Control of Reversible Activation Dynamics of [Ru{η6:κ1-C6H5(C6H4)NH2}(XY)]n+ and the Effect of Chelating-Ligand Variation

Article abstract of DOI:10.1002/chem.201701681

The potential use of organoruthenium complexes as anticancer drugs is well known. Herein, a family of activatable tethered ruthenium(II) arene complexes of general formula [Ru{η⁶:κ¹-C₆H₅(C₆H₄)NH₂}(XY)]ⁿ⁺ (closed tether ring) bearing different chelating XY ligands (XY=aliphatic diamine, phenylenediamine, oxalato, bis(phosphino)ethane) is reported. The activation of these complexes (closed- to open-tether conversion) occurs in methanol and DMSO at different rates and to different reaction extents at equilibrium. Most importantly, Ru^II-complex activation (cleavage of the Ru?N_tether bond) occurs in aqueous solution at high proton concentration (upon N_tether protonation). The activation dynamics can be modulated by rational variation of the XY chelating ligand. The electron-donating capability and steric hindrance of XY have a direct impact on the reactivity of the Ru?N bond, and XY=N,N′-dimethyl-, N,N′-diethyl-, and N,N,N′,N′-tetramethylethylenediamine afford complexes that are more prone to activation. Such activation in acidic media is fully reversible, and proton concentration also governs the deactivation rate, that is, tether-ring closure slows down with decreasing pH. Interaction of a closed-tether complex and its open-tether counterpart with 5′-guanosine monophosphate revealed selectivity of the active (open) complex towards interaction with nucleobases. This work presents ruthenium tether complexes as exceptional pH-dependent switches with potential applications in cancer research.

Full text of DOI:10.1002/chem.201701681

A Journal of
Accepted Article
Title: Control of Reversible Activation Dynamics of [Ru(η⁶:κ¹-
C₆H₅(C₆H₄)NH₂)(XY)]n+ and the Effect of Chelate Ligand
Variation
Authors: Francisco Martínez-Peña and Ana M Pizarro
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To be cited as: Chem. Eur. J. 10.1002/chem.201701681
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Control of Reversible Activation Dynamics of [Ru(η6:κ¹-
C6H5(C6H4)NH2)(XY)]n+ and the Effect of Chelate Ligand Variation
Francisco Martínez-Peña,[a] and Ana M. Pizarro*[a],[b]
Abstract: The potential use of organometallic ruthenium complexes
as anticancer drugs is well known. Here we show a family of
activatable tethered ruthenium(II) arene complexes of general
formula [Ru(η6:κ1-C6H5(C6H4)NH2)(XY)]n+ (closed tether-ring) bearing
Following the rationale behind the Warburg effect[4] whereby
high rates of aerobic glycolysis in cancer cells produce slightly
acidic extracellular pH,[5] Dyson and co-workers sought to exploit
the acidic microenvironment of the solid tumour by trapping a
RuII complex inside the cell upon protonation of the pta ligand in
[Ru(6-p-cymene)Cl2(pta)], where pta is 1,3,5-triaza-7-
phosphatricyclo[3.3.1.1]decane.[6]
We aim to exploit a drop in pH to control the activation of our
RuII complexes in a pH-dependent manner; i.e., by protonation
of a suitable Z upon cleavage of the RuZ bond. Most
importantly, we design the RuZ activation to be reversible by
tethering Z to our system, creating a switchable pH-responsive
compound (Chart 1).
different chelating XY ligands (XY
=
aliphatic diamine,
phenylenediamine, oxalato, bis(phosphino)ethane). The activation of
these complexes (closed-to-open tether conversion) occurs in
methanol and dimethylsulfoxide at different rates, and to different
reaction extents at equilibrium. Most importantly, RuII complex
activation (cleavage of the Ru–Ntether bond) occurs in aqueous
solution when the proton concentration is high (upon Ntether
protonation). The activation dynamics can be modulated by rational
variation of the XY chelating ligand. The electron-donating capability
and steric hindrance of XY have a direct impact on the Ru–N bond
reactivity, with XY = N,N'-dimethyl, N,N'-diethyl, and N,N,N',N'-
tetramethylethylenediamine affording complexes more prone to
activation. Such activation in acidic media is fully reversible, and
proton concentration also governs the deactivation rate; i.e., tether
ring closure slows down as the pH decreases. Interaction of a
closed-tether complex and its open-tether counterpart with 5'-GMP
indicates selectivity of the active (open) complex towards
nucleobase interaction. This work presents ruthenium tether
complexes as exceptional pH-dependent switches with potential
exploitation in cancer research.
Chart 1.
Interesting examples of aminophosphine chelate ring-opening in
Pt(II) and Pd(II) square-planar complexes where activation
occurs through PtN bond cleavage in aqueous solution (in a
pH- and chloride concentration-dependent manner) have been
reported to be dependent on the substituents and length of the
tether ring.[7] Rauchfuss produced ruthenium half-sandwich
Introduction
RuII arene organometallic compounds of the type [Ru(6-
arene)(XY)(Z)]n+ (where XY is a bidentate chelating ligand and Z
a monodentate ligand) are well known cytotoxic anticancer drug
candidates.[1] DNA metallation upon labilization of the RuZ
bond is an identified mechanism for the compounds' anticancer
activity. Various activation triggers for the Ru–Z bond have been
investigated.[2] They vary from a chloride concentration drop
resulting in the Ru–Cl/I bond hydrolysis (Z = halide), to ligand
oxidation of a thiophenolato ruthenium complex (Z = SPh)
followed by aquation; both scenarios leading to compound
activation and RuII cytotoxicity.[3]
tether
cyclopentadienyls
complexes
of
with
formula
thiophene-functionalised
[Ru(η5:κ1-C5H4(CH2)(2-
SC4H3)(PPh3)2]+. The tethering sulphur in the thiophene was
shown to be capable of chelating the metal as well as being
displaced by diatomic hydrogen and hydrogen sulphide.[8-9]
Miyaki
et
al.
prepared
complexes
[Ru(η6:κ1-
C6H5(CH2)nNH2)PPh3Cl]+ (n = 2 and 3) and related tethered
alcohols of the type [Ru(η6:κ1-C6H5(CH2)3OH)( N,N′-
chelate)]2+.[10] Despite very useful insights on the preferences of
tether binding and the stability of different-size tethers, the
dynamics of the Ruκ1-N/O bond remained uninvestigated.
Complexes
[Ru(6:1-C6H5(C6H4)NH2)Cl2]
and
[Ru(6:1-
C6H5(C6H4)NH2)(en)]Cl2 (en = ethylenediamine) have been
reported to be capable of undergoing RuNtether bond cleavage
under extreme acidic conditions (12 M HCl) overnight.[11]
[a]
F. Martínez-Peña, Dr. A. M. Pizarro
IMDEA Nanociencia
Faraday 9, Ciudad Universitaria de Cantoblanco, Madrid 28049,
Spain
Here we investigate the possibility of activating this class of RuII
[Ru(6:1-
E-mail: Ana.Pizarro@imdea.org
tether
complexes
of
general
formula
C6H5(C6H4)NH2)(XY)]n+, where XY is a bidentate chelating ligand,
within the acidic pH range. 2-Aminobiphenyl (C6H5(C6H4)NH2) is
chosen as the hemilabile ligand that chelates ruthenium by a 6-
arene and a 1-NH2 coordination bond. We demonstrate that pH-
activation (cleavage of the RuNtether bond) is not only possible
in the acidic pH scale but, most importantly, tunable, by using
chelating ligands with different electronic and steric properties.
