Luminescent ruthenium polypyridyl complexes with extended 'dppz' like ligands as DNA targeting binders and cellular agents

Article abstract of DOI:10.1039/c6dt03792e

Four new Ru(ii) polypyridyl complexes that contain an extended aromatic moiety derived from pyrazino[2,3-h]dipyrido[3,2-a:2′,3′-c]phenazine and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) have been synthesized, their solid state X-ray crystal structure determined and their photophysical and biological properties evaluated. Their interactions with DNA have been studied, and they have been tested for their potential as photodynamic therapeutic (PDT) agents in the treatment of cancer. A practical modification of a method by Carter, Rodriguez and Bard has been introduced and used to calculate binding parameters for the complexes which show a strong affinity for DNA with binding constants in the order of 10⁷ M¹ (in 10 mM phosphate buffer). The complexes containing phen as an ancillary ligand become emissive upon binding to DNA (“light switch effect”), but do not show selective cytotoxicity upon light irradiation. On the other hand, the TAP complexes, which show an inverse “light switch effect” (emission quenched upon binding to DNA), are strongly photo-toxic suggesting their use in Photodynamic Therapy (PDT). In HeLa cells the best PDT agent shows an IC₅₀ value (light) = 4 μM vs. IC₅₀ value (dark) = 62 μM.

Full text of DOI:10.1039/c6dt03792e

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2016, DOI: 10.1039/C6DT03792E.
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DOI: 10.1039/C6DT03792E
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Luminescent ruthenium polypyridyl complexes with extended
‘dppz’ like ligands as DNA targeting binders and cellular agents
Received 00th January 20xx,
Accepted 00th January 20xx
Bjørn C. Poulsen,a† Sandra Estalayo-Adrián,a† Salvador Blasco,a† Sandra A. Bright,a,b John M. Kelly, a
D. Clive Williamsb* and Thorfinnur Gunnlaugssona*
DOI: 10.1039/x0xx00000x
Four new Ru(II) polypyridyl complexes that contain an extended aromatic moiety derived from pyrazino[2,3-
h]dipyrido[3,2-a:2’,3’-c]phenazine and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) have
been synthesized, their solid state X-ray crystal structuree determiend and their photophysical and biological properties
evaluated. Their interactions with DNA have been studied, and they have been tested for their potential as photodynamic
therapeutic (PDT) agents in the treatment of cancer. A practical modification of a method by Carter, Rodriguez and Bard
has been introduced and used to calculate binding parameters for the complexes which show a strong affinity for DNA
with binding constants in the order of 107 M-1 (in 10mM phosphate buffer). The complexes containing phen as an ancillary
ligand become emissive upon binding to DNA ("light switch effect"), but do not show selective cytotoxicity upon light
irradiation. On the other hand, the TAP complexes, which show an inverse "light switch effect" (emission quenched upon
binding to DNA), are strongly photo-toxic suggesting their use in Photodynamic Therapy (PDT). In HeLa cells the best PDT
agent shows IC50 value (light)= 4 µM vs. IC50 value (dark) = 62 µM.
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as porphyrins, azadipyrromethenes,4 BODIPY based dyes and
more recently, Ru(II) polypyridyl complexes,5,6 that upon light
activation/irradiation can generate reactive species (such as
Introduction
Ruthenium (Ru(II)) polpyridyl complexes possess interesting
photophysical properties that can have applications in
supramolecular chemistry, biology and potentially in
medicine.1-3 In recent times, such complexes have been
developed as nucleic acid binders; the binding mode being
shown to be highly dependent on the structure of the
coordinating ligands.2 Recently the development of Ru(II)
complexes that have ligands possessing extended aromatic
regions such as dipyrido[3,2-a:2’,3’-c]phenazine (dppz) and
pyrazino[2,3-h]dipyrido[3,2-a:2’,3’-c]phenazine (pdppz) have
been developed and their ability to intercalate into
oligonucleotides has been studied, which includes the use of X-
ray crystallography in conjunction with ultra-fast spectroscopy,
such as transient IR.3 As the photophysical (i.e.
absorption/emission, lifetime, and quantum yield, etc.)
properties of such complexes are usually significantly
modulated upon DNA binding, their application as novel
photodynamic therapy (PDT) agents has also emerged.2a,2b,2c
PDT is a strategy to selectively kill cancer cells by modulation in
the toxicity of the therapeutic agent upon light irradiation, and
has been commonly employed using chemical structures such
reactive oxidative species) or partake in electron transfer
reactions, that results in the onset of apoptosis or necrosis
with concomitant cell death. We have recently explored the
properties of different Ru(II) complexes as imaging or anti-
cancer agents, and detailed biological profiling of two Ru(II)
polypyridyl complexes (1 and 2, Scheme 1)2a containing pdppz
as auxiliary ligands which, depends on the nature of the
remaining two polypyridyl ligands, function as luminescent
cellular probes (1) or as novel PDT agents (2).2a These results
Scheme 1: Structure of the ruthenium complexes: (1-2) 1,10-phenanthroline (phen)
and 1,4,5,8-tetraazaphenanthrene (TAP) ruthenium complexes with pyrazino[2,3-
h]dipyrido[3,2-a:2',3'-c]phenazine (pdppz). (3-4) phen and TAP ruthenium complexes
with 3-methylpyrazino[2,3-h]dipyrido[3,2-a:2’,3’-c]phenazine (mpdppz). (5-6) phen and
TAP ruthenium complexes with dipyrido[3,2-a:2’,3’-c][1,2,5]thiadiazolo[3,4-
4,3]phenazine (dtp).
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inspired us to make different variations in the structure of
these complexes and determine how such changes affect their
properties. Moreover, such complexes can be conjugated to
surfaces such as gold nanoparticles, as we have recently
demonstrated, and employed in cellular applications, such as
in luminescent imaging and theranostics.7,8 Herein we report a
new family of Ru(II) complexes that we have prepared with
extended aromatic groups derived from pdppz, Scheme 1,
using either phen or TAP ligands. We report their
characterisation, photophysical properties and anticancer
activity towards HeLa cancer cells. We also demonstrate that
the interactions of these with nucleic acids (e.g. DNA) can be
quantified by fitting the spectroscopic data using non-linear
regression analysis; this analysis demonstrating very high
affinity for the novel Ru(II) complexes presented herein.
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DOI: 10.1039/C6DT03792E
Results and Discussion
Synthesis of ligands and complexes
The synthesis of the ligand pdppz as well as the complexes 1
and 2 (see Scheme 1) has been previously reported by our
research group.9,2 The synthesis of 1,10-phenanthrolin-5,6-
dione,10-12
dipyrido[3,2-a:2’,3’-c][1,2,5]thiadiazolo[3,4-
4,3]phenazine (dtp),13 TAP,14 5-nitro-2,1,3-benzothiadiazole
and 4,5-diamino-2,1,3-benzothiadi-azole was by established
proceatures.15 Synthesis of complexes 3‒6 was achieved by
adapting the procedures for 1 and 2, as shown in Scheme 2.
