynamically preferred compared to those of 9-EtA. However, the bmp complexes
21
are inactive towards A2780 human ovarian cancer-cells. Calf-thymus (CT)-DNA
22
interactions for 1 and 11 consist of weak coordinative, intercalative, and monofunctional
arene
is
para-cymene
(p-cym),
biphenyl
(bip),
ethyl
benzoate
(etb),
2
1
coordination. Binding to biomolecules such as glutathione (GSH) may play a role in
2
deactivating the bpm complexes.
3
4
Key Words
Ruthenium, arene, bipyrimidine, hydrolysis, nucleobase, DNA.
5
Introduction
6
The well-established mechanism of action of the cytotoxic drug cisplatin is the alteration
7
of the secondary structure of DNA via coordination to the N7 atom of a guanine (G) or an
8
adenine (A) base, which requires its prior aquation in the cell to generate the more reactive
9
aqua complexes [Pt(NH3)2(OH2)Cl]+ and [Pt(NH3)2(OH2)2]2+ [1, 2]. In general, aquation
10
can be an important activation step for transition metal complexes prior to their
11
coordination to biomolecules [3]. Certain organometallic RuII complexes of the type [(η6-
12
arene)Ru(XY)Z]n+ where XY is a bidentate chelating ligand and Z is a leaving group,
13
exhibit promising cytotoxic activity against a variety of cancer cell lines, including
14
cisplatin-resistant cells [4, 5]. The nature of the arene, the chelating ligand, and the leaving
15
group can have a major influence on the rates of activation (towards hydrolysis and/or
16
binding to biomolecules) as well as on the cytotoxic activity [6]. It appears that the
17
presence of a more hydrophobic arene ligand along with a single ligand exchange site is
18
often associated with significant anticancer activity. Blocking ligand exchange reactions in
19
the remaining two coordination sites can usually be achieved by coordination of a stable
20
bidentate ligand; in this regard, particularly effective are those containing N,N'-heterocyclic
21
groups [7, 8, 9].
3
1
In the present work, we have studied and contrasted the chemical reactivity of a series of
2
organometallic RuII complexes of the type [(η6-arene)Ru(N,N')X][PF6] containing a N,N'-
3
chelating ligand, as well as various arenes, and different halides (X). Their aqueous solution
4
chemistry as well as the nucleobase binding (to 9-EtG and 9-EtA) were investigated. Their
5
potential as cytotoxic agents was explored not only by determining IC50 values against
6
A2780 (human ovarian), A2780cis (human ovarian cisplatin resistant), A549 (human lung)
7
or HCT116 (human colon) cancer cell lines but also by studying DNA interactions in cell-
8
free media. γ-Glutamyl-cysteinyl-glycine (glutathione, GSH) coordination to Pt(II) is
9
known to inhibit DNA binding contributing to cisplatin resistance in tumor cells [10] and
10
depending on its relative concentration [11, 12], it can both facilitate and/or inhibit
11
ruthenium interactions with DNA [13]. Reactions of GSH with a representative inactive
12
RuII arene complex (1) in aqueous solution at 310 K were therefore investigated in order to
13
establish whether GSH may play a role in the activity of this family of complexes.
14
4
1
Materials and Methods
2
Materials. RuCl3·3H2O was acquired from Precious Metals Online (PMO Pty Ltd) and
3
used
4
phenanthroline-5,6-dione (phendio), 4,7-diphenyl-1,10-phenanthroline (bathophen), 9-
5
ethylguanine (9-EtG), 9-ethyladenine (9-EtA), and KPF6 were obtained from Sigma-
6
Aldrich. KBr and KI (reagent grade) were obtained from Fisher. The RuII arene precursor
7
dimers [(6-arene)RuX2]2 where arene is para-cymene (p-cym), biphenyl (bip),
8
hexamethylbenzene (hmb), indane (ind), or tetrahydronaphthalene (thn) and X is Cl, Br, or
9
I were synthesized according to a previously reported method [14]. The dimer [(6-
10
etb)RuCl2]2 where etb is ethylbenzoate was synthesized following published literature [15].
11
The solvents used for UV-vis absorption spectroscopy were dry methanol (reagent grade)
12
and deionized water. For NMR spectroscopy, the solvents used were acetone-d6, DMSO-d6,
13
methanol-d4 and D2O obtained from Aldrich. All chemicals were used without further
14
purification. Cisplatin was obtained from Sigma-Aldrich (Prague, Czech Republic).
15
Chloridodiethylenetriamineplatinum(II) chloride ([PtCl(dien)]Cl) was a generous gift of
16
Professor Giovanni Natile from the University of Bari. Restriction endonucleases NdeI and
17
HpaI were purchased from New England Biolabs. Acrylamide, bis(acrylamide), and
18
ethidium bromide (EtBr) were obtained from Merck KgaA (Darmstadt, Germany). Agarose
19
was purchased from FMC BioProducts (Rockland, ME). Radioactive reagents were
20
obtained from Amersham (Arlington Heights, IL, U.S.A.). Stock aqueous solutions of
21
metal complexes (5 × 10–4 M) for the biophysical and biochemical studies were filtered and
22
stored at room temperature in the dark. The concentrations of ruthenium or platinum in the
23
stock solutions were determined by flameless atomic absorption spectrometry (FAAS). Calf
as
received.
2,2'-bipyrimidine
(bpm),
1,10-phenanthroline
(phen),
1,10-
5
1
thymus CT-DNA (42% G + C, mean molecular mass ca. 2 × 107) was also prepared and
2
characterized as described previously [16, 17]. pSP73KB (2455 bp) plasmid was isolated
3
according to standard procedures [23].
4
Synthesis of Ruthenium Complexes. Complexes [(η6-arene)Ru(N,N')X][PF6] where arene
5
is p-cym, bip, etb, ind, hmb, or thn; N,N' is bpm, phen, phendio, or bathophen; and X is Cl,
6
Br, or I were synthesized as previously described [6]. Typically, two mol equiv of the N,N'
7
chelating ligand and two mol equiv of KPF6 were added to a solution of one mol equiv of
8
the appropriate RuII arene dimer in of dry methanol (20 mL) with constant stirring over 48
9
h upon which the precipitate formed was collected by filtration. The remaining solution was
10
concentrated and portions of Et2O were added to further precipitate the product which was
11
again collected by filtration. Both solids were combined and washed with portions of Et2O
12
and MeOH and dried overnight under vacuum resulting in microcrystalline products.
13
Details of the amounts of reactants, volumes of solvents, color changes, and nature of the
14
products are described in the supporting information for the individual reactions, as well as
15
any variations in the synthetic procedure. Some complexes were also characterized by 13C
16
NMR spectroscopy.
17
X-ray Crystallography. Diffraction data were collected either on an Oxford Diffraction
18
Gemini four-circle system with a Ruby CCD area detector or on a Siemens SMART three-
19
circle system with CCD area detector equipped with an Oxford Cryosystem Cooler. All
20
structures were refined by full-matrix least squares against F2 using SHELXL 97 [18]. The
21
structures of complexes 3, 4, 6, 7 and 14 were solved by direct methods using SHELXS
22
[19] (TREF) with additional light atoms found by Fourier methods. Hydrogen atoms were
23
added at calculated positions and refined using a riding model with freely rotating methyl
6
1
groups. Anisotropic displacement parameters were used for all non-H atoms; H-atoms were
2
given isotropic displacement parameters equal to 1.2 (or 1.5 for methyl hydrogen atoms)
3
times the equivalent isotropic displacement parameter of the atom to which the H-atom is
4
attached.
5
NMR Spectroscopy. 1H and 13C NMR spectra were acquired in 5 mm NMR tubes at 298 K
6
(unless otherwise stated) on either a Bruker AV-400, Bruker DRX-500, Bruker AV III 600
7
or Bruker AV II 700 NMR spectrometers. All data processing was carried out using
8
XWIN-NMR version 3.6 (Bruker U.K. Ltd.). 1H NMR chemical shifts were internally
9
referenced to TMS via 1,4-dioxane (δ = 3.71) or residual MeOH (δ = 3.31). 1D spectra
10
were recorded using standard pulse sequences. Typically, data were acquired with 128
11
transients into 16 k data points over a spectral width of 14 ppm. 2D COSY or TOCSY and
12
NOESY spectra were recorded using standard pulse-pulse sequences. Typically, data were
13
acquired with 72 transients into 1024 k data points over a spectral width of 14 ppm using a
14
relaxation delay of 1.5 s and a mixing time of 0.06 s.
15
Elemental Analysis. Elemental analyses were performed by Exeter Analytical (U.K. Ltd.)
16
using an CE-440 Elemental Analyzer.
17
High Resolution Electrospray Mass Spectrometry (HR-MS). HR-MS data were
18
obtained on a Bruker MaXis UHR-TOF. All the samples were analyzed by positive-ion
19
ESI(+) mass spectra. Samples were prepared either in 100% H2O or 95% MeOH/5% H2O
20
mixture and typically injected at 2 μL min–1, nebulizer gas (N2) 0.4 bar, dry gas (N2) 4 L
21
min–1 and dry temp 453 K, Funnel RF 200V, Multiple RF 200, quadrupole ion energy 4 eV,
22
collision cell 5 eV, ion cooler RF settings, ramp from 50 to 250 V, unless otherwise stated.