[b]
Dr. A. M. Pizarro
Nanobiotecnología (IMDEA-Nanociencia), Unidad Asociada al
Centro Nacional de Biotecnología (CSIC)
Faraday 9, Ciudad Universitaria de Cantoblanco, Madrid 28049,
Spain
Supporting information for this article is given via a link at the end of
the document.
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The pH is a major factor that governs the activation and its
associated kinetic parameters. Since this type of organometallic
compounds has been reported to interact with DNA,[12-13] we also
investigated the interaction of our switchable system with a
model nucleobase, guanosine monophosphate (GMP). Finally,
and most importantly, we prove total reversibility, forcing the
activated form of the compound to reverse to its inactive form
(closed-tether), again showing pH-dependent inactivation
dynamics. We set the parameters that control the ruthenium
arene in/activation to further the design of pH-responsive
switchable anticancer metallodrug.
tether structures were confirmed by X-ray crystal structure
analysis (vide infra).
Table 1.
Tether ruthenium(II) arene complexes of general formula
[Ru(η6:κ1-C6H5(C6H4)NH2)(XY)]Cln (n = 0, 2) studied in this work, containing
different XY chelating ligands.
Complex
1
XY chelate
Complex
8
XY chelate
en
dpip
2
3
dap
9
tmen
Results and Discussion
dab
10
o-pda
Synthesis and Characterization
Eleven closed-tether ruthenium(II) arene complexes, 1–11, of
general formula [Ru(η6:κ1-C6H5(C6H4)NH2)(XY)]n+ containing
primary, secondary and tertiary aliphatic amines, ortho-
phenylenediamine, and oxalato as chelating ligands (Table 1)
were prepared following the synthesis described by Pizarro et
al.,[11] in good yields. The complexes were fully characterized by
1H NMR spectroscopy, and CHN analysis for 1–5 and 8–11.
Complexes 6 and 7 were characterized in the solution state by
1H NMR spectroscopy and mass spectrometry. [Ru(6-etb)Cl2]2
dimer was used for the arene exchange reaction leading to
complex [Ru(η6:κ1-C6H5(C6H4)NH2)Cl2].
4
5
6
dach
pipma
dmen
11
12
13
oxo
dmphe
dtben
7
deen
14
bipy
Upon reaction of [Ru(η6:κ1-C6H5(C6H4)NH2)Cl2] with the
appropriate chelating ligand (details in Supporting Information),
a mixture of two structural isomers, closed and open-tether RuII
complexes (Figure 1) was obtained for complexes 1–8, while
complexes 9–11 were obtained as closed-tether isomers only.
Observation of open-to-closed tether conversion in aqueous
solution, overnight at ambient temperature, allowed for the
separation of the closed-tether RuII complexes 1–8. The crude
product is re-dissolved in water, left to stand overnight at
ambient temperature and subsequently lyophilized, affording
quantitatively the closed-tether complexes 1–8 in good yields
(over 95%). Tether-ring-opened analogues 1Cl–10Cl, of general
structure [Ru(η6-C6H5(C6H4)NH2)(XY)Cl]Cl, where the κ1-bound
Ntether is released from the first coordination sphere of the metal
to allow a chloride ligand bind the ruthenium center, were
characterized in solution by 1H NMR and LC/MS. While attempts
to isolate complexes 12 and 13 were unsuccessful, it was
possible to isolate pure the opened tether-ring analogues 12Cl
and 13Cl (characterized by CHN and NMR analysis). Complex
11Cl was not observed in methanol, solvent in which 11 was
insoluble. Dissolution of 11 in HCl afforded the dichlorido
precursor [Ru(η6:κ1-C6H5(C6H4)NH2)Cl2]. Complex 14Cl could be
dissolved in [D6]DMSO, yet instability towards arene loss
afforded complex [Ru(bipy)3]2+.[14] Substitution of the chlorido
ligands by chelating ligands in methanol is considered a rational
strategy to afford Ru–arene stability,[11, 15-17] yet in this class of
Ru-tethered complexes, which contain a highly strained tether
ring, we have observed that this stabilization strongly depends
on the nature of the donor atoms in the X–Ru–Y chelate. For
example, in [D6]DMSO at 298 K, the complex stability toward
decomposition related to arene loss increases in the order
Figure 1. Ruthenium arene tether isomers of general formula [Ru(η6:κ1-
C6H5(C6H4)NH2)(XY)]Cl2, closed-tether complex (left), and [Ru(η6-
C6H5(C6H4)NH2)(XY)Cl]Cl, open-tether complex (right). RuII arene closed-
tether complex 11, where XY is oxalato, is neutral, and no chloride ions are
present in its structure; for the open tether, the vacant site would be occupied
by a solvent molecule, and the complex remains neutral.
Usually both isomers, closed- and open-tether species, showed
a typical set of three resonances attributable to the 6-bound
arene protons in the range ca. 5.20–6.20 ppm. The
characteristic chemical shift difference between the most
deshielded and most shielded η6-bound arene resonances are
ca. 0.70 ppm and 0.20 ppm for closed- and open- tether
complexes, respectively (Figure S1). Analysis of the 1H NMR
spectra in all solvents used in this work proved an instant and
reliable evidence for the open- versus closed-tether ring status
in RuII arene complexes in solution. Both closed- and open-
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dichloride < bipy < oxalate < phenylenediamine < aliphatic
diamine (Figure S2). Presence of -acceptor ligands (o-pda,
bipy) lead to decomposition as previously observed, to a lesser
extent, for other RuII non-tether biphenyl complexes containing
phenylazopyridines and bipyrimidines.[18-19] Decomposition
mediated by arene loss is attributed to the competition for the
electron density on the ruthenium centre between the 6-bound
tethered arene and the electron acceptor chelating ligand.
The complexes adopt the expected pseudo-octahedral “three-
legged piano-stool” geometry with the ruthenium η6-bonded to
the arene ligand occupying three of the six octahedral positions.
The other three positions in the first coordination sphere are
occupied by a bidentate chelating ligand and either a NH2 group
(pendant from the η6-bound arene) for 1, 4, 8 and 10, or a
chlorido ligand for 7Cl, 9Cl, and 9HCl. Closed-tether complexes
1, 8 and 10 crystallized with two molecules, and 4 with eight
X-crystal structures of complexes 1, 4, 8, 10, 7Cl, 9Cl, and 9HCl, molecules, in the unit cell. Structures corresponding to open-
were essential to unequivocally assign the open- and closed-
tether ring structures. Solution experiments with complexes 11–
14 were not performed due to their instability and/or insolubility
under the conditions used in this work.
tether complexes 7Cl, 9Cl and 9HCl crystallized with four, two
and four molecules in the cell unit, respectively.
X-ray Crystal Structures
We determined the X-ray crystal structure of the closed-tether
complexes 1, 4, 8 and 10 (Figure 2), and open-tether complexes
7Cl, 9Cl and 9HCl (Figure 3).
X-ray diffraction data of suitable crystals corresponding to
closed-tether complexes were collected for [Ru(η6:κ1-
C6H5(C6H4)NH2)(en)]Cl2 (1), [Ru(η6:κ1-C6H5(C6H4)NH2)(dach)]Cl2
(4) and [Ru(η6:κ1-C6H5(C6H4)NH2)(o-pda)]Cl2 (10) from aqueous
solutions. Single crystals of [Ru(η6:κ1-C6H5(C6H4)NH2)(dpip)]Cl2
(8) were obtained from the reaction mixture. Crystals suitable for
X-ray
analysis
of
complexes
(7Cl) and
(9Cl) were
[Ru(η6:κ1-
[Ru(η6:κ1-
C6H5(C6H4)NH2)(deen)Cl]Cl
C6H5(C6H4)NH2)(tmen)Cl]Cl
obtained
by
crystallisation from methanol. Single crystals of protonated-
open-tether complex [Ru(η6-C6H5(C6H4)NH3)(tmen)Cl]Cl2 (9HCl)
were obtained from a 1 M HCl solution of 9.