The extended polypyridyl ligands 3-methylpyrazino[2,3-
h]dipyrido[3,2-a:2’,3’-c]phenazine (mpdppz) and dtp were
synthesised by condensation of 1,10-phenanthroline-5,6-dione
with 2-methyl-5,6-diaminoquinoxaline or 4,5-diamine-2,1,3-
benzothiadiazole, respectively by refluxing in ethanol, yielding
mpdppz or dtp as a white and a yellow solid in 92% and 81%
yield, respectively. Microwave reactions of mpdppz and dtp
with the appropriate Ru(II) bispolypyridyl dichloride at 140 °C
followed by column chromatographic purification using neutral
alumina and MeCN:H2O (10:0 to 9:1) as eluent, yielded
complexes 3‒6 as orange-red solids in 74%, 59%, 30% and 44%
yield, respectively. The distinct units of the complexes were
readily discerned by NMR (CD3CN, 400 MHz) (See ESI Fig. S4,
S7, S13 and S16). CHN analysis and mass spectrometry showed
the formation of the desired products in high purity.
Photophysical characterisation of Ru(II) complexes
The photophysical properties of 3‒6 were studied in 10 mM
aqueous sodium phosphate buffer at pH 7.4 for complexes 4
and 6 and in acetonitrile for complexes 3 and 5 (as their
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chloride salts); these latter complexes were found to be non- measurements were undertaken and the results are
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emissive in aqueous solution.16, 17 As has been reported for summarised for each of the complexesDiOnI:T10a.b10le391/C.6TDhTe03f7o92uEr
other related Ru(II) polypyridyl complexes containing extended complexes exhibited similar emission properties compared to
aromatic ligands, similar characteristic bands were observed in dppz Ru(II) complexes described in the literature.18,19
the UV-Vis absorption and the emission spectra of 3‒6 (Figure Complexes 3 and 5, showed luminescence lifetimes of 166ꢀns
1 & ESI).18,19 Complexes 3 and 5 showed two absorption bands and 169ꢀns, respectively in air-saturated acetonitrile solution.
at 223 nm and 262 nm (ε(3) = 12.5×104 M-1 cm-1, ε(5) = These values are very close to the reported lifetime of
9.0×104 M-1 cm-1) which can be assigned to π-π* intra-ligand [Ru(phen)2dppz]2+ complex (177ꢀns).20 However, whilst the
transitions of the phen ligands. An absorption band was also lifetime of complex 5 (590 ns) in de-aerated acetonitrile
observed at 310 nm (ε(3) = 7.5×104 M-1 cm-1, ε(5) = 6.4×104 M-1 solution is close to the value of [Ru(phen)2dppz]2+ (663 ns), the
cm-1) which is characteristic of π-π* transitions within the value obtained for the lifetime of 3 is significantly shorter
extended aromatic ligand mpdppz and dtp. Further two (324 ns) than the others. However, this value is close to that
absorption bands were observed centred at 360 nm and reported previously by our laboratory for 1 (411ꢀns).2
A
380 nm, these being attributed to π-π* transition within the possible explanation of this observation could be the existence
phenazine part of mpdppz and dtp. Finally, a broad absorption of more than one luminescent state as has previously been
band centred at 440 nm (ε(3) = 2.41×104 M-1 cm-1, ε(5) = described for the similar complex [Ru(phen)2PHEHAT]2+,
1.83×104 M-1 cm-1) corresponding to the metal to ligand charge (PHEHAT = 1,10-phenanthrolino[5,6-b]1,4,5,8,9,12–hexaazatri-
transfer (MLCT) transition of the Ru(II) complex with an phenylene).21 For 4 and 6, which have TAP ligands, the excited
electron transfer from the metal centre to the extended ligand electron is located at TAP instead of at the extended aromatic
was also observed. Complexes 4 and 6 showed an absorption ligand and the luminescent properties should be less
band at 275 nm (ε(4)
=
8.8×104 M-1 cm-1, ε(6)
=
influenced by the extended ligand structure. Complexes 4 and
6.8×104 M-1 cm-1) characteristic of π-π* intra-ligand transitions 6 showed similar lifetimes values in air-saturated aqueous
of the ancillary TAP ligands; an absorption band at 310 nm solution (679ꢀns and 697ꢀns, respectively) and in deaerated
(ε(4) = 8.7×104 M-1 cm-1, ε(6) = 7.9×104 M-1 cm-1) corresponding aqueous solution (801ꢀns and 864ꢀns, respectively). These
to π-π* transition within the ligand mpdppz and dtp; and an values are slightly smaller than the corresponding reported
absorption band at 412 nm (ε(4) = 2.4×104 M-1 cm-1, ε(6) = values of [Ru(TAP)2dppz]2+ (820ꢀns and 1090ꢀns in air-saturated
2.0×104 M-1 cm-1) attributed to the typical MLCT transition of and de-aerated aqueous solution, respectively).22 The
this kind of complex where an electron transfer occurs from quantum yields of the four compounds reported here, 3 and 5
the metal centre to the TAP ligands. Concerning the emission showed values higher than seen for [Ru(phen)2dppz]2+ (φF =
properties, luminescence bands were observed with band 0.0073 in air-saturated acetonitrile solution), while quantum
maxima at 619 nm for complexes 3 and 5 (λexc = 440 nm), and yields of complexes 4 and 6 are quite close to the
at ca. 630 nm for complexes 4 and 6 (λexc = 415 nm).
Luminescence quantum yields (φem
corresponding value of the [Ru(TAP)2dppz]2+ complex (φF =
)
and lifetime 0.035 in air-saturated aqueous solution).20, 22 More detailed
studies are currently being undertaken in our laboratory these
complexes.
Crystallographic Studies
Having synthesised and photophysically characterised the
above complexes, we next initiated an investigation into their
solid-state structures.‡ Both the TAP complexes (4 and 6) were
successfully crystallised to give products that were suitable for
single crystal X-ray diffraction studies. Unfortunately, the phen
analogues (3 and 5) gave crystals that were not suitable for
such investigations. The crystal structures can be seen in
Figures 2 and 3.
Molecules of 6.2Cl crystallize in a monoclinic C2/c system in
which the unit cell contains 8 complexes of 6, with 16 chlorine
counterions along with water molecules. The asymmetric unit
contains one molecule of complex 6 in which the metal atom is
octahedrally coordinated with two TAP ligands and one dtp
moiety, and this coordination geometry is fairly regular for the
bond lengths (see Table 2) with Ru-N bond averaging 2.06 Å.
The N-Ru-N angles are not far from 90° and these deviations
are imposed by the geometry of the bidentate ligands. None of
[a] Air-saturated aqueous solution of [Ru(bpy)3]2+ as reference (φF = 0.028).1
Estimated errors ± 5%.
the external nitrogen atoms of the complex seem to be
engaged in hydrogen bonding from the solvent molecules
(water) except for N9 which receives one from O6 (cf. CIF file).
[b] The luminescence decays are monoexponential. Estimated errors ±10%.