7
1
UV-vis Absorption Spectroscopy. UV-vis absorption spectra were recorded on a Cary 50-
2
Bio spectrophotometer with a PTP1 Peltier temperature controller or on a Beckman DU
3
7400 UV-Vis spectrophotometer equipped with a thermoelectrically controlled cell holder,
4
in a 1-cm pathlength quartz cells (600 μL). Spectra were recorded at 310 K in deionized
5
water from 220 to 800 nm and were processed using UV-Winlab software for Windows 95
6
controller
7
pH* Measurement. pH values were measured at ambient temperature using a Corning 240
8
pH meter equipped with a micro combination KNO3 (chloride free) electrode calibrated
9
with Aldrich buffer solutions of pH 4, 7, and 10. The pH* values (pH meter reading
10
without correction for effects of deuterium (D) on glass electrode) of NMR samples in D2O
11
were measured at about 298 K directly in the NMR tube, before and after recording NMR
12
spectra, using the same method. The pH* values were adjusted with dilute NaOH or HNO3
13
solutions in D2O.
14
pKa* Values. For determinations of pKa* values (for solutions in D2O), the pH* values of
15
solutions of the aqua complexes in D2O were varied from ca. pH* 1 to 12 by the addition of
16
dilute NaOH or HNO3 solutions in D2O, and 1H NMR spectra were recorded. The chemical
17
shifts of the arene ring protons were plotted against pH* values. The pH* titration curves
18
were fitted to the Henderson-Hasselbalch equation using ORIGIN version 8.0, with the
19
assumption that the observed chemical shifts are weighted averages according to the
20
populations of the protonated and deprotonated species. These pKa* values can be converted
21
to pKa values by use of the equation pKa = 0.929 pKa*+ 0.42 suggested by Krezel and Bal
22
[20] for comparison with related values in the literature.
8
1
Aqueous Solution Chemistry. Hydrolysis of the RuII arene halido complexes was
2
monitored by UV-vis spectroscopy. The nature of the hydrolysis products as well as the
3
extent of the reactions were verified by 1H NMR spectroscopy or HR-MS. For UV-vis
4
spectroscopy the complexes were dissolved in methanol and diluted with H2O to give 100
5
μM solutions (5% MeOH/95% H2O). The absorbance was recorded at several time
6
intervals at the selected wavelength (at which the maximum changes in absorbance were
7
registered) over ca. 8–16 h at 310 K. Plots of the change in absorbance with time were
8
computer-fitted to the pseudo first-order rate equation, A = C0 + C1e–kt (where C0 and C1 are
9
computer-fitted constants and A is the absorbance corresponding to time) using Origin
10
version 8.0 (Microcal Software Ltd.) to give the half-lives (t1/2, min) and rate constant
11
values (k, min–1). For 1H NMR spectroscopy, the complexes were dissolved in MeOD-d4
12
and diluted with D2O to give 100 μM solutions (5% MeOD-d4/95% D2O). The spectra were
13
acquired at various time intervals on a Bruker DMX 700 spectrometer (1H = 700 MHz)
14
using 5 mm diameter tubes. All data processing was carried out using XWIN NMR version
15
2.0 (Bruker U.K. Ltd.). The relative amounts of RuII arene halido species or aqua adducts
16
(determined by integration of peaks in 1H NMR spectra) were quantified.
17
Rate of Arene Loss. The complexes were dissolved in MeOD-d4 and diluted with D2O to
18
give 100 μM solutions (5% MeOD-d4/95% D2O). Arene loss over time was followed by 1H
19
NMR spectroscopy at 310 K for 24 h.
20
Computational Studies. DFT calculations were carried out using the 2009 version of the
21
Amsterdam Density Functional (ADF) program [21]. Uncontracted Slater Type Orbital
22
(STO) basis sets comprised a triple-ζ plus 5p orbital set (TZP) on Ru with double-ζ plus
23
polarization (DZP) on all other atoms. Default convergence criteria were applied for Self9
1
Consistent Field (SCF) and cartesian geometry optimizations. For optimizations in internal
2
coordinates, in particular transition state (TS) searches, the angle threshold was set to 1.5°
3
(default = 0.5 º). This criterion was relaxed due to the long bond lengths at the transition
4
states, which make it harder to define torsional terms accurately. The same problem occurs
5
for reactant and product species because the respective entering and leaving groups are
6
included in the calculation, and their relatively weak interaction with the rest of the
7
complex again leads to less well defined torsional terms. However, the energetic
8
consequences of relaxing the angle constraints are negligible. The ADF program reported a
9
single negative eigenvalue in the Hessian matrix for all transition state optimizations. A
10
representative TS was confirmed as a first order saddle point with frequency calculations as
11
described earlier [22]. The conductor-like screening model (COSMO) as implemented in
12
ADF was used to simulate the aqueous environment with ε = 78.4, probe radius = 1.9 Å,
13
and the ND parameter which controls integration accuracy set to 4 (default 3). The atomic
14
radii (Å) used were Ru = 1.950, O = 1.517, C = 1.700, N = 1.608, H = 1.350, Cl = 1.725,
15
Br = 1.850, and I = 1.967.
16
DFT-Geometry Optimization of DNA Model Nucleobase Adducts. Geometry
17
optimizations were carried out for the 9-EtG and 9-EtA adducts of [(6-p-cym)Ru(bpm)(9-
18
EtG-N7)]2+ (1-9EtG), [(6-p-cym)Ru(bpm)(9-EtA-N7)]2+ (1-9EtA), [(6-hmb)Ru(bpm)(9-
19
EtG-N7)]2+ (8-EtG), [(6-hmb)Ru(bpm)(9-EtA-N7)]2+ (8-EtA) [(6-ind)Ru(bpm)(9-EtG-
20
N7)]2+ (9-EtG), [(6-ind)Ru(bpm)(9-EtA-N7)]2+ (9-EtA), [(6-thn)Ru(bpm)(9-EtG-N7)]2+
21
(10-EtG-N7), [(6-thn)Ru(bpm)(9-EtG-N3)]2+ (10-EtG-N3), [(6-thn)Ru(bpm)(9-EtA-
22
N7)]2+ (10-EtA), [(6-p-cym)Ru(phen)(9-EtG-N7)]2+ (11-EtG), [(6-p-cym)Ru(phen)(9-
23
EtA-N7)]2+ (11-EtA), [(6-p-cym)Ru(bathophen)(9-EtG-N7)]2+ (13-EtG), and [(6-p10
1
cym)Ru(bathophen)(9-EtA-N7)]2+ (13-EtA), for the free 9-ethylguanine and 9-ethyladenine
2
molecules, and for the RuII arene cations without the bound 9-EtG or 9-EtA. The energies
3
of the separate optimized fragments were subtracted from the energy of the whole RuII
4
arene nucleobase adducts to obtain the total binding energy of 9-EtG and 9-EtA in each
5
complex.
6
DNA Binding Kinetics. Calf thymus DNA (CT-DNA) and plasmid DNAs were incubated
7
with the RuII arene complexes or platinum complexes in 10 mM NaClO4 (pH ≈ 6) at 310 K
8
for 24 h. For each individual assay the values of rb (rb values are defined as the number of
9
atoms of the metal bound per nucleotide residue) were determined by Flameless Atomic
10
Absorption Spectrometry (FAAS).
11
DNA Transcription by RNA Polymerase In Vitro. Transcription of the (NdeI/HpaI)
12
restriction fragment of pSP73KB DNA with T7 RNA polymerase and electrophoretic
13
analysis of the transcripts were performed according to the protocols recommended by
14
Promega (Promega Protocols and Applications, 43−46 (1989/90)) as previously described
15
[23]. The DNA concentration used was 3.9 × 10−5 M (0.0125 g /L) (0.25 g/sample)
16
(related to the monomeric nucleotide content) and the concentration of complexes was ca.
17
1.17 × 10−6 M.
18
19
Unwinding of Negatively Supercoiled DNA. Unwinding of closed circular supercoiled
20
pUC19 plasmid DNA was assayed by an agarose gel mobility shift assay [24]. The mean
21
unwinding angle can be calculated from the equation Φ = −18σ/rb(c), where σ is the
22
superhelical density (representing the number of turns added or removed relative to the
23
total number of turns in the relaxed plasmid, indicating the level of supercoiling), and rb(c)
11
1
is the rb value at which the supercoiled and nicked forms comigrate [24]. Samples of
2
plasmid DNA at the concentration of 1.0 × 10−4 M (0.032 g/L) (0.5 g/ sample) (related
3
to the monomeric nucleotide content) were incubated with the RuII arene complexes at 310
4
K for 24 h. All samples were precipitated by ethanol and redissolved in the TAE (Tris-
5
aceate/EDTA, pH = 8.0) buffer to remove free, unbound RuII arene complexes. One aliquot
6
of the precipitated sample was subjected to electrophoresis on 1% agarose gels running at
7
298 K with TAE buffer and the voltage was set at 25 V. The gels were then stained with
8
ethidium bromide (EtBr), followed by photography with a transilluminator. Electron
9
Absorption Spectrometry (EAS) and FAAS were used for the determination of rb values.
10
Circular Dichroism (CD). Isothermal CD spectra of CT-DNA modified by the RuII arene
11
complexes at a concentration of 3.3 × 10–4 M were recorded at 298 K in 10 mM NaClO4 by
12
using a Jasco J-720 spectropolarimeter equipped with a thermoelectrically controlled cell
13
holder. The cell pathlength was 1 cm. CD spectra were recorded in the range of 230−600
14
nm in 0.5 nm increments with an averaging time of 0.5 s.