Figure 3. ORTEP diagrams and atom numbering schemes for (A) [Ru(η6-
C6H5(C6H4)NH2)(deen)Cl]Cl (7Cl), (B) [Ru(η6-C6H5(C6H4)NH2)(tmen)Cl]Cl (9Cl),
and (C) [Ru(η6-C6H5(C6H4)NH3(tmen)Cl]Cl2 (9HCl), with thermal ellipsoids
drawn at 50% probability. The hydrogen atoms (with the exception of the
tether NH2/NH3 protons), chloride counterions and solvent molecules have
been omitted for clarity.
Selected bond lengths and angles of closed-tether complexes 1,
4, 8 and 10 are shown in Table 2, and detailed crystallographic
data in Table S1. The Ru–Carene bond lengths are in the range of
2.108–2.221 Å. The Ru–N1 distances are 2.148–2.166 Å, and
the distances ruthenium-to-centroid are 1.631–1.669 Å. The
propeller twist of the pendant ring with respect to the
coordinated phenyl of 2-aminobiphenyl ranges from 82.6º in 4 to
89.9º in 10. The torsion angles (σ) of planes containing C7, C6,
RuII and N1 lie within the range 1.05 (10) to 3.82º (4).
Coordination of the NH2 group of the pendant arm to the
ruthenium center results in different Ru–centroid distances,
appearing to be shorter in 1 (1.659 Å), than the reported open-
tether analogue 1Cl (1.666 Å).[11] Additional differences are
observed in C7–offset distances (0.488 Å towards ruthenium for
1 and 0.055 Å for the corresponding open-tether counterpart
1Cl).
Importantly, the size of the tether arm may influence the strain
on the structure as indicated by bond lengths and angles. In
order to demonstrate the contribution of tether-ring size to cause
such strain we compare complex 1 with X-ray crystallographic
data of structurally related complexes, containing a five-member
Figure 2. ORTEP diagrams for X-ray structures for (A) [Ru(η6:κ1-
C6H5(C6H4)NH2)(en)]Cl2 (1), (B) [Ru(η6:κ1-C6H5(C6H4)NH2)(dach)]Cl2 (4), (C)
[Ru(η6:κ1-C6H5(C6H4)NH2)(dpip)]Cl2 (8) and (D) [Ru(η6:κ1-C6H5(C6H4)NH2)(o-
pda)]Cl2 (10), with thermal ellipsoids drawn at 50% probability. The H atoms
(except those on the nitrogen pendant from the tether), chloride counterions
and solvent molecules have been omitted for clarity.
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and a six-member ring with a tethering aliphatic amine. For
No significant differences are observed between the open-tether
9Cl and its corresponding protonated species 9HCl. However, a
longer distance for the N1–C12 bond is observed in 9HCl versus
9Cl, indicating nitrogen protonation and subsequent loss of
conjugation of the amine group (1.378 vs 1.462 Å).
In all complexes the chloride counterions are involved in weak
bonding interactions with the N(tether) hydrogen atoms and the
hydrogen atoms on the N(XY-chelate ligand) (Figure S3 and
Table S4). No intermolecular π-π stacking interactions were
observed.
example, in complex
1
and its analogue [Ru(η6:κ1-
C6H5(CH2)2NH2)(en)]Cl2,[20] both with two carbon atoms between
the η6-bound arene and the Ntether, centroid distances are similar
(1.659 vs 1.651 Å), and Ru–C6–C7 angles are in the same
range 113–115º. However, when the number of carbon atoms in
the
tether
arm
is
three,
as
in
[Ru(η6:κ1-
C6H5(CH2)3NH2)(deen)]Cl2,[20] Ru–centroid distance increases to
1.679 Å and the Ru–C6–C7 angle to 126º. This suggests that
the shorter the pendant arm, the higher the ring constraint, since
the non-strained Ru–C6–C7 angle in open-tether complexes is
ca. 130º.
Solution studies in non-aqueous solvents
It has been reported that when the tether pendant group is
poorly
coordinating,
such
as
alcohol
or
amine,
Table 2. Selected bond lengths (Å) and angles (deg) for complexes 1, 4, 8
and 10.
coordination/dissociation can occur depending on the
experimental conditions.[9-11, 17, 21] We report here the effects of
varying
the
XY
chelating
ligand
in
[Ru(η6:κ1-
C6H5(C6H4)NH2)(XY)]2+ on the tether ring opening in different
solvents (methanol and dimethyl sulfoxide) with the aim of
exploiting these systems as activatable organometallic switches,
in drug design or otherwise.
1H NMR or UV-visible spectroscopy were used to study the
lability of the Ru–Ntether bond of closed-tether RuII complexes in
different solvents over 24 h. All quantification data (extent of
open-tether species versus closed-tether complex at equilibrium)
were generated by NMR spectroscopy, while kinetic data were
obtained by UV-vis spectroscopy in the case of methanol, and
by NMR for DMSO.
Bond / angle
1
4
8
10
Ru-C6
Ru-C5
Ru-C4
Ru-C3
Ru-C2
Ru-C1
Ru-centroid
Ru-X
Ru-Y
Ru-N1
X-Ru-Y
X-Ru-N1
Y-Ru-N1
Propeller twist
Offset C7
σ
2.115(13)
2.184(13)
2.207(14)
2.221(15)
2.198(13)
2.191(13)
1.659
2.144(11)
2.148(11)
2.161(11)
80.4(4)
88.2(4)
88.0(4)
89.7
0.488 (+)
1.29
2.113(6)
2.180(6)
2.185(6)
2.219(6)
2.199(6)
2.190(6)
1.656
2.124(5)
2.147(5)
2.156(4)
79.17(18)
86.89(18)
89.72(18)
82.6
2.114(4)
2.188(5)
2.179(5)
2.211(5)
2.218(5)
2.203(4)
1.669
2.128(3)
2.131(4)
2.167(3)
79.45(14)
89.45(13)
88.15(16)
88.9
2.108(7)
2.163(8)
2.175(8)
2.189(7)
2.192(7)
2.170(8)
1.643
2.128(6)
2.126(6)
2.147(5)
79.6(2)
88.5(2)
87.7(2)
89.9
0.460 (+)
3.82
0.460 (+)
1.20
0.475 (+)
1.05
As expected, open-tether complexes 7Cl, 9Cl and 9HCl show
longer ruthenium-centroid distances (1.684–1.688 Å), in
agreement
with
a
non-strained
structure.
Detailed
crystallographic data are shown in Table S2. Selected bond
lengths and angles are shown in Table S3. The propeller twist of
the pendant ring with respect to the coordinated phenyl of 2-
aminobiphenyl is significantly smaller, ranging from 41 to 46º.
The Ru–Carene bond lengths are accordingly significantly higher
(2.168–2.273 Å) for the open tether complexes, and the Ru–Cl
distances are 2.400–2.410 Å.
Figure 4. 1H NMR low-field spectral region of 6 mM solutions of [Ru(η6:κ1-
C6H5(C6H4)NH2)(en)]2+ (1, ) showing resonances of the η6-bounded arene
ligand at various times in (A) [D4]MeOH at 298 K (Z = Cl, X = NH2), (B)
[D6]DMSO at 298 K (Z = DMSO, X = NH2), and (C) 1 M [D1]HCl solution at 310
K (Z = Cl, X = NH3+). New sets of peaks appear (), corresponding to an
open-tether species, in all solvents over 24 h, except in [D2]H2O.