[c] Cloonan et al.2
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DOI: 10.1039/C6DT03792E
Figure 2: X-ray crystal structure for complex 6. (First row) Crystal structure for 6 with
highlighting of the complex. (Second row) Dimeric structure of neighbouring Λ-6
molecules. Counter ions and solvent molecules are omitted for clarity. (Third row)
Arrangement of Λ (blue) and Δ (red) domains.
Figure 3: X-ray crystal structure for 4. (First row) Crystal structure for complex 4 with
highlighting of the complex and the bending of the mpdppz moiety. Only one of the
two units is shown. (Second row, left) Details of CH-π and CH-N bonds between
neighbouring molecules. (Second row, right) Details of the only interaction, CH3-π bond
between neighbouring molecules. (Third row, left) Stacking mode between
neighbouring molecules which form an angle of 87.6° (measured as the torsion angle of
Ru atoms and centroids of the central pyrazine within either mpdppz). (Third row,
right) Complexes are shown in red and blue. (Fourth row) Packing and chirality
domains. Hydrogen and chlorine atoms omitted for clarity.
The rest of the water molecules along with the counterions
form a hydrogen bond network. The dtp moiety is quite flat
and it shows face to face π stacking interactions with another
dtp moiety of a neighbouring complex with the same absolute
configuration (see Figure 2,middle) which results in complexes
that are paired together. The mean planes of each dtp units
are almost parallel and form a narrow angle of 4.01°; the
stacking distances range between 3.12‒3.67 Å and the shift
distance is 1.42 Å. At a higher level these pairs of ΛΛ or ΔΔ
complexes pack together forming layers that pile along the c
crystallographic axis.
Molecules of 4 crystallized in a triclinic P−1 system with two
chlorine counterions. This crystal was rather small, the
reflections very weak and the data collection was difficult.
Nonetheless the structure could be solved and refined. The
solvent molecules were very disordered and could not be
modelled properly. SQUEEZE tool23 was used to remove
electron density from the disordered areas and refinement
was continued with the recalculated data. The crystal structure
contains four molecules of 4 along with the corresponding
chlorine counterions. The asymmetric unit contains two
crystallographically different molecules of 4: one Λ-4 and one
Δ-4 along with four chlorine atoms that balance the charge
and as mentioned some very disordered solvent molecules. In
both cases the ruthenium core shows a fairly regular
octahedral geometry with N-Ru bond distance ranging from
1.99 to 2.12 Å for Ru1 and 2.03 to 2.12 Å for Ru2. The mpdppz
moiety is bent with respect to the plane defined by the Ru‒N
plane. We have tried to quantify this bending as the angle
between two planes: (1) the plane defined by the ruthenium
and the “phenanthroline” part of the mpdppz moiety and
(2) the mean planes defined by the “methyldiazine” part of the
mpdppz. This distortion is 14.13° for the Λ and 14.62° for the Δ
enantiomer. The mpdppz moieties pile up along the a axis
forming strands of alternate couples of isomers (see Figure 3,
fourth row) being the relative conformation different
depending on whether they are the same enantiomer or two
different ones. This can be explained in terms of the different
interaction modes between the ΔΛ and ΔΔ/ΛΛ isomers. In
Figure 3 (second and third row) it can be seen that one ArC-
H...π bond and one ArC-H...N bond hold the pair of complexes
in that particular conformation forming an angle of 87.6°
(measured as the torsion angle of the Ru atoms and centroids
of the central pyrazine ring within either mpdppz). This
conformation also explains the bending of the mpdppz moiety
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as these two bonds pull one of the TAP moieties in the
bending direction. For the ΔΛ stacking, we see that they form
an angle of 180° with each other due to an inversion centre
between them, and apart from the stacking, there is a CH3...π
bond. These bonds are weak, but their importance is not to be
neglected as they might be significant in explaining the overall
packing.24 These two features, the bending of the extended
aromatic moiety and the pattern of stacking, are missing in the
analogous crystal for 2 (CCDC number 1012983)2 where the
bending angle is 4.98°, the ΔΔ/ΛΛ stacking is missing, and only
ΔΛ pairs are present.
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DOI: 10.1039/C6DT03792E
Interactions with DNA
To explore the potential biological activity at the genome level
the binding of the complexes to DNA was next investigated.
Spectroscopic DNA titrations were carried out in 10 mM
phosphate buffered solution with pH at 7.4 and either with or
without 160ꢀmM NaCl by adding a solution of salmon testes
DNA (st-DNA) to the solution of the complexes in a 1 cm
emission quartz cuvette and by measuring the absorption and
emission after each addition.25 Titrations for all complexes
were repeated at least three times for each condition to
ensure reproducibility.
The increase in the concentration of st-DNA resulted in a
significant change in both the absorption and the emission
spectrum. The general behaviour for complexes 3‒6 was
similar to that previously reported for 1 and 22 as well as for
other intercalating compounds.16,22,25-28 The general trend was
an overall decrease in the absorption with increasing
concentrations of DNA. The decrease in the absorbance of the
MLCT band at 440ꢀnm for complexes 3 and 5, which contained
phen as ancillary ligand, was 17% and 18%, respectively, and
the decrease in the π-π* band for the extended aromatic
moiety at 310 nm was 44% and 52% for 3 and 5, respectively
(low ionic strength). Similarly for the TAP complexes the
decrease in the MLCT band at 412ꢀnm was 22% and 19%, and
the decrease in the π-π* band for the extended aromatic
moiety at 310 nm was 34% and 41% for 4 and 6, respectively
(low ionic strength). This hypochromic behaviour indicates
intercalation where particularly, the large decrease in the
absorbance at 310 nm can be explained by a strong interaction
between the chromophore of the extended aromatic moiety
and the chromophores of the base pairs in the form of π-π
stacking.29
We observe a fundamental difference in the change in the
emission spectra for complexes that have phen (3 and 5) and
TAP (4 and 6) as ligands.2 Complexes 3 and 5 exhibit the well-
known “light switch effect”,16, 30 as their emissions are fully
quenched in aqueous solution, but they become highly
emissive upon binding to DNA. This effect is explained by the
change in the environment due to the intercalation of the
extended aromatic moiety, which prevents solvent molecules
from interacting with N-atoms of the phenazine moieties, an
interaction that results in quenching of the emission.31 The
emission showed biphasic behaviour in solutions containing
only 10 mM phosphate buffer. In the first phase a strong
Figure 4: Changes in the UV-Vis and emission spectra of (top) 3 and (bottom) 4 with the
addition of increasing amounts of st-DNA (0-100ꢀµM base pairs) at pH 7.4 (10 mM
sodium phosphate). Similar spectra for the titration of 5 and 6 can be seen in
Supporting Information. Inset: Plot of the concentration corrected emission (Ea) at
increasing concentration of DNA base pairs and the corresponding non-linear fit of
equation (S3.5) (see ESI). Similar plots and fits have been made for the absorption data.
emission enhancement was observed, while in the second
phase the emission intensity began to decrease slowly. A
decrease was not observed at high ionic strength (160 mM
NaCl). In contrast to 3 and 5, TAP complexes 4 and 6 exhibit
80% and 81% quenching of the emission respectively upon
binding to DNA (at low ionic strength). This is common for
Ru(TAP)2 complexes as the excited state, instead of undergoing
radiative decay, can photo-oxidize guanine residues in DNA
resulting in an overall quenching of the emission.22,32
From the spectrophotometric data presented herein, we
have quantified the affinity of the complexes for DNA by
calculating values for the binding constant (Kb) and for the
binding site size (n). For this purpose we used a simple
modification of a method by Carter, Rodriguez and Bard.33 Our
method assumes the same binding model, however knowledge
of the molar absorbance or emission for bound complex is not
needed. A detailed description of the method and the
problems addressed by the method are given in Supporting
Information. The parameters were calculated using the
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changes in the absorbance of the MLCT band (440 nm or than the situation at low ionic strength. The phenVcieowmArpticlleexOenslin3e
DOI: 10.1039/C6DT03792E
and 5, and TAP complexes 4 and 6 show pairwise very similar
412 nm) as well as the changes of the integrated emission.