15
Flow Linear Dichroism (LD). Flow LD spectra were collected by using a flow Couette
16
cell in a Jasco J-720 spectropolarimeter adapted for LD measurements. The flow cell
17
consists of a fixed outer cylinder and a rotating solid quartz inner cylinder, separated by a
18
gap of 0.5 mm, giving a total pathlength of 1 mm. LD spectra of DNA at the concentration
19
3.3 × 10–4 M modified by the RuII arene complexes were recorded at 298 K in 10 mM
20
NaClO4.
21
Other Physical Methods. The FAAS measurements were carried out on a Varian AA240Z
22
Zeeman atomic absorption spectrometer equipped with a GTA 120 graphite tube atomizer.
12
1
The PAA gels were visualized by using a BAS 2500 FUJIFILM bioimaging analyzer, with
2
the AIDA image analyzer software (Raytest, Germany).
3
Cancer Cell Growth Inhibition. After plating, human ovarian A2780 and cisplatin-
4
resistant A2780cis cancer cells were treated with RuII arene complexes on day 3, and
5
human lung A549 and human colon HCT116 cancer cells on day 2, at concentrations
6
ranging from 0.1 to 100 μM. Solutions of the RuII complexes were made up in 0.125%
7
DMSO to assist dissolution (0.03% final concentration of DMSO per well in the 96-well
8
plate). Cells were exposed to the complexes for 24 h, washed, supplied with fresh medium,
9
allowed to grow for three doubling times (72 h), and then the protein content measured
10
(proportional to cell survival) using the sulforhodamine B (SRB) assay [25].
11
Reactions with Glutathione (GSH). A solution containing [(η6-p-cym)Ru(bpm)Cl][PF6]
12
(1) (100 μM) and GSH (10 mM) was incubated at 310 K in D2O and the changes monitored
13
by 1H NMR and UV-vis spectroscopy for 24 h.
14
Results and Discussion
15
Synthesis and Characterization. The [(6-arene)Ru(N,N')X]n+ complexes studied in this
16
work are shown in Figure 1. The monocationic RuII arene halido complexes 1−13 and the
17
9-EtG-N7 complex 14 were synthesized as PF6 salts in good yields (>50% in almost all
18
cases). All the complexes were fully characterized by 1D and 2D 1H NMR methods as well
19
as 1D 13C NMR. The molecular structures of complexes 3, 4, 6, 7 and 14 were determined
20
by single crystal X-ray diffraction. The molecular structure of complex 1 has previously
21
been published [14b]. Selected bond lengths and angles are given in Table 1, the structures
22
with numbering schemes are shown in Figure 2 and the crystallographic data are listed in
23
Table S1. In all cases, the complexes adopt the familiar pseudo-octahedral three-legged
13
1
piano stool geometry common to all other RuII arene structures [26] with the RuII atom π-
2
bonded to the corresponding arene ligand (p-cym in 3 and 14; bip in 4 and 6; or etb in 7),
3
coordinated to a chloride (4 and 7), to an iodide (3 and 6), or to N7 of 9-EtG (14), and to
4
two nitrogen atoms of the chelating ligand 2,2'-bipyrimidine (bpm) which constitute the
5
three legs of the piano stool.
6
The values for Ru–arene(centroid) bond lengths are comparable to analogous RuII arene
7
complexes containing N,N' chelated ligands [27, 56]. Neither the nature of the
8
corresponding N,N' chelating ligand, the arene nor the halogen greatly influences the
9
corresponding Ru–arene(centroid) distances (ca. 1.70 Å).The corresponding Ru–I bond lengths
10
in 3 and 6 are also within the same range (ca. 2.7 Å) and are slightly shorter compared to
11
other RuII arene complexes containing iodide as a leaving group [28, 29, 30]. Similarly, the
12
Ru−Cl bond lengths are almost the same (ca. 2.4 Å). The Ru(1)−N(1)(bpm) and
13
Ru(1)−N(8)(bpm) bond lengths in these arene complexes are ca. 2.09 Å. For the four halido
14
complexes, the Ru–N,N' bond lengths are significantly longer than those found in the
15
crystal structures of similar arene RuII arene bipyridine complexes [31]. The
16
N(1)−Ru(1)−N(8) bond angles in complexes 3, 4, 6, 7, and 14 do not differ significantly
17
from each other. In the case of complex [(6-bip)Ru(bpm)Cl][PF6] (4) , the RuII molecules
18
lay back-to-back with an adjacent complex in an intermolecular π-π stacking interaction.
19
(Figure S1). The X-ray crystal structures of compounds 3, 4, 6 and 7 show an increased
20
number of intra and/or intermolecular π-π stacking interactions, particularly for complexes
21
4 and 6 (Figure S2), which contain bip as the arene. Their crystal packing also displays
22
strong H-bonding throughout the unit cell, which is a common feature observed in similar
23
RuII arene complexes containing extended aromatic rings [32]. For complex 7, CH-π
14
1
interactions between the C−H protons of one of the pyrazine rings in the 2,2'-bipyrimidine
2
(bpm) chelating ligand and the centroid of one of the pyrazine rings in the bpm belonging
3
to a neighboring molecule were observed. (Figure S3). The occurrence of CH-π interactions
4
is now well established [33] and the interaction ranges from weak (CH···π centre 2.6–3.0
5
Å) to very strong (CH···π centre < 2.6 Å) [34]. Such interactions can play an important role
6
in protein stability and in recognition processes. The CH···π interactions observed for
7
complex 7 (2.9 Å) are within the weak-interaction range. The Ru–N7(9-EtG) bond distance in
8
the guanine adduct 14 (2.1125(19)Å) is similar to those in related organometallic RuII
9
guanine adducts [26]. There are multiple H-bonding interactions throughout the crystal.
10
The main fragments involved are bpm, 9-EtG, and solvent molecules (water), Figure S4.
11
Such aggregations have been observed in a number of RuII and PtII crystal structures
12
containing purine derivatives [35]. Water can play an important role in intercalation modes;
13
specific binding of water to DNA complexes can make a significant contribution to the free
14
energy of drug binding [36]. The 9-EtG adduct (14) was also characterized by 1H NMR
15
spectroscopy; Figure S5 shows its 2D 1H-1H NOESY spectrum. An NOE cross-peak
16
between H8 of bound 9-EtG and the 2,2'-CH in bpm was observed, suggesting that these
17
two atoms are in close proximity (as previously observed in analogous Ru II arene
18
complexes) [5, 37].
19
Aqueous Solution Chemistry. Dissolution of compounds 1–13 in 5% MeOH/95% H2O at
20
310 K gave rise to ligand exchange reactions as indicated by the concomitant changes in
21
UV-Vis absorption bands. The time-evolution spectra for all the RuII arene complexes at
22
310 K are shown in Figure S6. The time dependence of the absorbance of all the complexes
23
at selected wavelengths followed pseudo first-order kinetics in each case. The
15
1
corresponding rate constants and half-lives are listed in Table 2. The dependence of the
2
absorbance at 332 nm over ca. 16 h during aquation of [(6-p-cym)Ru(bpm)Cl][PF6] (1) at
3
310 K is shown in Figure S7.
4
In order to characterize the products of hydrolysis and to determine the extent of the
5
reactions, freshly-made 100 μM (5% MeOD-d4/95% D2O) solutions of complexes 1−13
6
were allowed to equilibrate for 24−48 h at 310 K and were then studied at the same
7
temperature using 1H NMR spectroscopy. The 1H NMR spectra of complexes 1−11 and 13
8
initially contained one major set of peaks (halido species) and then a second set of peaks
9
increased in intensity with time. The new set of peaks had the same chemical shifts as those
10
of the aqua adducts (prepared independently) under the same conditions (ca. 100 μM
11
solutions (5% MeOD-d4/95% D2O) at 310 K). The mass-to-charge ratios and isotopic
12
models obtained from HR-MS spectra were consistent with the formation of the aqua
13
adducts, Table S2. Table 3 summarizes the equilibrium constants (calculated by integration
14
of 1H NMR signals) after 24 h of reaction for complexes 1−11 and 13. For complexes [(6-
15
bip)Ru(bpm)Cl][PF6] (4), [(6-bip)Ru(bpm)Br][PF6] (5), [(6-etb)Ru(bpm)Cl][PF6] (7),
16
and [(6-p-cym)Ru(phendio)Cl][PF6] (12) an additional set of peaks was also observed
17
corresponding to the products which had undergone arene loss during the aquation. In the
18
case of complex 12, it was observed that it displayed a complicated 1H NMR spectrum
19
upon dissolution which could not be explained by hydrolysis alone.
20
Within the series of complexes having p-cym as arene and bpm as chelating ligand,
21
the hydrolysis reactions of the chlorido (1) and bromido (2) complexes are more
22
thermodynamically favored (K = 790.6 and 280.5 μM, respectively) compared to that of the
23
iodido complex (3) (K = 14.0 μM). Similarly, in the bip/bpm series it was found that the
16
1
chlorido (4) and bromido (5) complexes hydrolyzed to a larger extent (K = 9.0 and 10.4
2
μM, respectively) than the analogous iodido (6) complex (K = 0.2 μM). For complexes 1,
3
11, and 12−13 where p-cym (arene) and Cl (leaving group) are kept constant but the
4
chelating ligand is varied, the amount of aqua adduct (and equilibrium constant) determined
5
by 1H NMR increases in the order bathophen (13) < phen (11) < bpm (1). When the
6
chelating ligand is bpm and the leaving group as Cl, the extent of hydrolysis decreases with
7
arene in the order p-cym (1) > thn (10) > ind (9) > hmb (8) > etb (7) > bip (4).