The structural analysis shows a pronounced tilt in the η6-bound
arene for closed-tethers in comparison to open-tether complexes,
as indicated by the differences in the RuC3 versus RuC6
bond lengths. Additional tether-ring strain is defined by the offset
of C7 with regard to the plane that contains the η6-bound arene
(Table 2). Comparison between the propeller twist in closed
complexes (83–90º) versus open tether structures (40–46º)
furthers the evidence of the constraint that the tether ring holds,
which has been related to instability of the complexes towards
arene loss.[17]
Activation in methanol of the Ru–Ntether bond can provide
important information about the tuneability of the ring-opening
process since methanol readily dissolves our complexes and
appears to be non-coordinating. Ring-opening dynamics were
followed with complexes 110 by 1H NMR spectroscopy. 1H
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NMR data of freshly prepared 6–7 mM solutions of complexes
110 were recorded directly after the complex was dissolved (t ≤
10 min) and at 24 h, at 298 K in [D4]MeOH. The 1H NMR spectra
initially contained one major set of peaks attributable to the
closed-ring form. A second set of peaks appeared over time
(Figure 4A), increasing in intensity until reaching equilibrium (2–
24 h). The new set of peaks were assigned to the open-tether
peaks corresponding to the intact closed-tethered species, as
well as some low-intensity peaks assignable to RuII open-tether
complexes (5–65%). After 24
h at 298 K, the peaks
corresponding to the closed-tether precursor [Ru(η6:κ1-
C6H5(C6H4)NH2)(XY)]2+ disappeared while the intensity of the
signals corresponding to the open ring-tether complex reached
100% (Figure 4B) in all cases. The new open-tether RuII arene
counterparts,
1Cl–10Cl,
of
general
formula
[Ru(η6-
species
were
identified
as
[Ru(η6-
C6H5(C6H4)NH2)(XY)Cl]+, formed to different extents at
equilibrium. The crystallographic structures of 7Cl and 9Cl were
obtained from methanol and unequivocally confirmed the
chlorido open-tether complexes. The extent of the conversion of
closed-to-open-tether at equilibrium varied depending on the
chelating ligand (Figure 5). Only complexes bearing a secondary
or tertiary aliphatic diamines as chelating ligand XY (6–9)
underwent full conversion over 24 h at 298 K. Chelating ligands
binding through primary amines RNH2 (1–5 and 10) showed
pecentages of the open-tether complex at equilibrium in the
range of 57–71%. Since the basicity of the chelating amines is
similar and seems not to govern this trend (Table S5), we
suggest that this effect might be attributable to a higher steric
hindrance effect by the secondary and tertiary XY amines.
C6H5(C6H4)NH2)(XY)(DMSO)]n+,
1dmso–11dmso.
The
conversion of closed-tether RuII complexes into the open-tether
dmso-adducts occurred at different rates, and the rate constants
1
were determined based on data obtained by H NMR (Table 3
and Figure S5). For complexes 2, 3, 6, 7, 9 and 10 the tether
opening rates in DMSO were too rapid to be determined.
The observed trend for the tether-ring opening process in DMSO
is similar to that of methanol throughout the entire series (Table
3). We observed rapid reactivity in complexes 6, 7 and 9, which
bear sterically demanding XY diamines of the type
RR'NCH2CH2NRR', where at least one of the R substituents is a
carbon chain (methyl or ethyl) with free 360 rotation along the
N–C(R) bond. This observation would be in agreement with an
associative mechanism[22] for the substitution reaction.
According to this mechanism, a molecule of solvent enters the
ruthenium coordination sphere before the tether unbuckles from
the RuII center, and therefore a more crowded intermediate
would favor rapid dissociation of the Ntether from the ruthenium.
Complex 4 shows - in both solvents - the slowest k and the
longest half-life times of the series, which is in contrast with the
rapid ring-opening rate of its aromatized counterpart, complex
10, demonstrating how the tether ring dynamics can be finely
tailored by the nature of XY. In their crystal structure a longer
Ru–Ntether bond (2.156 Å) in 4 is observed, as opposed to a
shorter (2.148 Å) bond in 10. This is in agreement with electron
back donation by phenylenediamine in 10, thus rendering the
ruthenium center more electrophilic and subsequently more
prone to nucleophilic attack.[16] Such attack could be enhanced
in 10 due to the less sterically demanding sp2 ligand
phenylenediamine in comparison to sp3 diaminocyclohexane.
Table 3. Opening-tether kinetic data for complexes 1–11 in methanol,
dimethylsulfoxide and 1 M HCl.
Activation under acidic conditions
Since our aim was to prove the pH-dependent on-and-off
switching capability and tuneability of our system, we
investigated the possibility of activating the Ru–Ntether bond in
aqueous solution at 310 K.
In order to determine the ring opening rate constants in
methanol, 0.2 mM solutions of the RuII complexes were
monitored over 24 h by UV-visible spectroscopy (Table 3 and
Figure S4). The rate of tether-ring opening for complexes 2, 3, 6,
7, 9 and 10 was too fast to be determined (equilibrium reached
in < 5 min).
In DMSO, the high coordinating nature of the solvent results in
total tether-opening for all complexes in the series, following
nucleophilic attack by dimethyl sulfoxide and subsequent
formation of the DMSO adduct within 12 h. 1H NMR data of
freshly prepared 6–7 mM solutions of complexes 1–11 were
recorded directly after the complex was dissolved (t ≤ 10 min)
and at 24 h, at 298 K in [D6]DMSO. The initial 1H NMR spectra of
complexes 1–11 after sample preparation (ca. 7 min) showed
Very few studies where protonation of the binding functional
group in the tether plays an important role in the dynamics of
metal–tether bond in aqueous solution have been reported.[7, 21,
23]
Pizarro et al. reported the synthesis of [Ru(η6-
C6H5(C6H4)NH3)Cl3] from its closed-tether counterpart, [Ru(η6:η1-
C6H5(C6H4)NH2)Cl2], by suspending the former in concentrated
HCl (ca. 12 M) at ambient temperature for 18 h. The tethered
nitrogen dissociated from the ruthenium upon protonation.[11]
Activation of a Ru–Ntether bond was also proved in the RuII tether
complex [Ru(η6:κ1-C6H5(C6H4)NH2)(en)]Cl2 in 12 M HCl for 18 h,
yielding [Ru(η6:κ1-C6H5(C6H4)NH3)(en)Cl]2+. Acidic activation had
been deemed unsuccessful at pH as low as 2. In this work we
tailor the tethered RuII arene complex by incorporating a variety
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of XY chelating ligands to (i) confer extraordinary stability to the
[Ru(η6:κ1-arene:N)(XY)]n+ system and (ii) demonstrate the
possibility of fine tuning the activation of the Ru–Ntether bond at
acidic pH.
or ethyl groups, freely rotating along the NC(R) axes in the
chelating RR'NCH2CH2NRR' ligand.
In order to investigate the behavior of the Ru–NH2(tether) bond
under milder acidic conditions, complexes 1–10 in 0.1 M [D1]HCl
solutions were followed over time at 310 K by 1H NMR. A
mixture of closed- and protonated open-tether RuII complexes,
[Ru(η6:κ1-C6H5(C6H4)NH2)(XY)]2+
and
[Ru(η6-
C6H5(C6H4)NH3)(XY)Cl]2+, was observed for all complexes at
equilibrium. In general, higher percentages of open-tether RuII
species are observed with increasing steric hindrance on the
nitrogen atoms of the N,N-chelating diamine (Figure 5).