Both the absorption corresponding to the MLCT band and binding constants where the phen complexes bind strongest.
the integrated emission indicated a very strong interaction The emission data gave Kb for 3 and 5 at 2.6x106 and
between the complexes and DNA at low ionic strength (10 mM 1.4x106 M-1, respectively and Kb was 0.33x106 M-1 for both 4
sodium phosphate) with binding constants in the order of and 6. A similar trend between phen and TAP complexes have
107 M-1 (see Table 2 and Table S1 in the ESI). The data showed previously been shown for 1 and 2 in 100 mM NaCl (see
that values of n for all complexes except 3 were 1.7 ± 0.2 base Table 2).2
pairs (bp). It should be noted that fitting to this binding
Circular dichroism studies
isotherm is empirical and interpretation is necessarily
approximate. It implicitly assumes that the binding of both
enantiomers is similar, that the affinity for the ten discrete two
base-pair binding sites34a is the same, and that binding occurs
only from one of the grooves. In the crystal structures so far
reported for [Ru(phen)2dppz]2+ and [Ru(TAP)2dppz]2+ with DNA
the complex intercalates via the minor groove,35 although
some solution experiments indicate that entry from the major
groove is possible.36 Crystal structures have also shown that at
the high concentrations present in the crystal semi-
intercalation of the ancillary ligand of the lambda-enantiomer
is possible. However in solution this binding mode is likely to
be much weaker than intercalation. The work of Lincoln and
co-workers has shown that the DNA binding of the
enantiomers [Ru(phen)2dppz]2+ and [Ru(bpy)2dppz]2+ is
complex. The complexes show some preference for binding to
particular sequences and the enantiomers show contrasting
behaviour.34b For [Ru(phen)2dppz]2+ both cooperative and anti-
cooperative interactions have been demonstrated.34c Similar
effects may be expected here and could be the reason for the
relatively low site size recorded for the complexes here.
Circular dichroism (CD) is a powerful spectroscopic technique
in the study of binding of ligands (here ruthenium complexes)
to DNA with respect to study the binding modes as well as the
conformational changes of DNA.38 DNA is an optically active
molecule as a result of the chiral sugar-phosphate backbone
and its helicity. B-DNA form exhibits a typical CD spectrum
characterized by a positive band around 280 nm and a
negative band around 245 nm.52 In addition chiral and achiral
molecules bound to DNA can have induced CD (ICD) signals.
Strong ICD indicates groove binding since this will place the
molecule close to the chiral sugar backbone, where as
intercalation will place the molecules between the achiral base
pairs.39 ICD is known to be an order of magnitude stronger for
groove binders than for intercalators.
Binding constants for the four complexes are still high, in
160 mM NaCl, but decrease by an order of magnitude to about
106 M-1 (see Table 2) while n-values hardly change. These
results indicate a similar binding mode under both conditions,
however with a significantly weaker binding at increased ionic
strength. A good agreement between the size of the values of
Kb and n are seen when the values calculated from the
absorption and emission data, respectively, are compared. This
indicates a more specific and well-defined mode of binding
Table 2 Binding parameters obtained from emission data for the binding between
the complexes and st-DNA in 10 mM sodium phosphate buffered solution at pH 7.4
and either with or without 160 mM NaCl (high and low ionic strength respectively).
The parameters and SEM are for the mean of three replicates. Parameters obtained
from the absorption data can be seen in Supporting Information.
Low ionic strength
Kb (106 M-1)
High ionic strength
Complex
n (#bp)
Kb (106 M-1)
n (#bp)
3
4
27 ±4
42 ± 9
2.5 ± 0.1
1.8 ± 0.1
1.8 ± 0.1
1.9 ± 0.1
1.5 ± 0.1
1.6 ± 0.1
2.6 ± 0.2
0.33 ± 0.02
1.35 ± 0.1
0.33 ± 0.07
4.6 ± 0.5
2.7 ± 0.1
1.6 ± 0.1
1.6 ± 0.1
1.8 ± 0.2
2.0 ± 0.1
1.9 ± 0.1
5
11 ± 0.8
28 ± 0.4
12 ± 3
6
1[a]
2[a]
Figure 5: Circular dichroism spectra of st-DNA (150 μM nuclear phosphate) in 10 mM
sodium phosphate buffered aqueous solution at pH 7.4, in the absence and presence
of (a) 3 and (b) 6 at different nuclear phosphates to dye (P/D) ratios.
5.4 ± 0.4
0.84 ± 0.1
[a]Cloonan et al.2
6 | J. Name., 2012, 00, 1-3
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In order to evaluate the ability of complexes 3-6 to bind to 3‒6 displayed an increase of Tm for st-DNA from 69.8 °C to
View Article Online
DOI: 10.1039/C6DT03792E
DNA, CD titrations were carried out by keeping the 76.4 °C, 76.1 °C, 76.5 °C and 76.3 °C, respectively, indicating
concentration of st-DNA constant (150 µM nucleotide) and stabilisation of the DNA helix by DNA-Ru complex interaction.
measuring the CD spectrum for different concentrations of These values are smaller than those observed for the complex
complex (Figure 5). Racemic mixtures of the complexes were [Ru(phen)2dppz]2+ (increase of 9.1 °C) with calf thymus DNA at
used in the titrations and therefore they show no optical similar P/D ratio but similar to ΔTm values determined for
activity. However, the CD spectra of st-DNA show significant other Ru(II) complexes.44, 45
changes in the presence of complexes 3–6. For wavelengths
higher than 300 nm some CD signals were observed that could
Viscometry Studies
be assigned to ICD of the complexes since this was outside of
In order to determine whether the complexes interact with
the range where DNA absorb or that one of the enantiomers
DNA through intercalation we measured the viscosity of st-
(Δ or Λ) is demonstrating preferential binding to DNA.