8
Complexes 2, 5, 7−9 and 11−13 undergo relatively fast hydrolysis with half-lives of
9
< 60 min at 310 K. The reported half-lives of aquation of the previously reported chlorido
10
ethylenediamine
11
tha)Ru(en)Cl][PF6] and [(6-bip)Ru(en)Cl][PF6] [38] are 10–80 times smaller than those of
12
these complexes under comparable conditions. Within the p-cym/Cl series containing
13
various chelating ligands, the presence of a better π-acceptor chelating ligand reduces the
14
electron-density on the RuII centre, making it less favorable for the chlorido ligand to leave,
15
thus slowing down the hydrolysis reaction. Thus the rates increase in the order 1 (bpm) <
16
12 (phendio) < 11 (phen) < 13 (bathophen). The fact that the substituent heteroatoms on the
17
chelating ligands (such as the extra pair of nitrogens in 2,2'-bipyrimidine (bpm) or two
18
oxygens in 1,10-phenanthroline-5,6-dione (phendio)) which are electron donors [39] may
19
contribute to stabilization of the Ru−Cl bonds by π-back donation. Previous work [40] has
20
shown that an N,N' chelating group such as 2,2'-bipyridine (bpy) slows down substitution of
21
the aqua ligand in [(η6-C6H6)Ru(bpy)(OH2)]2+ just as the replacement of ethylenediamine
22
(en) by acetylacetonate (acac) to form [(η6-arene)Ru(acac)]+ complexes accelerates
23
hydrolysis. Within the RuII arene bpm/Cl series, it was observed that the incorporation of
(en)
RuII
arene
complexes,
[(6-dha)Ru(en)Cl][PF6],
[(6-
17
1
arenes with either an increased aromatic character or electron-withdrawing substituents in
2
the coordinated ring, significantly decreases the rate of the hydrolysis reaction in the order
3
7 (etb) > 8 (hmb) > 9 (ind) > 10 (thn) > 1 (p-cym) > 4 (bip). Bip has a high aromaticity and
4
competes as a π-acceptor [41] with the chelating ligand (bpm) for electron density. This
5
leads to a weakening of the corresponding Ru−arene bonds and consequently to the
6
complete loss of the bip in the case of complex 4. No arene loss is observed in the case of
7
the complexes bearing other arenes, all of which have electron donating aliphatic
8
substituents on the ring. A similar arene loss was previously observed for other RuII bip
9
complexes containing phenylazopyridines as π-acceptor ligands [42]. Within this same
10
series of complexes 1−6, it can also be noticed that a combination of a large leaving group
11
(such as I) and a large arene (such as bip) in complexes 3 and 6 together make the RuII
12
centre less accessible to an incoming ligand. This effect corresponds to the experimental
13
observation of a very slow hydrolysis rate. The inclusion of a more electronegative halide
14
(like Cl or Br in 1 and 2 or 4 and 5) leads to an increase in the hydrolysis rate when
15
compared to the iodido derivatives, which is enhanced when the arene is replaced by a less
16
sterically-demanding ligand such as p-cym in complexes 1−3. Arene ligands such as
17
benzene (bz) are reported to exhibit a strong trans-labilizing effect for the aqua ligand in
18
[(η6-bz)Ru(OH2)3]2+ [43]. This class of strong π-acid ligands is able to accept electron
19
density from the central RuII atom giving rise to a higher charge on the metal. Acidic
20
hydrolysis of RuIII complexes such as [Ru(NH3)4(X)2]+ and [Ru(NH3)5X] 2+ (X is Cl, Br,
21
and I) occurs via an associative pathway in which bond-making is more important than
22
bond-breaking [44].
23
Additionally, the changes in the 1H NMR chemical shifts of the aromatic protons in
24
either the corresponding arene rings or the corresponding N,N' chelating ligands of the aqua
18
1
adducts of complexes 1, 8-11 and 13 present in an equilibrated 100 μM (D2O) solution at
2
310 K were followed with change in pH* over the range ca. 1 to 12. Figure S8 shows how
3
the peaks shift to higher field due to deprotonation of the bound water molecule in the aqua
4
adduct of complex 1, [(η6-p-cym)Ru(bpm)(OH2)]2+, but do not change in intensity as an
5
indication that no other species are being formed. The pKa* values for complexes 1, 8,9,
6
and 11 are listed in Table 4. For complexes 1 (p-cym/bpm), 8 (hmb/bpm), 9 (ind/bpm), and
7
11 (p-cym/phen) the pKa* values are in the range from 6.91 to 7.32 and are all significantly
8
lower (ca. 1.5 units) than those reported for analogous [(η6-arene)Ru(N,N')(OH2)]2+
9
complexes [6b, 45]. Such a decrease in acidity has been attributed before to an increased
10
electron density on the metal centre favored by a combination of electron-donating/π-
11
acceptor−arene/chelating ligands. Complexes 1, 8, 9, and 11 will therefore be present as a
12
mixture of aqua and (less reactive) hydroxido adducts at pH 7.4.
13
Mechanism of Hydrolysis. Density Functional Theory (DFT) computational methods were
14
employed to obtain information about the influence of the leaving group on the mechanism
15
of hydrolysis for the p-cym/bpm series of RuII arene complexes 1−6. A test of the structural
16
accuracy of the functional PW91 with COSMO solvation was performed by comparing the
17
fully optimized structures of the cations in complexes 1–6 with the corresponding X-ray
18
crystal structures of 1 [14b], 3, 4 and 6. The functional PW91 was found to overestimate
19
the RuII bond lengths by ca. 0.01−0.04 Å, particularly for the computed Ru−X distances
20
(~2.44 Å), which were ca. 0.05 Å longer than those found in the solid state. However, the
21
overall agreement with the experimental data was satisfactory. A scheme for the reaction
22
modeled is shown in Figure 3. For each of the resting states ([RS] = {[(6-
23
arene)Ru(bpm)X]+·H2O]})
and
the
corresponding
products
([P]
=
{[(6-
19
1
arene)Ru(bpm)(OH2)]2+·X–]}), the entering (H2O) and the leaving groups (X–) are retained
2
within the second coordination sphere of the RuII centre. Complex 1 was chosen as a model
3
system; based on previously reported work [6b] on analogous RuII arene complexes. A full
4
geometry optimization of its transition state ([TS]) was performed starting from Ru–Cl and
5
Ru–OH2 distances of 3.200 Å and 2.899 Å, respectively, in the initial geometry. The
6
optimised structure for the [TS] for the RuII arene cation [(6-p-cym)Ru(bpm)Cl]+ is shown
7
in Figure 4. The geometry-optimized structure in the [TS] of the RuII arene cation in
8
complex 1, gave Ru–Cl and Ru–OH2 distances of 3.11 Å and 2.68 Å, respectively. The
9
corresponding energy value determined for the [TS] (–6657.51 kcal mol–1) was found to be
10
20.1 kcal mol–1 larger than that for the chlorido compound in the [RS]. Given that the
11
hydrolysis reaction could be assumed to be either an associative, a dissociative, or an
12
interchange (Ia or Id) process, a frequency calculation was performed for the [TS] of the
13
RuII arene cation in complex 1. Two imaginary frequencies were retrieved from the
14
computation, –152 and –20 cm–1. The latter is small and arises from the numerical noise
15
inherent in the finite difference method required when a COSMO field is enabled. The
16
former (and more significant) frequency value (–152 cm–1) gave a vibrational mode where
17
the entering water molecule (H2O) and leaving halido ligand (Cl–) are moving in a
18
concerted process consistent with an associatively activated reaction. The corresponding
19
scaled displacement vectors are shown in Figure S9. Under the assumption that the same
20
associative hydrolysis mechanism applies for other related systems, the effect of varying X
21
and the arene was explored. The results are listed in Table 5, from where it can be seen that
22
the corresponding barrier heights do not vary significantly when the arene p-cym (in
23
complexes 1, 2, and 3) is substituted by bip (in complexes 4, 5, and 6). The forward
20
1
reaction barriers and overall reaction energies for the aquation of the corresponding halido
2
ligand (Cl, Br, or I) follow the increasing order Cl ≈ Br < I, and p-cym < bip.