Strikingly, complex 9 (XY = tetramethylethylenediamine, tmen),
with the highest rate constant for the opening process of the
series, showed the least percentage of open species at
equilibrium, perhaps attributable to the lack of H-bond formation
capability by tmen.[24]
A decrease in the concentration of [D1]HCl (0.01 M) resulted in a
modest extent of activation of the Ru–Ntether bond (ca. 2–5% of
open-tether species at equilibrium). Further dilutions of [D1]HCl
did not lead to the activation of the Ru–NH2(tether) bond as
observed by NMR spectroscopy.
Figure 5. Percentage of RuII open species at equilibrium, corresponding to the
activation of the complexes 1–10 in [D4]MeOH (light grey bars) and in 0.1 M
[D1]HCl (dark grey bars).
Complexes 1–10 were dissolved in 1 M [D1]HCl and their 1H
NMR spectra were recorded at 310 K over 24 h (Figure 4C). In
1H NMR experiments of complexes 1, 6, 7 and 9 in [D1]HClO4
(0.1 M) were also carried out in presence and absence of 0.1 M
NaCl, in order to investigate the role of the chloride ions in the
opening process in acidic conditions. In the absence of NaCl,
the percentage of protonated open species 1HCl, 6HCl, 7HCl
and 9HCl were 17, 77, 50 and 3%, respectively, significantly
lower than in the presence of sodium chloride (56, 79, 65 and
18%, respectively) as seen in Figure S7. Complex 6 and 7
showed the lowest activation dependency to the presence of
excess chloride ions. The importance of the pH was
demonstrated when complex 6 afforded merely 15% of the open
tether complex 6Cl when exposed to a 2 M NaCl solution for 24
h at 298 K. When experiments of tether ring activation of 1, 6, 7
and 9, using a mixture of 0.1 M [D1]HClO4 and 0.1 M NaCl were
carried out, identical extent of protonation of the open species to
that of activation in 0.1 M [D1]HCl solutions were obtained after
24 h.
all
cases
the
open-ring
tethered
cation
[Ru(η6-
C6H5(C6H4)NH3)(XY)Cl]2+, 1HCl–10HCl, was identified as the
only species at equilibrium. The 1H NMR data showed that
complexes 1–10 were fully converted into the corresponding
open-tether ruthenium arene hydrochloride salt upon
dissociation of the NH2(tether) group from the ruthenium center,
position now occupied by a chlorido ligand. Under these acidic
+
conditions, the NH2 is protonated to become NH3 . X-ray
crystallographic structure of complex 9HCl allowed the
unequivocal assignment of these complexes (Figure 3C). The
conversion of closed-tethered species into the protonated open-
tether RuII complexes occurred at different rates as determined
by UV-visible spectroscopy (shown in Table 3 and Figure S6).
The trend of the rates of activation for the series 1–9 shared
similarities with those in methanol and DMSO (Table 3).
Complex 9, bearing tetramethylethylenediamine as a chelating
ligand, showed the highest rate constant for the pH-dependent
tether-opening process. Complexes 6 and 7 were converted too
rapidly into their protonated open-tether form (fully opened within
5 min), so their rate constants could not be determined by 1H
NMR nor UV-visible spectroscopy. The slowest rates of ring-
opening in strong acidic conditions were for complexes 1, 4, 5, 8,
and 10, where the XY chelating ligand forms a 5-member
chelating ring, which bears no freely rotating substituents in the
N,N-chelating diamines. As observed for the tether/opening
dynamics in non/aqueous solvents, this is in agreement with an
associative mechanism for the substitution reaction (vide supra).
Complexes 5 and 8, bearing secondary amines with restricted
rotation of the NC bond in the chelating ligand, present the
pKa values and reversibility of the activation process
The activation (opening) and deactivation (closing) of the
ruthenium complex can be understood as the unbuckling and
buckling of the pendant NH2(tether) to RuII. For this reason,
control over the availability of this group to bind to the metal
+
center through its acid/base equilibrium (unavailable NH3
versus available for metal coordination NH2) appears as an
elegant way of controlling compound activation. For this purpose
we have determined the acid/base equilibrium (pKa values) of
the pendant tether-nitrogen for complexes 6HCl and 9HCl.
1
The pH dependency of the H NMR chemical shifts of the 6-
bound arene of [Ru(η6-C6H5(C6H4)NH3)(dmen)Cl]2+ (6HCl) and
[Ru(η6-C6H5(C6H4)NH3)(tmen)Cl]2+ (9HCl) was probed over the
pH range 010 and the pKa of the amine group of the pendant
arm was determined. Complexes 6 and 9 were dissolved in 1 M
[D1]HCl to obtain 6HCl and 9HCl. At pH 0, the species in
slowest rate constants, perhaps due to
a more stable
intermediate in the substitution reaction. This rational would
explain why 6, 7 and 9 show the most rapid tether-ring opening,
attributable to the high steric hindrance imposed by the methyl
solution
was
identified
as
[Ru(η6-
C6H5(C6H4)NH3)(dmen/tmen)Cl]2+. With increasing pH, the 1H
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NMR triplet resonance corresponding to Hm (meta- proton of the
6-bound arene) shifted to high-field accompanied with a
decrease in intensity (see Figure 6 for 9HCl). A plot of the
change in chemical shift of the Hm(6HCl) and Hm(9HCl) versus
pH is shown in Figure 7. The data were fitted to the Henderson-
Hasselbach equation which yielded pKa values of 2.51 and 2.64
for 6HCl and 9HCl, respectively. These values are ca. 1.5 units
lower than that of the free ligand (pKa 3.81). This suggests that
electron density from the aniline pendant ring is more readily
delocalized when 2-aminobiphenyl is η6-coordinated to RuII. The
higher pKa for 9HCl versus 6HCl may be understood on the
basis of the more electron rich tmen in comparison with dmen,
perhaps ultimately conferring more basicity to the tether NH2
through the arene ligand on the RuII.
the electrophilic ruthenium center for the nucleophilic free
dangling amine.
Figure 7. 1H NMR chemical shift dependency of the Hm proton of the 6-bound
arene on pH of the protonated open-tether complexes [Ru(η6-
C6H5(C6H4)NH3)(dmen)Cl]2+ (6HCl) and [Ru(η6-C6H5(C6H4)NH3)(tmen)Cl]2+
(9HCl). The curve represents the best fit to the Henderson-Hasselbalch
equation and correspond to pKa value of 2.51 and 2.64, respectively.
Figure 6. pH-dependence of the signals corresponding to the η6-bound arene
spectral region of [Ru(η6-CH5(C6H4)NH3)(tmen)Cl]2+ () in [D1]HCl/[D2]H2O.
Since our aim is to design systems capable of reversible
switching, we investigated the pH effect in the reverse tether-
ring closure. This process, to our understanding mediated by
hydrolysis,[11] was followed for complex 6Cl in water at different
pH values above the pKa of the amine tether group. Open-tether
complex 6Cl (6 mM) was dissolved in different pH buffered
aqueous solutions at 310 K. The closure rate to form 6 in these
solutions was monitored by 1H NMR spectroscopy. Data were
fitted to the Henderson-Hasselbach equation which yielded half-
life values of 84, 75, 63 and 15 min for pH 3.4, 4.3, 5.8 and 7.2,
respectively. The rate constants were calculated as 8.2, 9.3,
11.0 and 50.8 × 10–3 min–1. The data revealed that inactivation
(ring closure) of the RuII complex is slower at the more acidic pH.
The results, shown in Figure 8, clearly illustrate that tether-ring
fastening is governed by pH.