DNA with increasing ratio of complexes 3—6. The plot of
At short wavelength, the evolution of a strong negative band
⅓
(η/η0) against D/P (concentration of ruthenium divided by
concentration of nuclear phosphate) gave a straight line with
positive slope for all complexes (see Figure 6) which can be
considered a positive proof for intercalation. The slope of this
plot quantifies the contribution of the intercalator to the
elongation of the DNA. For complexes 3 and 4 which contain
mpdppz as extended aromatic ligand we have found the
was observed with a maximum at about 275 nm for complex 3
and 5, and about 295 nm for complex 4 and 6. These bands
could be attributed to π-π* intra-ligand transitions of the
ancillary phen and TAP ligands, respectively and the strong
signal indicated that the phen and TAP complexes were
positioned in the grooves that also is the expected placement
of the auxiliary ligands when mpdppz or dtp are intercalating.
slopes are almost identical: 1.60 ± 0.05 and 1.58 ± 0.04
At high P/D ratio, a very small negative CD signal was observed
respectively. Similarly, for complexes 5 and 6 the values found
for the four complexes at about 415 nm corresponding to the
typical MLCT absorption band of the Ru(II) polypyridyl
complexes. The exact binding modes cannot be determined
from these observations, however the existence of an
interaction between complexes 3-6 and the DNA molecule can
be concluded. These results are similar to other Ru(II)
complexes reported in the literature.38-43
Thermal denaturation studies
Evidence that a compound is bound to DNA can be deduced by
an increase in the melting temperature (Tm) of the double-
stranded structure, the temperature at which 50% of the
duplex has dissociated to a single stranded form.41-43 Thereby,
thermal denaturation studies are useful to evaluate the ability
of a compound to bind to DNA. Thermal denaturation curves
of st-DNA (150 μM nuclear phosphates) in the presence of
complexes 3‒6 in 10 mM phosphate buffer at P/D ratios of 50,
20 and 10 are shown (See ESI). The Tm was determined as the
were 1.27 ± 0.07 and 1.12 ± 0.04 respectively which are also
temperature corresponding to the steepest increase in the
close to each other. It can be seen that the complexes with the
same extended aromatic ligand show similar behaviour, which
suggests that the intercalation takes place preferentially
through that moiety.
absorption by finding the maximum in the first derivative. In
the absence of Ru(II) complexes, st-DNA exhibited a Tm of 69.8
°C. At P/D of 50 and 20, there was a small increase in the Tm
for the four complexes (Table 3). At P/D ratio of 10, complexes
Cellular uptake and viability studies
The uptake of the four complexes 3‒6 into HeLa (cervical
cancer) cells in the dark was investigated using confocal
ΔTm (°C)
P/D=20
microscopy (see Figure 7). The results demonstrated that the
cells successfully internalised all complexes at 100 µM within
24ꢀhours. The complexes appeared to localise within the
cytoplasm of cells with red fluorescent emission observed
outside the nucleus. These results are in agreement with
previous results observed for complexes 1 and 2.2 Some
toxicity against the cells was observed during confocal imaging
(The generate of singlet oxygen by 3-6 to was not quantified).
Complex
P/D=50
P/D=10
3
4
5
6
0.7 ± 0.1
0.3 ± 0.4
1.2 ± 0.8
0.8 ± 0.0
1.2 ± 0.8
2.1 ± 0.1
1.5 ± 0.9
2.3 ± 0.5
6.6 ± 1.3
6.3 ± 0.4
6.7 ± 0.4
6.5 ± 0.5
This journal is © The Royal Society of Chemistry 20xx
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Journal Name
View Article Online
DOI: 10.1039/C6DT03792E
Figure 7: Complexes were successfully taken up by HeLa cells and localised in the cytoplasm.
Next the complexes were assessed for cytotoxicity against those observed previously for phen complex 1. These results
HeLa cells under both dark and visible light (from an HgXe arc suggest that the introduced modifications of pdppz have
lamp) (photoactivated) conditions (see Table 4). 25×103 increased the overall cytotoxicity relative to the phen
cells/well were seeded into 96-well plates and treated with reference complex. This increase in toxicity has been
compound (ruthenium complexes – see Table 4) for 24 hours confirmed by comparison of the viability of the cells at
at 37 °C. Following this, the cells were either irradiated with 100 µM. The cell viability in the case of 1 was 35% while for 3
18 J cm−2 of light for one hour or maintained in the dark. After and 5 it was 6% and 5%, respectively. However, light activation
further 24 hours incubation, each well was treated with 20 μL resulted in a more than 10 fold increase in the toxicity of the
of Alamar Blue and left to incubate for 4−5 hours. Emission TAP complexes 4 and 6, similar to what is seen for TAP
was read at 590 nm (excitation at 544 nm). The complex 2. The IC50 values for 4 and 6 in the dark were 60 µM
chemotherapeutic drug paclitaxel (Taxol®) and the previously and 63 µM, respectively, decreasing to 5 µM and 4 µM after
studied phen complex 1 have been used as references. The photoactivation. These IC50 values following light activation are
studies demonstrated that the phen complexes 3 and 5 did not marginally lower than the value reported for 2 (9ꢀµM), which
show light-dependent cytotoxicity yielding similar IC50 values possessed a similar toxicity in the dark (IC50 value at 70 µM).
under both dark and light conditions.
These IC50 values were, however, significantly lower than
Conclusions
Table 4. Effects of Ru(II) Complexes 1-6 on HeLa cervical cancer cells with or without
light activation. Results are presented as the mean ± SEM.
We have reported the synthesis of four new DNA binding and
cytotoxic ruthenium polypyridyl complexes with two new
IC50, μM
ligands derived from dppz (mpdppz and dtp) and phen or TAP
as ancillary ligands. Crystal structures were obtained for two of
the complexes that incorporated the TAP ancillary ligand
(complexes 4 and 6). Their photophysical properties have been
studied, and they have shown MLCT based luminescence
properties, which were modulated in the presence of st-DNA.
Complexes with phen as ancillary ligand (complexes 3 and 5)
have shown no emission in aqueous medium. However, in the
presence of DNA the luminescence was switched on, a
Complex
Dark
37 ± 6
60 ± 15
34 ± 3
Light
48 ± 8
4.8 ± 0.5
33 ± 6
4.0 ± 1.5
>100
3
4
5
6
63 ± 8
1[a]
2[a]
Taxol®
>100
70 ± 6
0.0028± 0.0005
8.8 ± 2.9
0.0026 ± 0.0006
[a] Cloonan et al.2
8 | J. Name., 2012, 00, 1-3
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Page 9 of 14
PleaDsaeldtoonnTortaandsjuascttimonasrgins
Journal Name
ARTICLE
property known as the "light switch" effect and assigned to Electrothermal 9100 melting point apparatus. Elemental
View Article Online
intercalation.
analyses were conducted at the MicroDanOaI:l1y0t.i1c0a3l9/LCa6bDoTr0a3t7o9r2yE,
The complexes with TAP as ancillary ligand (4 and 6) have School of Chemistry and Chemical Biology, University College
shown the opposite behaviour. In aqueous medium these Dublin (UCD).