3
The transition state obtained from density functional theory (DFT) calculations,
4
suggested that aquation of the [(6-arene)Ru(bpm)X]+ complexes where arene is p-cym
5
(1−3) or bip (4−6) and X is Cl, Br, or I, proceeds via a concerted interchange (associative)
6
mechanism rather than a stepwise dissociation/coordination process (dissociative). For the
7
p-cym/bpm series of complexes (1−3) the reaction does not appear to be strongly
8
associatively nor dissociatively activated, because the corresponding Ru–X bonds at the
9
transition state extend by ~0.66, 0.71, and 0.81 Å for Cl, Br, and I, respectively, relative to
10
the reactant species. In the case of the corresponding Ru–O bonds in the transition state, it
11
was found that they are ca. 0.50 Å longer than in the aqua products in the three cases. The
12
results for the bip/bpm series of complexes (4−6) showed that the hydrolysis might not be
13
strongly associatively nor dissociatively activated either. The corresponding Ru–X bonds at
14
the transition state extend by ca. 0.58, 0.61, and 0.69 Å for Cl, Br, and I, respectively,
15
relative to the reactant species, whereas the Ru–O bonds in the transition state were found
16
to be ca. 0.55 Å longer than in the aqua products. Given that Ru–X bond-breaking alone is
17
not the rate-controlling step in the associative pathway, a heavier (and larger) halide will
18
impede the access of the H2O molecule to the central RuII atom in associative states. This
19
hypothesis is in good agreement with the experimental observation that complexes 3 and 6
20
(bearing I as the leaving group) display the slowest rates of hydrolysis within the
21
corresponding series. This assumption has also been suggested for Ru complexes
22
displaying higher coordination numbers (i.e. seven) [46]. The calculated reaction barriers
23
and overall reaction energies for the aquation of the halido complexes 1–6 follow the
21
1
increasing order Cl ≈ Br < I. However, the effect of different halides on the experimental
2
hydrolysis rates of these RuII arene complexes differs from the calculation and follows the
3
increasing order Br < Cl < I. This trend has been experimentally observed before for
4
platinum compounds of the type [PtXn(OH2)4−n](2−n)+, for which Br analogues of Cl
5
complexes hydrolyze faster in all three hydrolysis steps [27]. The calculated higher
6
activation energies might be responsible for the observed slower hydrolysis of the iodido
7
complexes during the associative ligand interchange in each series. Furthermore, the
8
electron-accepting effect of strong π-acid arene ligands might be responsible for the shift
9
toward a more associative pathway in the Id ↔ Ia mechanistic continuum for the [(η6-p-
10
cym)Ru(N,N')Cl]+ complexes studied herein. The accuracy of the calculation was not
11
sufficient to account for the differences found experimentally in the hydrolysis rates
12
between the chlorido and bromido complexes.
13
Interactions with Nucleobases. Interactions of several complexes with 9-EtG and 9-EtA
14
were studied by multidimensional 1H NMR spectroscopy and the nature of the products
15
was verified by HR-MS. All the reactions were carried out in NMR tubes in D2O and
16
followed over 48 h at 310 K. Figure 5 shows the reaction of complex 11 with 9-EtG as an
17
example. The 1H NMR peaks corresponding to H8 in all the 9-EtG-N7 adducts are shifted
18
to high field (ca. 0.5 ppm) relative to free 9-EtG under the same conditions. Often,
19
metallation at the N7 site of purine bases produces a low field shift of the H8 resonance by
20
about 0.3–1 ppm [47, 48]. This effect has also been observed before for analogous Ru II
21
arene complexes containing bpyError! Bookmark not defined. or acac [6a] as the chelating ligands.
22
The compounds studied in this work showed significant and rapid binding to 9-EtG-N7
23
(detectable after ca. 10 min and to ca. 34–94% extent). The reactions of complexes [(η6-p-
22
1
cym)Ru(bpm)Cl)]+ (1), [(η6-thn)Ru(bpm)Cl)]+ (10) and [(η6-p-cym)Ru(bathophen)Cl)]+
2
(13) required ca. 8 h to reach equilibrium in each case. However, a different behavior was
3
observed for complexes [(η6-hmb)Ru(bpm)Cl)]+ (8) and [(η6-ind)Ru(bpm)Cl)]+ (9), which
4
reacted with 9-EtG much faster, reaching equilibrium after 56 and 52 min, respectively.
5
Table S6 lists the percentage of species present in solution for the reactions of complexes 1,
6
8–11, and 13 with 9-EtG after selected times. The reactions of complexes 8, 9, and 10 with
7
9-EtG were found to produce higher yields. Interestingly, for complex [(η6-
8
thn)Ru(bpm)Cl][PF6] (10) ca. 12% of a second guanine-bound species (possibly [(η6-
9
thn)Ru(bpm)(9-EtG-N3)]2+) was also detected at equilibrium (ca. 510 min), Figure S10.
10
The mass-to-charge ratios and isotopic models obtained from HR-MS spectra were
11
consistent with the formation of the guanine adducts as the corresponding products of the
12
individual reactions, Table S3. The addition of an equimolar amount of 9-EtA (100 μM) to
13
freshly-prepared D2O solutions of the complexes at 310 K resulted in no new species even
14
after 48 h.
15
Since nucleobase binding is likely to require initial hydrolysis, the slow aquation
16
rates and reduced extent of hydrolysis of these complexes at equilibrium may account for
17
the observed extent of nucleobase binding. The calculated binding energies for 9-EtG in the
18
corresponding nucleobase adducts appear to be related to the trend determined for the
19
extent of nucleobase binding (vide infra). None of the RuII arene complexes 1, 8–11, and 13
20
showed evidence of binding to 9-ethyladenine. These complexes display a more
21
discriminating behavior towards binding to purine bases when compared to cisplatin, for
22
which binding to adenine is also observed [49]. It has been found that H-bonding from C6O
23
in guanine to N–H protons in the bidentate chelating ligand ethylenediamine (en),
23
1
contributes to the high preference for binding of {(η6-arene)Ru(en)}2+ to guanine versus
2
adenine. However, replacement of en (NH as H-bond donor) by bpm (no NH) in these
3
series of complexes did not change the selectivity for guanine bases.
4
In order to gain further insight into the nature and relative stabilities of the guanine
5
and adenine adducts of the RuII arene complexes 1, 8−11, and 13, their optimized
6
geometries were obtained using DFT calculations. Their minimum energy structures are
7
shown in Figures S11 and S12 (for the 9-EtG-N7 and the 9-EtA-N7 adducts, respectively).
8
The total binding energies for both nucleobases are shown in Table 6. The binding energies
9
include a COSMO contribution which simulates an aqueous environment. Under these
10
conditions, the binding of 9-ethylguanine was found to be more favorable than that of 9-
11
ethyladenine by ca. 10.0 kcal mol–1. Furthermore, the nucleobase 9-ethylguanine shows
12
significant binding energies towards all compounds (≥38.5 kcal mol–1), the largest value
13
being for the adduct [(6-thn)Ru(bpm)(9-EtG-N7]2+ (10-9EtG) with a value of 41.0 kcal
14
mol–1. In the case of 9-ethyladenine, a smaller binding energy towards all compounds
15
(≤34.4 kcal mol–1) was found; the largest energy was calculated for the adduct [(6-
16
thn)Ru(bpm)(9-EtA-N7]2+ (10-9EtA). The binding of 9-EtG to the N3 position in complex
17
10 was also investigated. The minimum energy structures are shown in Figure S13 for both
18
the 9-EtG-N7 and 9-EtG-N3 adducts. The calculated total binding energies are 41.0 and
19
18.5 kcal mol–1 for the 9-EtG-N7 and the 9-EtG-N3 adduct, respectively.
20
Despite the lack of cytotoxic activity, compounds 1, 8, 9, and 10 showed a
21
significant calculated binding energy for 9-EtG-N7 (ca. 38.8 kcal mol–1). As would be
22
expected, the binding of 9-EtG to complex 10 through N7 is ca. 20 kcal mol-1 more stable
23
than the binding through N3. The calculations were also able to reproduce the H-bond
24
1
distance (CH(N,N’-chelating)···O(9-EtG)) found in the X-ray crystal structure of the 9-EtG adduct,
2
complex 14 within sufficient accuracy. Therefore, it is assumed that the analogous values
3
from the DFT-optimised geometries for the rest of the 9-EtG adducts of complexes 1, 8–11
4
and 13 will also be within the expected ranges. The calculations predict CH(N,N’-
5
chelating)···O(9-EtG) distances within the range of 2.20–3.11 Å and C–H(N,N’-chelating)···O(9-EtG)
6
angles within 114.93–134.97º. The shortest H-bond distance was found for the 9-EtG
7
adduct of complex 10, which might explain the high binding energy calculated for this
8
adduct.
9
DNA Binding Reactions in Cell-Free Media. In order to explore the possibility of DNA
10
as a potential target, two complexes [(6-p-cym)Ru(bpm)Cl][PF6] (1) and [(6-p-
11
cym)Ru(phen)Cl][PF6] (11) were selected for further studies of CT-DNA interactions in
12
cell-free media. The results of the DNA binding experiments are summarized in Table 7.
13
Both complexes reacted with CT-DNA to a moderate extent and the reactions were
14
complete after ca. 20 h. Complex 11 was found to bind much faster and to a larger extent
15
than complex 1, with equilibrium for complex 11 being reached within the first 1.5 h. After
16
24 h of reaction both complexes has reacted to a similar, ca. 60%, Figure S14. The dialysis
17
experiments against two different sodium salts indicate that the coordination of the Ru II
18
arene complexes to CT-DNA is reversible and dependent on the nature of the salt. In the
19
case of [(6-p-cym)Ru(phen)(Cl)][PF6] (11), dialysis either against 10 mM NaClO4 or 0.1
20
M of NaCl resulted in a decrease in the percentage of complex bound to DNA by ca. 20%.
21
For complex 1, dialysis against 10 mM NaClO4 did not change the percentage of complex
22
bound to DNA whereas dialysis against 0.1 M of NaCl reduced the amount to the same
23
extent as for complex 11 (ca. 20%).
25
1
Further investigations were aimed at identifying the Ru binding sites in natural DNA for
2
the reactions of [(6-p-cym)Ru(bpm)Cl][PF6] (1) and [(6-p-cym)Ru(phen)Cl][PF6] (11).