Figure 8. The rate of reversion
of the open-tether species [Ru(η6-
C6H5(C6H4)NH2)(dmen)Cl]Cl (6Cl), to [Ru(η6:κ1-C6H5(C6H4)NH2)(dmen)]2+ (6) in
four different citrate/phosphate buffered solutions over time at 310 K.
Interaction with nucleobases and biological activity
Irreversible DNA damage is often believed to be the mechanism
of action of metal-based anticancer complexes, with guanine N7
nitrogen being the preferred metal binding site on DNA.[13, 25-27]
We propose that open (activated) tether RuII complexes are
prone to nucleobase interaction, while the corresponding closed
tether complex, with no vacant site in the first coordination
Proton concentration plays a key role both in the activation and
in the deactivation process, as protons appear to compete with
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sphere around the ruthenium centre, should not interact with the
model nucleobase.
more importantly, in acidic water by varying the N,N-chelating
ligand. In general, both electronic and steric effects play a role in
Ru–Ntether activation in a substitution reaction that appears to
occur via an associative mechanism.
We were interested in knowing if closed-to-open tether RuII
arene activation could lead to DNA interaction, hence the
reaction of 6Cl with guanosine monophosphate (GMP) was
investigated. The binding of the open-tether complex 6Cl,
[Ru(η6-C6H5(C6H4)NH2)(dmen)Cl]+, to 5'-GMP in either water or
methanol was monitored by 1H NMR spectroscopy. Upon mixing
6Cl with 5'-GMP (1:5 metal:nucleobase ratio, 5 mM RuII) in
[D4]methanol, total disappearance of the peaks corresponding to
complex 6Cl and the appearance of new peaks assignable to
Ru-guanine adducts, including three new peaks in the H8 region
We demonstrate that organometallic drug activation via
dissociation of the Ru–Ntether bond in acidic water solution is
possible within the pH scale. Importantly, protonation of the
tethering group locks the complex in its activated form.
Reversibly, deprotonation triggers metal binding thus rendering
the complex unsuited for biomolecule interaction, conveniently
inactivating the drug. The dynamics of tether ring opening
(activation) and closure (inactivation) are obligingly pH
dependent as well. The process is fully reversible. Our future
work aims to synthesize RuII tether complexes whose buckling
tether group presents a pKa value nearer to neutrality by
combining a variety of tethered arenes with the adequate XY
chelating ligand to fine tune the activation process in the slightly
acidic tumor microenvironment, providing a creative strategy to
trigger selective cell cytotoxicity.
1
of guanine, was observed in the H NMR spectra at 298 K over
24 h (equilibrium). The three new peaks (Figure S8) were
tentatively assigned to the Ru–N7GMP, Ru–O(PO3)GMP and
Ru–N1GMP adducts, according to literature.[28-29] We hoped to
investigate the pH-dependency of the binding of complex 6Cl to
+
nucleobases. However, the pKa of the tether NH3 (ca. 2.5) is
inconveniently close to that of the N7 of guanine (pKa 2.57).[28, 30]
This implies that prevention of Ru–Ntether bond formation by
+
carrying out the reaction at pH < pKa(NH3 tether) would also
render the guanine unable for interaction with the ruthenium.
Reaction of closed-tether 6 with 5 mol equiv of 5'-GMP in
[D2]H2O at 310 K did not afford any new H8 signals over 24 h
Experimental Section
Materials. RuCl33H2O was acquired from Precious Metals Online.
1
(Figure S8) as observed by H NMR. These results support that
Ethylendiamine
(en),
1,3-diaminopropane
(dap),
trans-1,2-
opening of the tether-ring equals RuII complex activation, while a
closed tether RuII complex is rendered unreactive towards
nucleobases. These results are in agreement with the lack of
cytotoxic activity in our cell viability experiments in the human
ovarian cancer cell line A2780 for complexes 111 (Table S6).
At the maximum concentration tested (100 M), well within the
range of cytotoxic activity for the type of drugs of general
structure [Ru(η6-arene)(XY)Cl]Cl as reported in literature,[31] no
significant inhibition of cell growth was observed (with the
exception of complex 10, perhaps attributable to redox-active o-
pda ligand) and thus the complexes were deemed inactive. This
demonstrates the inactivated state of the compounds at
physiological pH, and represents the first step towards rational
design of a pH-responsive organometallic pro-drug.
diaminocyclohexane
(dach), 2-piperidinemethanamine
(pipma),
N,N,N′,N′-tetramethylethylenediamine (tmen), sodium oxalate (oxo), 2,2'-
bipyridyl (bipy), ethyl benzoate, and 5'-guanosine monophosphate were
purchased from Sigma-Aldrich. N,N′-dimethylethylenediamine (dmen),
N,N′-diethylethylenediamine
(deen),
and
1,2-
bis(dimethylphosphino)ethane (dmphe) were acquired from Alfa Aesar.
1,4-Diaminobutane (dab), 2,2'-bipiperidine (dpip), and N,N'-di-tert-
butylethylenediamine (dtben) were purchased from TCI Europe.
Dichloroethane, magnesium, 2-aminobiphenyl, and o-phenylenediamine
(o-pda) were acquired from Acros Organics, and iodine from Fisher.
Ethanol, dry methanol, diethyl ether, tetrahydrofuran, dimethylsulfoxide,
sodium metal, and sodium chloride were purchased from Sharlau. The
solvents used for UV-visible absorption spectroscopy were dry methanol,
and deionized water. For NMR spectroscopy, the solvents used were
[D4]methanol, [D6]DMSO, [D1]HCl, [D1]HClO4 and [D2]H2O obtained from
VWR International.
Preparation of complexes. The RuII dimer [Ru(6-etb)Cl2]2 where etb is
ethyl benzoate was synthesized following the reported synthesis by
Habtemariam et al.,[32] including a modification of the esterification step
whose details are given in the Supporting Information. The dichlorido
complex [Ru(η6:κ1-C6H5(C6H4)NH2)Cl2] was synthesized via thermal
displacement of ethyl benzoate in the RuII dimer [Ru(6-etb)Cl2]2 by 2-
aminobiphenyl in 1,2-dichloroethane under pressure overnight as
reported by Pizarro et al.[11] Complexes 1–11, 12Cl and 13Cl (Table 1)
were isolated as chloride salts using slight variations of a reported
procedure.[11] Typically, 1 mole equivalent of the bidentate chelating
ligand (XY) was added to a suspension of the dichlorido ruthenium
complex [Ru(η6:κ1-C6H5(C6H4)NH2)Cl2] in methanol or methanol/water to
afford [Ru(η6:κ1-C6H5(C6H4)NH2)(XY)]2+. Complexes 1Cl10Cl, 12, 13
and 14Cl were characterized in solution. Details of the amount of
reactants, volume of solvents, color changes, stirring times, and nature of
the product are given in the supplementary section for the individual
reactions, as well as any variation in the synthetic procedure.
Conclusions
In this work we present a series of RuII arene complexes
capable of on-and-off switching in a pH-dependent manner. The
complexes have
a
general structure of [Ru(η6:κ1-
C6H5(C6H4)NH2)(XY)]Cln, and show structural stability in solution.
We have used this ruthenium platform to understand the
chelate-based ring-opening and ring-closing reactions in view of
development of newer pH activatable inorganic drugs.