complexes showed a relatively strong emission that was
UV-Vis-NIR and luminescence measurements were done in a
however almost totally quenched in the presence of DNA. This 1.0ꢀcm optical path quartz cell at room temperature. The
was assigned to photo-induced oxidation of guanine when the absorption spectra were recorded using a Varian Cary 50 UV-
compounds were bound to DNA as has been studied in detail Visible spectrophotometer. The emission and excitation
with [Ru(TAP)2dppz]2+. A modification of a method by Carter, spectra were recorded on a Varian Cary Eclipse fluorescence
Rodriguez and Bard has been used to calculate binding spectrophotometer. Fluorescence lifetime experiments were
parameters from the spectroscopic data showing binding carried out on a Horiba Scientific FluoroLog - Modular
constants in the order of 107 and 106 M-1 at low and high ionic Spectrofluorometer equipped with Time Correlated Single
strength, respectively. The binding was significantly stronger Photon Counting (TCSPC) capability. Circular Dichroism (CD)
for the phen complexes than for the TAP complexes. Further, spectra were recorded at a concentration corresponding to an
CD spectroscopy and DNA denaturation studies have been optical density of approximately 1.0 for the DNA absorbance at
used to investigate the interaction between the complexes and 260 nm (150 ꢀM) on
DNA. In accordance with previous studies the phen and TAP spectropolarimeter.
a
Jasco J-810-150S CD
complexes show differences in their toxicity upon light
X-ray diffraction data were collected on a Bruker APEX 2 DUO
activation. The phen complexes did not show increased CCD diffractometer using graphite-mono-chromatized Mo-Ka
toxicity upon exposure to light while the TAP complexes (0.71073 Å). Crystals were mounted in a cryoloop/MiTeGen
showed a good therapeutic window with a more than 10-fold micromount and collected at 100(2) K using an Oxford
increase in toxicity upon exposure to light. It was Cryosystems Cobra low temperature device. Data were
demonstrated with confocal microscopy that HeLa cells took collected using omega and phi scans and were corrected for
up the compounds with emission in the cytoplasm outside the Lorentz and polarization effects.46 Structures were solved with
nucleus indicating that cell death is unlikely to be due to SHELXS-2014 and further refined with SHELXL-2014.47 In the
interaction with nuclear DNA. In conclusion phen complexes 3 case of the crystal 4!2Cl!xH2O, highly disordered solvent
and 5 have shown to be candidates for use in fluorescence molecules were removed using SQUEEZE48 software and then
imaging while TAP complexes 4 and 6 have shown to be refined as indicated.
candidates for use in photodynamic therapy.
Viscosity was measured with a Cannon-Manning semi-micro
viscometer at precisely 25.0 °C. Constant DNA concentration of
1.2 mM and total sample volume of 1 mL were used. DNA was
sheared with ultrasound to obtain fragments of ca. 200 bp.
Ruthenium/nuclear phosphate concentration ratios (D/P)
ranging from 0.02 to 0.25 were measured. Viscosity (η) was
calculated as sample flow time minus buffer flow time. Finally
Experimental
Materials and Methods
All chemicals were purchased from Sigma-Aldrich, Acros
Organics or TCI and used as received without further
purification. The DNA used for all the experiments was double-
stranded salmon testes DNA sodium salt purchased from
Sigma-Aldrich which was dissolved in a buffered aqueous
solution and then filtered to remove insoluble impurities. DNA
concentration in base pairs was determined by UV using
ε260nm = 13200 M−1cm−1.50
⅓
(η/η0) values (where η0 is the viscosity of DNA alone) were
plotted against D/P.49
General Biological Procedures
Cell culture reagents were obtained from Greiner Bio-one and
all other chemicals were obtained from Sigma unless
otherwise stated.
NMR spectra were recorded either using a Bruker DPX-400
Avance spectrometer, which operates at 400.13 MHz for H
1
Cell culture. HeLa cells were grown in a cell culture flask using
low-glucose Dulbecco’s Modified Eagle Medium supplemented
with 10% fetal bovine serum and 50 µg/mL penicillin/
streptomycin at 37 °C in a humidified atmosphere of 5% CO2.
NMR and 100 MHz for 13C NMR, or a Bruker AV-600
spectrometer, operating at 600.1 MHz for 1H NMR and
150.2 MHz for 13C NMR. Chemical shifts (δ) were referred
relative to the internal solvent signals and reported in ppm.
Electrospray ionisation (ESI) mass spectra were recorded on a
Mass Lynx NT V 3.4 with a Waters 600 controller connected to
Viability assay. 25×103 cells/well were seeded in a 96-well
plate and treated as indicated. Each well was then treated with
20 µL of Alamar Blue (BioSource) and left to incubate at 37 °C
in the dark for 4‒5 hour. Fluorescence was read at 590 nm
(excitation 544 nm). The background fluorescence of the
media without cells + Alamar Blue was taken away from each
group, and the control untreated cells represented 100% cell
viability. Data points represent the mean ± SEM of triplicate
treatments performed on three independent days with activity
a
996 photodiode array detector using HPLC-grade
acetonitrile, methanol, dichloromethane or chloroform as
carrier solvents. All high resolution (HR) masses were reported
within ±10 ppm of the expected mass. Infrared spectra were
recorded on a Perkin Elmer Spectrum One FT-IR spectrometer
fitted with a Universal ATR Sampling Accessory. Melting points
were obtained in an unsealed capillary tube using an
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J. Name., 2013, 00, 1-3 | 9
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Page 10 of 14
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Journal Name
expressed as percentage cell viability compared to vehicle 8.26-8.22 (1H, m), 8.16 (2H, d, J = 5.2 Hz), 8.05 (2H, d, J =
View Article Online
13
treated controls. IC50 values for the toxicity of the compounds 5.2 Hz), 7.85-7.80 (2H, m), 7.68-7.63 (4H, m), 3.59 (3H, s).
DOI: 10.1039/C6DT03792CE
were calculated for each of the three independent NMR (CD3CN, 100 MHz, δ): 158.38, 155.48, 155.36, 154.32,
experiments by interpolation to concentration corresponding 154.23, 153.98, 151.64, 151.58, 148.89, 148.87, 148.81,
to 50% viability and this were used to calculate the mean 145,57, 144.95, 142.42, 141.13, 140.32, 139.43, 137.98,
±SEM. For photoactivation studies, cells were subjected to 137.96, 137.92, 135.18, 134.41, 132.20, 132.09, 132.07,
18 J cm–2 using a Hamamatsu L2570 200 W visible light form a 132.05, 131.63, 131.23, 129.10, 129.06, 128.20, 128.14,
HgXe arc lamp equipped with a NaNO2 filter.
126.93, 126.90, 126.87, 22.77 (CH3). IR (ATR, cm-1): 3059
(aromatic C-H stretch), 1426 and 1361 (C-N stretches). MALDI-
Confocal microscopy. HeLa cells were seeded at a density of HRMS (m/z) calculated for C45H28N10Ru: 810.1542. Found:
5×104 cells/mL and treated as indicated. Cells were then 810.1567 [M]+.
washed two times with fresh media, nuclei were stained blue
with Hoechst and cells were imaged by live confocal Bis(1,4,5,8-tetraazaphenanthrene)(3-methyl-pyrazino[2,3-
microscopy using an Olympus FV1000 point scanning h]dipyrido[3,2-a:2’,3’-c]phenazine)ruthenium(II) chloride (4).
microscope with a 60× oil immersion lens with an NA [Ru(TAP)2Cl2] (93 mg, 173 μmol, 1 eq) and mpdppz (59 mg,
(numerical aperture) of 1.42. The software used to collect 0.169 mmol, 1 eq) were suspended in a mixture EtOH/H2O
images was FluoView Version 7.1 software.