3
The autoradiogram of the inhibition of RNA synthesis by T7 RNA polymerase on
4
pSP73KB DNA containing adducts of the RuII arene complexes or cisplatin is shown in
5
Figure 7. The bands corresponding to the transcription of DNA modified by complexes 1
6
and 11 yielded fragments of newly synthesized RNA of defined sizes, which indicates that
7
RNA synthesis on these templates was prematurely terminated. The major stop sites
8
occurred at similar positions in the gel and were solely at guanine residues, for both Ru II
9
arene complexes, Figure 8.
10
Intensities of the bands corresponding to the transcription of DNA modified by complex 11
11
are considerably weaker than those of the bands corresponding to the transcription of DNA
12
modified by complex 1 (Figure 7). This may indicate that efficiency of DNA adducts of 11
13
to prematurely terminate RNA synthesis by T7 RNA polymerase is lower than that of DNA
14
adducts of 1. We can speculate that this reduced efficiency is associated with lesser
15
distortion of DNA conformation exerted by DNA adducts of 11 (compared to DNA adducts
16
of 1) as deduced from the results of CD spectroscopy of DNA modified by these complexes
17
(Figure 10). Another possibility might be through the labilization of DNA-metal adducts by
18
1,10-phenanthroline chelating ligand in 11 being greater than that exerted by 2,2'-
19
bipyrimidine chelating ligand in 1 due to the introduction of electron withdrawing
20
substituents in the 4,4' positions of bpm in complex 11 (which contains 1,10-phenanthroline
21
chelating ligand). As a consequence, some molecules of 11 originally bound to DNA might
22
be displaced by T7 RNA polymerase during transcription of the template strand containing
23
them and consequently would be unable to prematurely terminate RNA synthesis by this
26
1
enzyme. The labilization of other metal-biologically relevant molecules by spectator
2
ligands (in metal complexes) has already been demonstrated for Pt(II) complexes[50].
3
The rate of binding to DNA for complex 1 is slower than that determined for the anticancer
4
drug cisplatin (t1/2 ca. 2 h under similar conditions) [51], for which DNA binding is thought
5
to be responsible for its cytotoxic properties. Interestingly, the corresponding binding rate
6
for complex 11 was found to be in the same range as that of cisplatin. In contrast, other Ru II
7
arene analogues i.e. [(η6-bip)Ru(en)Cl]+, which has also been shown to be cytotoxic to
8
cancer cells [26a, 52], react much more rapidly with DNA under similar conditions (t1/2 ca.
9
10 min).
10
The native agarose gels resulting from DNA modified by complexes [(6-p-
11
cym)Ru(bpm)Cl][PF6] (1) and [(6-p-cym)Ru(phen)Cl][PF6] (11) are shown in Figure 9.
12
The DNA unwinding angles produced by the adducts of 1 and 11 were determined to be
13
7.7° and 6.6°, respectively, which is consistent with only a small reduction of the intensity
14
of the negative CD band at ca. 245 nm of DNA modified by 1 or 11 (vide infra) [53]. This
15
is smaller than that observed for the RuII arene complexes [(η6-arene)Ru(en)Cl]+ (range 7–
16
14°) [30] and resembles more those produced by monofunctional cisplatin adducts (6° and
17
13° for mono or bifunctional adducts, respectively) [54]. The co-migration point of the
18
modified supercoiled and nicked DNA (rb(c)) was reached at rb = 0.13 and 0.15 (for 1 and
19
11, respectively) as shown in Table 8
20
The DNA binding after hydrolysis of the RuII arene complexes 1 and 11 results in a mild
21
degree of unwinding (7–8°). This relatively small DNA unwinding is very similar for the
22
two complexes, but even smaller than that observed for [(η6-arene)Ru(en)Cl]+ complexes
23
(range 7–14°) [30].
27
1
CD spectra of CT-DNA modified by complexes (1) and (11) (at 298 K in 10 mM
2
NaClO4) were also recorded at rb values in the range of 0.013−0.047. As can be seen from
3
Figure 10, small changes in the CD spectrum at wavelengths below 300 nm are observed
4
upon interaction of complex 1 (and to a much lesser extent of complex 11) with CT-DNA.
5
As a consequence of the ruthenation of CT-DNA, the intensity of the positive CD band at
6
around 280 nm increases for complex 1 whereas the CD spectrum recorded for CT-DNA
7
modified by 11 remains unchanged. The signature of complexes 1 and 11 bound to CT-
8
DNA includes no ICD. The changes in CD spectra of CT-DNA (monitored at 246 and 278
9
nm) modified by RuII arene complexes 1 and 11 (at different rb values) are shown in Table
10
S4.
11
CD spectra showed that the binding of 1 (and to a lesser extent of 11) to DNA results in
12
subtle conformational alterations in DNA that could be related to a denaturational
13
character, similar to those induced in DNA by clinically ineffective transplatin. It is
14
possible that these changes could also be associated with the bound Ru arene fragment
15
given that these RuII arene complexes show maxima in the proximity of a DNA maximum.
16
Overall, these combined results might suggest the presence of combined covalent
17
(coordinative), non-covalent intercalative, and monofunctional coordination binding modes
18
of DNA binding for complexes 1 and 11 upon hydrolysis.
19
Binding of the RuII arene complexes to CT-DNA was also monitored by linear dichroism
20
spectroscopy (LD). It is well established that the magnitude of the LD signal measured
21
within the DNA absorption band (i.e. at the 258 nm maximum) is a function of its
22
persistence length [55]. The magnitudes of the LD signals at 258 nm decrease as a function
23
of rb for the RuII arene complexes 1 and 11, Figure 11. The changes in LD spectra of CT-
24
DNA modified by the RuII complexes at different rb values were monitored at 258.5 nm,
28
1
Table S5. It can be seen that both complexes behave similarly and their changes are within
2
the same range. These results might suggest that the formation of DNA adducts could be
3
eventually accompanied by the appearance of flexible hinge joints at the site of the lesion.
4
Cancer Cell Growth Inhibition The halido complexes 1−6 (Figure 1) were tested against
5
the A2780 human ovarian, A2780 cisplatin resistant human ovarian, A459 human lung, and
6
HCT116 human colon cancer cell lines, whereas the remaining halido complexes 8−11 and
7
13 were tested against the A2780 human ovarian cancer cell line. Strikingly, all the bpm-
8
containing complexes 1−6 and 8−10 displayed IC50 values larger than 100 μM against the
9
corresponding cell lines tested (IC50 value for cisplatin was 1.0 μM under the same
10
conditions). Complexes 11 and 13, bearing phen and bathophen as the chelating ligand,
11
respectively, were cytotoxic to A2780 human ovarian cancer cells, Table 9. The most active
12
complex is [(6-p-cym)Ru(bathophen)Cl][PF6] (13) with an IC50 value of 0.5 μM,
13
comparable to that of cisplatin (IC50 for cisplatin 1.1 μM under the same conditions). The
14
IC50 for [(6-p-cym)Ru(phen)Cl][PF6] (11) against this cancer cell line was 23 μM.
15
A loss of cytotoxicity towards cancer cells has been previously observed for complexes of
16
the type [(6-arene)Ru(en)Cl]+ when en, a σ-donor, is replaced by 2,2'-bipyridine [56], a
17
strong π-acceptor. Changing the electronic features of the chelating ligands by
18
incorporating electron donating heteroatoms in the 4,4' positions of bpm (such as in the
19
phendio complex 12) did not restore the cytotoxic activity. From a structural point of view,
20
loss of activity in these derivatives could arise from the absence of N(sp3)H groups, which
21
are known to stabilize nucleobase adducts through strong H-bonding between an NH of en
22
and C6O from the guanine (G) nucleobase [57]. The electronic properties of the complexes
23
might also account for the observed loss of activity; metal-DNA bonds have been shown to
29
1
be labilized by heteroarene ligands..After substitution of two water molecules by thiourea
2
(tu) for instance, labilization of the Pt–N bond in the trans position forms a ring-opened
3
trisubstituted [Pt(tu)3(N-Nopen)]2+ species [Error! Bookmark not defined.]. Similar
4
reactions are also known in the biotransformation pathway of cisplatin;where the resulting
5
products are inert to further substitution reactions and therefore limit the active
6
concentration of the drug.[58]
7
Interactions with GSH. 1H NMR and UV-vis absorption spectra of solutions containing
8
the inactive complex [(6-p-cym)Ru(bpm)Cl][PF6] (1) (100 μM) and a 100-fold molar
9
excess of GSH (10 mM, to mimic intracellular conditions) were acquired over 24 h at 310
10
K. The time evolution spectra for the RuII arene complex 1 are shown in Figure S15. The
11
1
H NMR spectra of complex 1 initially contained one major set of peaks (chlorido species)
12
and then a second set of peaks assignable to the aqua adduct [(6-p-cym)Ru(bpm)OH2]2+,
13
increased in intensity with time. A third set of peaks attributable to the GS-bound
14
ruthenium adduct was also detected [(6-p-cym)Ru(bpm)GS]+ (1-GS), Figure 12. The
15
mass-to-charge ratio and isotopic model obtained from HR-MS spectra were consistent
16
with the formation of the tripeptide-substituted RuII product; and the calculated m/z value
17
for C28H36N7O6RuS (700.1508), found m/z (700.1493). Some sulfur-bound thiolate adducts
18
with platinum anticancer drugs are formed irreversibly and are also largely unreactive (e.g.
19
towards DNA binding) [59].