The complexes bear a η6:κ1-arene:N tether-ring capable of
locking and unlocking by the Ru–Ntether bond. We have
demonstrated that once activated (tether-ring open) the RuII
complexes are prone to interaction with nucleophiles, such as
chloride ions, as well as with model DNA nucleobases. The
activation kinetics and thermodynamic equilibria of the Ru–Ntether
bond can be finely tuned in methanol, dimethylsulfoxide and,
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Instrumentation. Single crystal X-ray Diffraction. Suitable crystals of
compounds 1, 4, 7Cl, 8, 9Cl, 9HCl, and 10, were coated with mineral oil
and mounted on Mitegen MicroMounts. The samples were measured in a
Bruker D8 KAPPA APEX II diffractometer with CCD area-detector,
equipped with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å).
The substantial redundancy in data allowed empirical absorption
corrections (SADABS)[33] to be applied using multiple measurements of
symmetry-equivalent reflections. Raw intensity data frames were
integrated with the SAINT program, which also applied corrections for
Lorentz and polarization effects. The Bruker SHELXTL Software
Package was used for space group determination, structure solution, and
refinement.[34] The space group determination was based on a check of
the Laue symmetry, and systematic absences were confirmed using the
structure solution. The structures were solved by direct methods
of closed- and open-tether species in [D6]DMSO, [D4]MeOH (298 K) and
[D1]HCl solutions (1, 0.1, and 0.01 M [D1]HCl) at 310 K at equilibrium (1–
1
8 h) were quantified by integration of peaks in the H NMR spectra. The
extent of the ring opening process by 1H NMR spectroscopy in acidic
conditions at equilibrium was also determined in 1 M [D1]HClO4/[D2]H2O
and 0.1 M [D1]HClO4/[D2]H2O (with and without 0.1 M NaCl) at 310 K in
ca. 6 mM RuII solutions for selected complexes of the series (vide infra).
In order to determine the rate of the ring-opening process in [D6]DMSO,
1H NMR spectra were acquired at 10 minutes intervals over 12 h at 298 K.
The data following time-course dynamics of the DMSO-adduct formation,
based on peak integrals, were fitted to a pseudo-first-order kinetic
equation. OriginPro was used to fit the exponential decay and to obtain
the rate constant (k, min–1). Experiments with 6 and 7 were performed by
adding a 10 µL aliquot of a 15 mM heavy water solution to 690 µL of
[D6]DMSO. In methanol and 1 M HCl ([RuII] = 200 M), the rate constants
were calculated from the time-course data obtained by following the
opening process by UV-visible spectroscopy. Experiments with
complexes 6 and 7 were performed by adding an aliquot of a RuII 6 mM
water solution to either methanol or 1 M HCl for a final RuII concentration
of ca. 100 M. The absorbance was recorded at several time intervals
over 24 h at 298 K for methanol and at 310 K for 1 M HCl. A plot of the
change in absorbance with time was fitted to the appropriate equation for
pseudo-first-order kinetics using OriginPro to give the half-lives (t1/2, min)
and rate constants (k, min–1). The tether ring-closure of open tether
complex 6Cl was monitored by 1H NMR using different buffered aqueous
solutions. Citric acid/Na2HPO4 buffer was used to prepare pH 3.4, 4.3,
5.8 and 7.2 [D2]H2O solutions. Complex 6Cl was dissolved (ca. 6 mM) in
each buffered solution and the spectra were acquired at 10 min intervals
over 12 h at 310 K. The data of the time-course dynamics to form the
closed-tether complex (6) was determined by integration of the 1H NMR
peaks and data were fitted to the equation for pseudo-first-order kinetics.
(SHELXL-2014/7),[35-36] completed with different Fourier syntheses, and
2
refined with full-matrix least-squares using SHELXS minimizing ω(Fo
–
Fc2)2. Weighted R factors (Rw) and goodness of fit (S) are based on F2;
conventional R factors (R) are based on F. All non-hydrogen atoms were
refined with anisotropic displacement parameters. Hydrogen atom
positions were geometrically calculated and allowed to ride on their
parent carbon or nitrogen atoms with fixed isotropic U. All scattering
factors and anomalous dispersion factors are contained in the SHELXTL
6.10 program library.
The crystal structures of compounds 1, 4, 7Cl, 8, 9Cl, 9HCl and 10 have
been deposited at the Cambridge Crystallographic Data Centre with
deposition numbers CCDC 1522976, 1522978, 1522989, 1522990,
1522991, 1522992, and 1522997, respectively.
NMR Spectroscopy. 1H NMR spectra were acquired in 5 mm NMR
tubes using a Bruker DPX 400 MHz spectrometer. 1H NMR chemical
shifts were internally referenced to 1,4-dioxane (3.75 ppm) for aqueous
solutions, (CHD2)(CD3)SO (2.50 ppm) for [D6]DMSO and CD2HOD (3.34
ppm) for [D4]methanol. Data processing was carried out using
MestreNova, version 6.0 (Mestrelab Research, S.L.).
Calculation of pKa values. In order to determine the pKa values of the
pendant NH2 group in open tether complexes, the pH of aqueous
solutions of complexes 6HCl and 9HCl was varied from pH 0 to 10 by the
addition of dilute aliquots of [D1]NaOH and [D1]HCl, measuring the
solution pH before and after recording the 1H NMR spectra. The chemical
shifts of the bound-arene ring protons were plotted against pH. The pH
titration curves were fitted to the Henderson-Hasselbach equation using
the program OriginPro, with the assumption that the observed chemical
shifts are weighted averages according to the populations of the
protonated (6HCl and 9HCl) and the deprotonated (6Cl and 9Cl) species,
i.e., the NH3+/NH2 equilibrium on the pendant NH2 tether arm.
Mass Spectrometry. Electrospray ionization mass spectra (ESI-MS)
were obtained by infusing the samples into an Agilent 6120 Single
Quadrupole LC/MS. The cone voltage was varied between 40 and 200 V
depending on sensitivity. The mass spectra were recorded with a scan
range of m/z 50–500 for positive ions.
UV-visible Spectroscopy. A Cary 5000 UV-Vis-NIR spectrophotometer
(Agilent Technologies, CA), was used with 1 cm path length cuvettes
(850 L) provided with a Temperature Controller. Spectra (500–200 nm)
were recorded at 310 K when the samples were in water and at 298 K for
methanol samples. The data were processed using OriginPro version 9.0
(OriginLab, Northampton, MA).
Interaction with nucleobases. 6Cl was allowed to react with 5'-GMP (9
mol equiv) in [D4]methanol at 298 K. The reaction was monitored by 1H
NMR over 24 h, when equilibrium had been reached. In addition, reaction
of closed-tether complex 6 with 5'-GMP (5 mol equiv) was followed in
[D2]H2O at 310 K for 24 h. Their 1H NMR spectra were recorded at
various time intervals.
Elemental Analysis. Elemental analysis was carried out using a Leco
analytical elemental analyzer CHNS-932.
pH Measurements. The pH* values (pH meter readings in [D2]H2O
solutions) of NMR samples in [D2]H2O were measured at 298 K directly in
the NMR tube, using a FiveEasy pH meter equipped with a Crison micro-
electrode calibrated with Crison buffer solutions at pH 4 and 7. The pH*
values can be converted to pH values by use of the equation pH = 0.929
pH* + 0.42, suggested by Krezel and Bal,[37] for comparison with related
values in the literature.
Acknowledgements
We thank Dr. M. W. Davies for helpful scientific discussion on
organic synthesis, and Dr. J. Perles and Dr. M. Ramírez for
collection and analysis of the X-ray data. Dr. A. Arnáiz (Cell
Culture Facility at IMDEA Nanociencia) is gratefully
acknowledged for assistance with cell culture techniques. We
acknowledge funding from the EC (FP7-PEOPLE-2013-CIG (no.