(1:1, 8 mL). The mixture was degassed by bubbling with Argon
for 15 minutes and then heated at 140 °C for 40 minutes using
microwave irradiation. Solvent was evaporated and the
resulting red solid was purified by alumina chromatography
using MeCN:H2O (10:0 to 9:1) as eluent. Solvent was removed
at reduced pressure yielding a red solid which was dried in
vacuo (88 mg, 59%). Calculated for C41H24Cl2N14Ru+ 2.2H2O+
2.5NaCl: C, 46.00; H, 2.67; N, 18.32%. Found: C, 46.23; H, 2.67;
N, 18.16%. m.p.: > 250 °C (decomp). 1H NMR (CD3CN, 600 MHz,
δ): 9.99 (1H, dd, J = 8.3 Hz, 1.1 Hz), 9.82 (1H, dd, J = 8.3 Hz,
1.3 Hz), 9.16 (1H, s), 9.00-8.97 (4H, m), 8.63 (4H, s), 8.60 (1H,
d, J = 9.5 Hz), 8.50 (1H, d, J = 9.5 Hz), 8.32-8.30 (2H, m), 8.26-
8.23 (4H, m), 7.94-7.89 (2H, m), 2.90 (3H, s). 13C NMR (CD3CN,
100 MHz, δ): 157.62, 156.74, 156.66, 153.67, 150.73, 150.68,
150.58, 150.57, 150.45, 150.44, 150.07, 146.93, 146.51,
146.50, 146.45, 143.31, 143.30, 143.22, 140.88, 139.83,
139.75, 135.98, 133.81, 133.73, 132.64, 131.44, 131.32,
128.63, 128.59, 127.74. IR (ATR, cm-1): 3058 (aromatic C-H
stretch), 1486, 1385 and 1273 (C-N stretches). ESI-HRMS (m/z)
calculated for C45H28N10Ru: 407.0676. Found: 407.0679 [M]2+.
Synthesis of ligands and complexes
3-Methylpyrazino[2,3-h]dipyrido[3,2-a:2’,3’-c]phenazine
(mpdppz).
2-Methyl-5,6-diaminoquinoxaline
(74
mg,
0.43 mmol, 1 eq) and 1,10-phenanthroline-5,6-dione (94 mg,
0.45 mmol, 1 eq) were suspended in a mixture of ethanol and
H2O (1:1, 25 mL). The mixture was heated in a high-pressure
tube at 140 °C overnight followed by cooling to room
temperature. The suspension was filtered, and the grey filter
cake was washed with CH2Cl2 (25 mL) leaving the product as a
white solid that was dried in vacuo (137 mg, 92%). Calculated
for C21H12N6+2.25H2O: C, 64.84; H, 3.12; N, 21.60%. Found: C,
64.84; H, 3.41; N, 21.99%. m.p.: > 250 °C. 1H NMR (CDCl3/TFA,
400 MHz, δ): 10.69 (1H, d, J = 7.3 Hz), 10.16 (1H, d, J = 8.0 Hz),
9.77 (1H, s), 9.40 (2H, bs), 8.95 (1H, d, J = 9.3 Hz), 8.79 (1H, d, J
= 9.3 Hz), 8.38 (2H, bs), 3.27 (3H, s, CH3). 13C NMR (CDCl3/TFA,
100 MHz, δ): 158.26, 150.48, 148.91, 145.24, 144.67, 142.06,
141.83, 140.36, 140.27, 140.00, 139.86, 138.52, 136.63,
136.39, 129.59, 129.22, 129.15, 128.14, 127.87, 21.04 (CH3). IR
(ATR, cm-1): 1392 and 1367 (C-N stretches). ESI-HRMS (m/z)
calculated for C21H13N6: 349.1196. Found: 349.1202 [M+H]+.
Dipyrido[3,2-a:2’,3’-c][1,2,5]thiadiazolo[3,4-h]phenazine
(dtp). 4,5-Diamine-2,1,3-benzothiadiazole (121 mg, 0.73 mmol,
1 eq) and 1,10-phenanthroline-5,6-dione (156 mg, 0.74 mmol,
1 eq) were suspended in a mixture of ethanol and H2O (1:1,
20 mL). The mixture was heated in a high-pressure tube at
140 °C overnight followed by cooling to room temperature.
The resulting yellow precipitate was filtered, washed with H2O,
ethanol and diethyl ether, and dried in vacuo (200 mg, 81%).
Bis(1, 10-phenanthroline) (3-methylpyrazino[2, 3-h] dipyrido
[3, 2-a:2’, 3’-c]phenazine) Ruthenium(II) chloride (3).
[Ru(phen)2Cl2] (160 mg, 0.301 mmol, 1 eq) and mpdppz
(105 mg, 0.301 mmol, 1 eq) were suspended in 8 mL of a 50%
mixture EtOH/H2O. The mixture was degassed by bubbling
with Argon for 15 minutes and then heated at 140 °C for
40ꢀmin using microwave irradiation. Solvent was evaporated
and the resulting red solid was purified by column
chromatography with neutral alumina using MeCN:H2O (10:0
to 9:1) as eluent. Solvent was removed at reduced pressure
yielding a red solid which was dried in vacuo (198 mg, 74%).
Calculated for C45H28N10RuCl2+1.3NaCl: C, 53.86; H, 3.33; N,
13.96%. Found: C, 53.68; H, 3.33; N, 13,83; Cl, 10.47%. m.p.: >
1
m.p.: > 250 °C. H NMR (CDCl3, 400 MHz, δ): 9.87 (1H, dd, J =
8.1 Hz, 1.8 Hz), 9.70 (1H, dd, J = 8.1 Hz, 1.8 Hz), 9.35 (2H, m),
8.33 (2H, syst AB), 7.88 (1H, dd, J = 4.4 Hz, 8.1 Hz), 7.86 (1H,
dd, J = 4.4 Hz, 8.1 Hz). 13C NMR (CDCl3, 100 MHz, δ): 156.21,
152.96, 152.89, 152.81, 144.98, 141.26, 140.16, 137.94,
135.08, 134.63, 132.19, 127.37, 127.32, 125.30, 124.74. IR
(ATR, cm-1): 2984 (aromatic C-H stretch), 1494 (aromatic C=C
stretch), 1368 (C=N stretch). ESI-HRMS (m/z) calculated for
C18H9N6S: 341.0609. Found: 341.0610 [M+H]+.