20
21
Conclusions
have
shown
here
that
several
2,2'-bipyrimidine
(bpm)
complexes
[(η6-
22
We
23
arene)Ru(bpm)Cl][PF6] are inactive as anticancer agents towards human ovarian cancer
30
1
cells. However a change in the chelating ligand from bpm to 1,10-phenanthroline (phen) or
2
4,7-diphenyl-1,10-phenanthroline (bathophen) leads to activity. Significant changes in the
3
chemical reactivity of the compounds towards hydrolysis are also observed; the hydrolysis
4
rates of [(η6-arene)Ru(N,N')X]+ complexes vary over a wide range, from half-lives of
5
minutes (14.5 min for complex [(η6-etb)Ru(bpm)Cl]+ (7)) to hours (12 h for complex [(η6-
6
bip)Ru(bpm)I]+ (6)) at 310 K. Density functional theory calculations on bpm complexes 1–
7
6 suggest that aquation occurs via a more associative pathway in an Ia ↔ Id mechanistic
8
continuum for which bond-making is of greater importance than bond-breaking. For both p-
9
cym and bip bpm-containing complexes 1–6, the calculated reaction barriers and overall
10
reaction energies follow the order I > Br ≈ Cl which may explain the slow hydrolysis rate
11
determined by UV-vis spectroscopy for iodido complexes 3 and 6.
12
In general, we were not able to establish a correlation between hydrolysis rates and
13
anticancer activity which implies that the mechanism of action for these series of
14
complexes does not depend solely on this process. The half-sandwich RuII arene complexes
15
containing phenanthroline (11) or bathophenanthroline (13) as N,N'-chelating ligands are
16
more cytotoxic towards A2780 human ovarian cancer cells, in contrast to the analogous
17
complexes containing bpm (1‒6). X-ray crystal structures show that bip complexes (4 and
18
6) can form strong inter- and intra-ligand π-π interactions which enforces planarity on the
19
bpm ligand, particularly in the case of complex 4. An interesting feature of the structure of
20
complex 7 is the presence of aromatic CH-π (bpm) interactions. Strong binding to 9-EtG,
21
but not to 9-EtA, was observed for complexes containing N,N' chelating ligands such as
22
bpm, phen and bathophen as well as different arenes such as p-cym, hmb, ind and thn. By
23
the use of DFT calculations, the binding energies for model DNA nucleobases were
31
1
assessed. DFT calculations show that the 9-EtG nucleobase adducts of all complexes are
2
thermodynamically preferred compared to their 9-EtA adducts by ca. 10 kcal mol–1,
3
explaining the guanine-specific binding observed experimentally for complexes 1, 8–11
4
and 13. DNA binding studies show that complexes 1 and 11 bind to DNA, suggesting that
5
it could be target for these complexes, though the induced conformational changes are not
6
significant. The reduced cytotoxic potency of the bpm-containing complexes might be due
7
to the weakness of lesions on DNA or side reactions with other biomolecules such as
8
glutathione (GSH). The formation of a presumably largely unreactive RuII-GS adduct might
9
contribute to the lack of cytotoxicity [59].
10
Acknowledgements. S.B.-L. thanks WPRS/ORSAS (UK) and CONACyT (Mexico) for
11
funding a research studentship. B. L., O. N. and V. B. were supported by the Czech Science
12
Foundation (Grants P301/10/0598 and 301/09/H004).We also thank EDRF and AWM
13
(Science City) and ERC (grant. no 247450) for funding, and Dr Ivan Prokes and Dr Lijiang
14
Song and Mr. Philip Aston of the University of Warwick for their help with NMR and MS
15
instruments, respectively.
16
Supporting Information Available.
17
Details of the preparation and characterization of all the complexes in this work.
18
Crystallographic data for 3, 4, 6, 7, 14; mass-to-charge ratios obtained from HR-MS spectra
19
for the products of hydrolysis of RuII arene complexes 1−14; mass-to-charge ratios
20
obtained from HR-MS spectra for the products of interactions of Ru II arene complexes 1,
21
8–11, and 13 with 9-EtG; changes in CD and LD spectra of CT-DNA modified by RuII
22
arene complexes 1 and 11; X-ray crystal structure of 4 showing a - stacking interaction;
23
CH-π interaction in the crystal structure of 7; bis-water bridged interaction in the X-ray
32
1
crystal structure of 14; 1H-1H NOESY NMR spectrum of 14 in D2O (aromatic region only);
2
time evolution of the hydrolysis reactions of complexes 1–13; dependence of the
3
absorbance during aquation of 1 at 310 K; 1H NMR spectra recorded during a pH* titration
4
of a solution of the aqua adduct of complex 1; DFT-optimised geometry in the transition
5
state [TS] during the hydrolysis reaction of the RuII arene cation 1; 1H NMR spectra of the
6
reaction of 10 with 9-EtG in D2O at 310 K after 510 min; optimised geometries for the
7
guanine and adenine adducts; kinetics of the binding of complexes 1 and 11 to CT-DNA;
8
hydrolysis reaction of complex 1 in the presence of 100-fold excess of GSH followed by
9
UV-vis spectroscopy.
10
X-ray crystallographic data for complexes 3, 6, 14, 4 and 7 are available as Supporting
11
Information and have been deposited in the Cambridge Crystallographic Data
12
Centre under the accession numbers CCDC 872981, 872982, 872983, 872984, 872985,
13
respectively. Copies of the data can be obtained free of charge from the CCDC (12 Union
14
Road, Cambridge CB2 1EZ, UK; tel: (+44) 1223-336-408; fax: (+44) 1223-336-003; e-
15
mail: deposit@ccdc.cam.ac.uk; website link: (http://www.ccdc.cam.ac.uk/).
16
17
18
33
1
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Tables
Table 1. Selected bond lengths (Å) and angles (o) for [(6-p-cym)Ru(bpm)I][PF6] (3), [(6bip)Ru(bpm)Cl][PF6] (4), [(6-bip)Ru(bpm)I][PF6] (6), [(6-etb)Ru(bpm)Cl][PF6] (7), and
[(6-p-cym)Ru(bpm)(9-EtG-N7)][PF6]2 (14).
Bond length/angle
Ru−arene(centroid)
Ru(1)−I(1)
Ru(1)−Cl(1)
Ru(1)−N(13)
Ru(1)−N(1)
Ru(1)−N(8)
C(6)−C(7)
N(8)−Ru(1)−N(1)
I(1)−Ru(1)−N(8)
Cl(1)−Ru(1)−N(8)
I(1)−Ru(1)−N(1)
Cl(1)−Ru(1)−N(1)
N(13)−Ru(1)−N(8)
N(13)−Ru(1)−N(1)
3
1.704
4
1.691
6
1.693
7
1.684
14
1.693
2.706(3)
–
2.091(2)
–
2.402(8)
–
2.092(2)
2.093(2)
1.476(4)
76.72(9)
--83.64(6)
–
83.00(7)
–
–
2.70476(16)
–
–
2.0901(12)
2.0833(12)
1.477(2)
76.90(5)
82.60(3)
–
88.00(3)
–
–
–
–
2.3743(9)
–
2.073(3)
2.081(3)
1.472(5)
77.06(12)
–
83.36(8)
–
84.67(8)
–
–
–
–
2.1125(19)
2.0972(18)
2.0941(18)
1.477(3)
77.05(7)
–
–
–
–
86.59(7)
88.64(7)
2.0833(19)
1.472(4)
76.78(8)
86.38(6)
–
85.79(6)
–
–
–
40
Table 2. Hydrolysis data for complexes 1−13 determined by UV-vis spectroscopy as 100
μM solutions (5% MeOH/95% H2O) at 310 K.
Compound
t1/2 (min)
k × 10−3 (min−1)a
(1)
92.3
7.51 ± 0.07
[(6-p-cym)Ru(bpm)Cl][PF6]
6
[( -p-cym)Ru(bpm)Br][PF6]
(2)
22.4
31.0 ± 0.91
6
(3)
234.8
2.95 ± 0.08
[( -p-cym)Ru(bpm)I][PF6]
6
b
(4)
175.9
3.94 ± 0.04
[( -bip)Ru(bpm)Cl][PF6]
(5)
39.7
17.0 ± 0.31
[(6-bip)Ru(bpm)Br][PF6]b
6
b
(6)
714.6
0.97 ± 0.04
[( -bip)Ru(bpm)I][PF6]
6
b
(7)
14.5
50.0 ± 0.05
[( -etb)Ru(bpm)Cl][PF6]
(8)
40.2
17.2 ± 1.32
[(6-hmb)Ru(bpm)Cl][PF6]
6
(9)
43.3
16.0 ± 0.15
[( -ind)Ru(bpm)Cl][PF6]
6
(10) [( -thn)Ru(bpm)Cl][PF6]
89.9
7.71 ± 0.44
(11) [(6-p-cym)Ru(phen)Cl][PF6]
22.8
30.5 ± 0.43
6
b
(12) [( -p-cym)Ru(phendio)Cl][PF6]
59.6
11.6 ± 0.10
6
(13) [( -p-cym)Ru(bathophen)Cl][PF6]
16.9
40.8 ± 0.86
a
The errors are fitting errors
b
The rate constants for complexes that underwent arene loss detected by 1H NMR (4, 5, 7,
and 12) were determined over the period of time before the onset of arene loss.
41
Table 3. Equilibrium constants (K, μM) and percentage of arene loss of Ru II arene
complexes at equilibrium after 24 h of the hydrolysis reaction in a 100 μM (5% MeODd4/95% D2O) solution at 310 K of complexes 1−11 and 13 followed by 1H NMR.