631396), and FP7-PEOPLE-2011-COFUND AMAROUT II), from
Methods. Solution studies. The tether ring-opening process of closed-
1
tether complexes was monitored by H NMR or UV-visible spectroscopy
in methanol, dimethylsulfoxide and acidic aqueous solutions. The
concentration of RuII for the NMR studies was 6–7 mM, and 0.2 mM for
UV-visible spectroscopy, unless otherwise stated. The relative amounts
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[30] B. Lippert, in Prog. Inorg. Chem., John Wiley & Sons, Inc., Hoboken,
NJ, 2007, pp. 1-97.
the MINECO of Spain (RYC-2012-11231, CTQ2014-60100-R),
and from the Comunidad Autónoma de Madrid (S2013/MIT-
2850).
[31] R. E. Aird, J. Cummings, A. A. Ritchie, M. Muir, R. E. Morris, H. Chen,
P. J. Sadler, D. I. Jodrell, Br. J. Cancer 2002, 86, 1652.
[32] A. Habtemarian, S. Betanzos-Lara, P. J. Sadler, in Inorg. Synth., Vol.
35 (Ed.: T. B. Rauchfuss), John Wiley & Sons, Hoboken, NJ, 2010, pp.
160-163.
Keywords: ruthenium tether; pH activation; bio-organometallics;
anticancer complexes; bioinorganic chemistry
[33] Bruker, in APEX2,SAINT and SADABS, Bruker AXS Inc., Madison, WI,
2008.
[1]
[2]
[3]
A. L. Noffke, A. Habtemariam, A. M. Pizarro, P. J. Sadler, Chem.
Commun. 2012, 48, 5219-5246.
[34] B. AXS, in SHELXTL Version 6.10, Structure Determination Package,
Vol. Madison, WI, Bruker Analytical X-ray Instruments, 2000.
[35] G. M. Sheldrick, Acta Crystallographica Section A 2008, 64, 112-122.
[36] G. M. Sheldrick, Acta Crystallographica Section C 2015, 71, 3-8.
[37] A. Krȩżel, W. Bal, J. Inorg. Biochem. 2004, 98, 161-166.
A. M. Pizarro, A. Habtemariam, P. J. Sadler, Top. Organomet. Chem.
2010, 32, 21-56.
F. Wang, A. Habtemariam, E. P. L. van der Geer, R. Fernández, M.
Melchart, R. J. Deeth, R. Aird, S. Guichard, F. P. A. Fabbiani, P.
Lozano-Casal, I. D. H. Oswald, D. I. Jodrell, S. Parsons, P. J. Sadler,
Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 18269-18274.
O. Warburg, Science 1956, 123, 309-314.
[4]
[5]
M. G. Vander Heiden, L. C. Cantley, C. B. Thompson, Science 2009,
324, 1029-1033.
[6]
[7]
[8]
[9]
C. S. Allardyce, P. J. Dyson, D. J. Ellis, S. L. Heath, Chem. Commun.
2001, 1396-1397.
A. Habtemariam, B. Watchman, B. S. Potter, R. Palmer, S. Parsons, A.
Parkin, P. J. Sadler, Dalton Trans. 2001, 1306-1318.
M. Draganjac, C. J. Ruffing, T. B. Rauchfuss, Organometallics 1985, 4,
1909-1911.
J. Amarasekera, T. B. Rauchfuss, Inorg. Chem. 1989, 28, 3875-3883.
[10] Y. Miyaki, T. Onishi, H. Kurosawa, Inorg. Chim. Acta 2000, 300–302,
369-377.
[11] A. M. Pizarro, M. Melchart, A. Habtemariam, L. Salassa, F. P. A.
Fabbiani, S. Parsons, P. J. Sadler, Inorg. Chem. 2010, 49, 3310-3319.
[12] H.-K. Liu, F. Wang, J. A. Parkinson, J. Bella, P. J. Sadler, Chem. Eur. J.
2006, 12, 6151-6165.
[13] A. M. Pizarro, P. J. Sadler, Biochimie 2009, 91, 1198-1211.
[14] L. Ruhlmann, C. Costa-Coquelard, J. Hao, S. Jiang, C. He, L. Sun, I.
Lampre, Can. J. Chem. 2008, 86, 1034-1043.
[15] A. Habtemariam, M. Melchart, R. Fernández, S. Parsons, I. D. H.
Oswald, A. Parkin, F. P. A. Fabbiani, J. E. Davidson, A. Dawson, R. E.
Aird, D. I. Jodrell, P. J. Sadler, J. Med. Chem. 2006, 49, 6858-6868.
[16] T. Bugarcic, A. Habtemariam, R. J. Deeth, F. P. A. Fabbiani, S.
Parsons, P. J. Sadler, Inorg. Chem. 2009, 48, 9444-9453.
[17] T. Reiner, D. Jantke, X.-H. Miao, A. N. Marziale, F. J. Kiefer, J.
Eppinger, Dalton Trans. 2013, 42, 8692-8703.
[18] S. J. Dougan, M. Melchart, A. Habtemariam, S. Parsons, P. J. Sadler,
Inorg. Chem. 2006, 45, 10882-10894.
[19] S. Betanzos-Lara, O. Novakova, R. Deeth, A. Pizarro, G. Clarkson, B.
Liskova, V. Brabec, P. Sadler, A. Habtemariam, J. Inorg. Biochem.
2012, 17, 1033-1051.
[20] F. Martínez-Peña, A. M. Pizarro, Unpublished results.
[21] R. Stodt, S. Gencaslan, Iris M. Müller, William S. Sheldrick, Eur. J.
Inorg. Chem. 2003, 2003, 1873-1882.
[22] F. Wang, H. Chen, S. Parsons, I. D. H. Oswald, J. E. Davidson, P. J.
Sadler, Chem. Eur. J. 2003, 9, 5810-5820.
[23] T. G. Scrase, M. J. O’Neill, A. J. Peel, P. W. Senior, P. D. Matthews, H.
Shi, S. R. Boss, P. D. Barker, Inorg. Chem. 2015, 54, 3118-3124.
[24] M. Ito, Y. Endo, T. Ikariya, Organometallics 2008, 27, 6053-6055.
[25] R. C. Todd, S. J. Lippard, Metallomics 2009, 1, 280-291.
[26] S. J. Dougan, P. J. Sadler, Chimia 2007, 61, 704-715.
[27] A. M. Pizarro, N. P. E. Barry, P. J. Sadler, in Comprehensive Inorganic
Chemistry II (Second Edition) (Ed.: K. Poeppelmeier), Elsevier,
Amsterdam, 2013, pp. 751-784.
[28] H. Chen, J. A. Parkinson, R. E. Morris, P. J. Sadler, J. Am. Chem. Soc.
2003, 125, 173-186.
[29] A. F. A. Peacock, A. Habtemariam, S. A. Moggach, A. Prescimone, S.
Parsons, P. J. Sadler, Inorg. Chem. 2007, 46, 4049-4059.
This article is protected by copyright. All rights reserved.

10.1002/chem.201701681
Chemistry - A European Journal
FULL PAPER
FULL PAPER
A
new family of activatable
F. Martínez-Peña, A. M. Pizarro*
ruthenium arene tethered complexes
have
characterized.
been
synthesized
These
and
metal
Page No. – Page No.
Control of Reversible Activation
complexes can reversibly open and
close vacant position for
Dynamics
of
[Ru(η6:κ¹-
a
C6H5(C6H4)NH2)(XY)]n+ and the Effect
of Chelate Ligand Variation
interaction with biological molecules.
Both the activation and inactivation
processes are pH-dependent. This
class of RuII tether complexes are
presented as the first step towards
rational design of organometallic pH-
dependent switches with promising
applications in cancer research.
This article is protected by copyright. All rights reserved.