1
250 °C. H NMR (CD3CN, 600 MHz, δ): 9.86 (1H, d, J = 8.1 Hz),
9.70 (1H, d, J = 8.1 Hz), 9.15 (1H, s), 8.63 (2H, bs), 8.61 (2H, bs),
8.58 (1H, d, J = 9.4 Hz), 8.47 (1H, d, J = 9.4 Hz), 8.27 (5H, bs),
10 | J. Name., 2012, 00, 1-3
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PleaDsaeldtoonnTortaandsjuascttimonasrgins
Journal Name
Bis(1,10-phenanthroline)(dipyrido[3,2-a:2’,3’-
c][1,2,5]thiadiazolo[3,4-4,3]phenazine)ruthenium(II) chloride (MSCA, to SB), and Trinity College Dublin (TCD) for financial
(5). [Ru(phen)2Cl2] (0.158g, 0.297mmol, 1 eq) and dtp (0.101 g, support. We thank Dr. Fergus Poynton and Dr. Susan J. Quinn
0.297 mmol, 1 eq) were suspended in a mixture EtOH/H2O for their help and valuable discussion and guidance during this
(1:1, 8 mL). The mixture was degassed by bubbling with argon project. We thank Dr. Martin Feeney and Dr. Garry Hessman
for 15 minutes and then heated at 140 °C for 40 minutes using for the help with mass spectrometry studies, and Dr. John E.
microwave irradiation. Solvent was evaporated and the O’Brien and Dr. Manuel Ruether for the help with NMR
resulting red solid was purified by alumina chromatography studies.
ARTICLE
(IRC); PhD Scholarship B.C.P.), Marie Skłodowska-VCieuwriAerticalectOionlnines
DOI: 10.1039/C6DT03792E
using MeCN:H2O (10:0 to 9:1) as eluent. Solvent was removed
at reduced pressure yielding a red solid which was dried in
vacuo (77 mg, 30%). Calculated for C41H24Cl2N14Ru+3.5H2O: C,
Notes and references
52,40; H, 3,01; N, 14,29; S, 3,94; Cl, 8,03%. Found: C, 53,91; H,
3,34; N, 14,97; S, 3,43; Cl, 7,58%. m.p.: > 250 °C. H NMR
† These authors contributed equally to the work herein.
‡ CCDC numbers: 1489207 1489206
1
1. a). F. E. Poynton, J. P. Hall, P. M. Keane, C. Schwarz, I. V.
Sazanovich, M. Towrie, T. Gunnlaugsson, C. J. Cardin, D. J.
Cardin, S. J. Quinn, C. Long and J. M. Kelly, Chem. Sci., 2016, 7,
3075-3084. b) A. Byrne, C. S. Burke and T. E. Keyes, Chem. Sci.,
2016, 7, 6551-6562. c) B.-Z. Zhu, X.-J. Chao, C.-H. Huang and Y.
Li, Chem. Sci., 2016, 7, 4016-4023. d) V. Pierroz, R. Rubbiani, C.
Gentili, M. Patra, C. Mari, G. Gasser and S. Ferrari, Chem. Sci.,
2016, 7, 6115-6124. e) R. B. P. Elmes, J. A. Kitchen, D. C.
Williams and T. Gunnlaugsson, Dalton Trans., 2012, 41, 6607-
6610. f) J. A. Kitchen, E. M. Boyle and T. Gunnlaugsson, Inorg.
Chem. Acta, 2012, 381, 236-242. g) A. M. Nonat, C. Allain, S.
Faulkner and T. Gunnlaugsson, Inorg. Chem., 2010, 49, 8449–
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i) M. R. Gill, J. Garcia-Lara, S. J. Foster, C. Smythe, G. Battaglia
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(CD3CN, 400 MHz, δ): 9.86 (1H, d, J = 8.1 Hz), 9.70 (1H, d, J =
8.1 Hz), 9.15 (1H, s), 8.63 (2H, b), 8.61 (2H, m), 8.58 (1H, d, J =
9.4 Hz), 8.47 (1H, d, J = 9.4 Hz), 8.27 (5H, b), 8.23 (2H, m), 8.16
(2H, d, J = 5.2 Hz), 8.05 (2H, d, J = 5.2 Hz), 7.85‒7.80 (2H, m),
7.68‒7.63 (4H, m), 3.59 (3H, s). 13C NMR (CD3CN, 100 MHz, δ):
157.59, 155.66, 155.58, 154.28, 153.98, 153.69, 151.60,
151.55, 148.86, 148.81, 146.76, 141.20, 140.07, 139.67,
137.98, 137.94, 134.55, 132.64, 132.07, 132.05, 131.25,
131.12, 129.07, 128.26, 128.22, 127.58, 126.92, 126.87; IR
(ATR, cm-1): 3066 (aromatic C-H stretch), 1496 (aromatic C=C
stretch), 1360, 1336 (C=N stretch). MALDI-HRMS (m/z)
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c][1,2,5]thiadiazolo [3,4-4,3]phenazine)ruthenium(II) chloride
(6). [Ru(TAP)2Cl2] (140 mg, 262 µmol, 1 eq) and dtp (892 mg,
262 µmol, 1 eq) were suspended in a mixture EtOH/H2O (1:1,
8 mL). The mixture was degassed by bubbling with Argon for
15 minutes and then heated at 140 °C for 40 minutes using
microwave irradiation. Solvent was evaporated and the
resulting red solid was purified by alumina chromatography
using MeCN:H2O (10:0 to 9:1) as eluent. Solvent was removed
at reduced pressure yielding a red solid which was dried in
vacuo (100 mg, 44%). Calculated for C38H20N14Cl2SRu+0.5NaCl:
C, 47.88; H, 2.33; N, 20.57; S, 3.36%. Found: C, 46.73; H, 2.09;
1
N, 19.69; S, 6.10%. m.p.: 162-165 (decomp). H NMR (CD3CN,
600 MHz, δ): 9.83 (1H, dd, J = 8.2 Hz, 1.2 Hz), 9.77 (1H, dd, J =
8.2 Hz, 1.2 Hz), 9.01 (2H, 2d, J = 2.8 Hz), 8.99 (2H, d, J = 2.7 Hz),
8.62 (4H, s), 8.46 (2H, syst AB), 8.40 (2H, 2d, J = 2.9 Hz), 8.39
(2H, d, J = 2.7 Hz), 8.34 (2H, 2dd, J = 5.4 Hz, 1.3 Hz), 7.92 (2H,
2dd, J = 8.3 Hz, 5.4 Hz). 13C NMR (CD3CN, 100 MHz, δ): 157.62,
156.74, 156.66, 153.67, 150.73, 150.68, 150.58, 150.57,
150.45, 150.44, 150.07, 146.93, 146.51, 146.50, 146.45,
143.31, 143.30, 143.22, 140.88, 139.83, 139.75, 135.98,
133.81, 133.73, 132.64, 131.44, 131.32, 128.63, 128.59,
127.74. IR (ATR, cm-1): 3075 (aromatic C-H stretch), 1488
(aromatic C=C stretch), 1385, 1362 (C=N stretch). ESI-HRMS
(m/z) calculated for C38H20N14SRu: 806.0760. Found: 403.0387
[M]2+.
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Acknowledgements
We thank Science Foundation Ireland (SFI PI Awards 10/45
IN.1/B2999 and 13/IA/1865), and the Irish Research Council
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52. Y.-M. Chang, C. K.-M. Chen and M.-H. Hou, Int. J. Mol. Sci.,
2012, 13, 3394-3413.
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