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(13)
Compound
[( -p-cym)Ru(bpm)Cl][PF6]
[(6-p-cym)Ru(bpm)Br][PF6]
[(6-p-cym)Ru(bpm)I][PF6]
[(6-bip)Ru(bpm)Cl][PF6]
[(6-bip)Ru(bpm)Br][PF6]
[(6-bip)Ru(bpm)I][PF6]
[(6-etb)Ru(bpm)Cl][PF6]
[(6-hmb)Ru(bpm)Cl][PF6]
[(6-ind)Ru(bpm)Cl][PF6]
[(6-thn)Ru(bpm)Cl][PF6]
[(6-p-cym)Ru(phen)Cl][PF6]
[(6-p-cym)Ru(bathophen)Cl][PF6]
6
K (μM)
280.5
790.6
14.0
9.0
10.4
0.2
4.5
34.1
79.6
231.1
61.6
52.1
% Arene loss
0.0
0.0
0.0
48.1
68.3
0.0
2.9
0.0
0.0
0.0
0.0
0.0
42
Table 4. pKa* values for the aqua adducts of complexes 1, 8,9 and 11 at 298 K.
Compound
6
1
[( -p-cym)Ru(bpm)OH2]2+
8
[(6-hmb)Ru(bpm)OH2]2+
9
[(6-ind)Ru(bpm)OH2]2+
11
[(6-p-cym)Ru(phen)OH2]2+
pKa
6.96
7.04
6.91
7.32
43
Table 5. Selected bond lengths, forward reaction barriers, and overall reaction energies
from Density Functional Theory (DFT) calculations for the modeled reaction {[(6-pcym)Ru(bpm)X]+·H2O]} → [TS] →{[(6-p-cym)Ru(bpm)OH2]2+·Cl–]}.
Ru–X/Ru–OH2
[RS] (Å)
p-cym/bpm
1 Cl
2.44736/3.87080
2 Br
2.58087/3.93448
3I
2.76784/4.08973
bip/bpm
4 Cl
2.43726/3.82308
5 Br
2.57062/3.88138
6I
2.76183/4.01231
ΔE‡
(kcal mol–1)
ΔEreacc
(kcal mol–1)
3.1069/2.67606 4.00637/2.17416
3.29409/2.67608 4.13680/2.17987
3.58007/2.68230 4.42902/2.19029
20.09
21.01
22.79
5.5
7.28
9.61
3.01943/2.71710 4.01419/2.16185
3.18568/2.73201 4.15467/2.16741
3.45171/2.74296 4.41027/2.17431
19.96
20.94
22.28
6.16
7.51
10.28
Ru–X/Ru–OH2
[TS] (Å)
Ru–X/Ru–OH2
[P] (Å)
[RS] = Resting state
[TS] = Transition state
[P] = Product
ΔEreacc values relative to reactant species at zero
44
Table 6. Solution (COSMO) 9-EtG and 9-EtA binding energies for adducts of RuII arene
complexes 1, 8–11 and 13.
Compound
1
8
9
10
11
13
9-EtG (kcal mol–1)
38.5
39.3
39.3
41.0
39.8
38.7
9-EtA (kcal mol–1)
30.8
30.3
32.4
34.4
33.7
32.3
45
Table 7. Percentage binding of complexes 1 and 11 to CT-DNA (1.0 × 10−4 M) in 10 mM
NaClO4 at 310 K as determined by FAAS after 24 h.
% RuII bound
Method
DNA precipitation by EtOH
Dialysis against 10 mM NaClO4
Dialysis against 0.1 M NaCl
a
(1)a
61.0
77.0
21.6
(11)a
62.0
19.6
17.3
Data are the average of two independent experiments
46
Table 8. Unwinding of supercoiled pUC19 DNA by RuII arene complexes [(6-pcym)Ru(bpm)Cl][PF6] (1) and [(6-p-cym)Ru(phen)Cl][PF6] (11).
(1)
(11)
Cisplatin
Compound
[( -p-cym)Ru(bpm)Cl][PF6]
[(6-p-cym)Ru(phen)Cl][PF6]
6
rb(c)
0.13
0.15
0.08
Unwinding Angle (°)
7.7±1.7
6.6±1.7
13.0±0.4
47
Table 9. IC50 values for RuII arene complexes 11 and 13 against the A2780 human ovarian
cancer cell line.
(11)
(13)
a
Compound
6
[( -p-cym)Ru(phen)Cl][PF6]
[(6-p-cym)Ru(bathophen)Cl][PF6]
Cisplatin
IC50 μM (A2780)a
22.9
0.5
1.1
Complexes 1−6, and 8−10 had IC50 values larger than 100 μM against the cell lines tested (cisplatin 1.0 μM
under the same conditions)
48
Figures Caption
Figure 1. General structures of the complexes studied in this work, synthesized as PF6
salts.
Figure 2. X-ray structure of the cations in [(6-p-cym)Ru(bpm)I][PF6] (3), [(6bip)Ru(bpm)Cl][PF6] (4), [(6-bip)Ru(bpm)I][PF6]2 (6), [(6-etb)Ru(bpm)Cl][PF6] (7), and
[(6-p-cym)Ru(9-EtG-N7)][PF6]2 (14). Thermal ellipsoids show 50% probability. The
hydrogen atoms and counter ions have been omitted for clarity.
Figure 3. Hydrolysis reaction modeled for the RuII arene cations [(6-arene)Ru(bpm)X]+ of
complexes 1–6. [TS] is transition state.
Figure 4. DFT-optimised geometry of the transition state [TS] during the hydrolysis
reaction of the RuII arene cation [(6-p-cym)Ru(bpm)Cl]+ (1).
Figure 5. Time dependence of the 1H NMR spectra of a 100 μM solution of [(6-pcym)Ru(phen)Cl][PF6] (11) in D2O at 310 K in the presence of an equimolar amount of 9EtG. Blue = [(6-p-cym)Ru(phen)Cl]+, Green =[(6-p-cym)Ru(phen)(OH2)]2+, Magenta =
[(6-p-cym)Ru(phen)(9-EtG-N7)]2+; = phen, ● = p-cym; ♦ = bound 9-EtG-N7.
49
Figure 6. Numbering scheme for the nucleobases 9-EtG and 9-EtA.
Figure 7. Autoradiogram of 6% polyacrylamide/8 M urea sequencing gel showing
inhibition of RNA synthesis by T7 RNA polymerase on the NdeI/HpaI fragment containing
adducts of RuII arene complexes and cisplatin. Lanes: control, unmodified template; A, U,
G and C, chain terminated marker DNAs; cisplatin, 1 and 11, the template modified by
cisplatin at rb = 0.02, RuII arene complexes 1 at rb = 0.02 or 11 at rb = 0.015, respectively.
Figure 8. Schematic diagram showing the portion of the sequence used to monitor
inhibition of RNA synthesis by RuII arene complexes. The arrow indicates the start of the
T7 RNA polymerase, which used as template the bottom strand of the NdeI/HpaI fragment
of pSP73KB. The closed bullets represent major stop sites for DNA modified by complex 1
or 11, respectively. The numbers correspond to the nucleotide numbering in the sequence
map of the pSP73KB plasmid.
Figure 9. The unwinding of supercoiled pUC19 plasmid DNA by complexes 1 (top) and 11
(bottom). The plasmid was incubated with RuII arene complexes in 10 mM NaClO4, at pH 6
for 24 h at 310 K. Lanes in the top panel: 1 and 10, control, unmodified DNA; 2, rb = 0.06;
3, rb = 0.08; 4, rb = 0.09; 5, rb = 0.12; 6, rb = 0.14; 7, rb = 0.16; 8, rb = 0.18; 9, rb = 0.20.
Lanes in the bottom panel: 1 and 10, control, unmodified DNA; 2, rb = 0.05; 3, rb = 0.06; 4,
rb = 0.07; 5, rb = 0.08; 6, rb = 0.09; 7, rb = 0.11; 8, rb = 0.13; 9, rb = 0.15. The top bands in
50
each panel correspond to the form of nicked plasmid and the bottom bands to the closed,
negatively supercoiled plasmid.
Figure 10. Circular dichroism spectra of CT-DNA modified by RuII arene complexes 1 and
11. CD spectra were recorded for DNA in 10 mM NaClO4. The concentration of DNA was
3.3 × 10–4 M. The values of rb were in the range of 0.013−0.047.
Figure 11. Linear dichroism spectra of CT-DNA modified by RuII arene complexes 1 (top)
and 11 (bottom). LD spectra were recorded for DNA in 10 mM NaClO4. The concentration
of DNA was 3.3 × 10–4 M. The values of rb were in the range of 0.013−0.047.
Figure 12.
1
H NMR spectra of the reaction of a 100 μM solution of [(6-p-
cym)Ru(bpm)Cl][PF6] (2) with 100-fold excess of GSH in D2O at 310 K after 24 h. Blue =
[(6-p-cym)Ru(bpm)Cl]+; Green = [(6-p-cym)Ru(bpm)OH2]2+; Yellow = [(6-pcym)Ru(bpm)(GSH)]+; = bpm, ● = p-cym.
51
Figure 1.
52
Figure 2.
53
Figure3.
54
Figure 4.
55
Figure 5.
56
Figure 6.
57
Figure
7.
Figure 8.
58
Figure 9.
59
Figure 10.
60
Figure 11.
61
Figure 12.
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62