Design of photoactivatable metallodrugs: Selective and rapid light-induced ligand dissociation from half-sandwich [Ru([9]aneS3)(N-N′)(py)] 2+ complexes

Article abstract of DOI:10.1016/j.ica.2012.06.031

The synthesis of the inert Ru(II) half-sandwich coordination compounds, [Ru([9]aneS₃)(bpy)(py)][PF₆]₂ (1, [9]aneS ₃ = 1,4,7-trithiacyclononane, bpy = 2,2′-bipyridine, py = pyridine), [Ru([9]aneS₃)(en)

Full text of DOI:10.1016/j.ica.2012.06.031

Inorganica Chimica Acta 393 (2012) 230–238
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Design of photoactivatable metallodrugs: Selective and rapid light-induced
ligand dissociation from half-sandwich [Ru([9]aneS3)(N–N0)(py)]2+ complexes
b,
b
Giulio Ragazzon a, Ioannis Bratsos a, Enzo Alessio a, , Luca Salassa , Abraha Habtemariam ,
⇑
⇑
Ruth J. McQuitty b, Guy J. Clarkson b, Peter J. Sadler b,
⇑
a Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Trieste, 34127 Trieste, Italy
b Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 6 July 2012
The synthesis of the inert Ru(II) half-sandwich coordination compounds, [Ru([9]aneS3)(bpy)(py)][PF6]2
(1, [9]aneS3 = 1,4,7-trithiacyclononane, bpy = 2,20-bipyridine, py = pyridine), [Ru([9]aneS3)(en)(py)][PF6]2
(2, en = 1,2-diaminoethane), and [Ru([9]aneN3)(en)(dmso-S)][PF6]2 (3, [9]aneN3 = 1,4,7-triazacyclo-
nonane), is reported along with the X-ray crystal structure of 1. We investigated whether these com-
plexes have photochemical properties which might make them suitable for use as pro-drugs in
photochemotherapy. Complexes 1 and 2 underwent rapid (minutes) aquation with dissociation of the
pyridine ligand in aqueous solution when irradiated with blue light (k = 420 or 467 nm). The photode-
composition of 3 was much slower. All complexes readily formed adducts with 9-ethylguanine (9-EtG)
when this model nucleobase was present in the photolysis solution. Similarly, complex 1 formed adducts
with the tripeptide glutathione (GSH), but only when photoactivated. HPLC and MS studies of 1 showed
that irradiation promoted rapid formation of 1:1 (major) and 1:2 (minor) adducts of the oligonucleotide
d(ATACATGCTACATA) with the fragment {Ru([9]aneS3)(bpy)}2+. Density functional theory (DFT) calcula-
tions and time-dependent DFT reproduced the major features of the absorption spectra and suggested
that the lowest-lying triplet state with 3MLCT character, which is readily accessible via intersystem cross-
ing, might be responsible for the observed dissociative behavior of the excited states. These complexes
are promising for further study as potential photochemotherapeutic agents.
Metals in medicine Special Issue
Keywords:
Ruthenium
Photochemistry
Macrocycle
Coordination complex
Pyridine ligand
Bioinorganic
Ó 2012 Published by Elsevier B.V.
1. Introduction
effective only for surface cancers whereas longer wavelengths
(e.g. red light) penetrate more deeply [2].
Typically, anticancer metal compounds show non-specific tox-
icity, not limited to tumor cells. A strategy for overcoming this dis-
advantage would be a rational design that involves their selective
activation. In this approach a non-toxic precursor of the active spe-
cies is administered, and activated exclusively in the tumor region.
Provided that activation occurs selectively, this strategy would
have the clear advantage of limiting the undesired effects of the
drug, thus increasing its therapeutic index. In ideal conditions,
even if the prodrug distributes equally in the body, only the part
activated at the tumor site would be highly cytotoxic. The use of
light for activating an anticancer compound is one of the strategies
that is being investigated, and for this reason is termed photoacti-
vated chemotherapy (PACT) [1]. The effective wavelength range of
light in photobiological and phototherapeutic processes is between
300 and 900 nm. Shorter wavelengths (UVA, blue light) would be
One potential objective in the context of PACT is the develop-
ment of complexes capable of killing cancer cells by binding to
DNA as a target. This might occur upon photo-induced dissociation
of one or more ligands from the coordination sphere of the metal.
The irradiation of the tumor site with a light source would lead to
the release of active metal fragments directly into cancer cells [3].
After developing a large series of anticancer Ru(II)-arene orga-
nometallic compounds, whose mechanism of action is believed to
involve monofunctional coordination to DNA after the hydrolysis
of a labile monodentate ligand (e.g. Clꢀ from [( 6-arene)Ru(en)Cl]+,
g
en = 1,2-diaminoethane) [4], Sadler and co-workers have demon-
strated that irradiation of inert half-sandwich Ru(II) compounds
of the type [(g6-arene)Ru(N–N0)(L)]2+ (where N–N0 is a nitrogen
chelating ligand, L is typically a nitrogen monodentate ligand such
as pyridine) with visible light leads to the selective dissociation of
the monodentate ligand (i.e. activation). For example, the complex
[(g
6-p-cymene)Ru(bpm)(py)][PF6]2 (bpm = 2,20-bipyrimidine, py =
pyridine) is inert in aqueous solution over a wide range of pH
values (2–12) if kept in the dark. However, upon irradiation with
⇑
Corresponding authors.
E-mail address: p.j.sadler@warwick.ac.uk (P.J. Sadler).
0020-1693/$ - see front matter Ó 2012 Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.ica.2012.06.031

G. Ragazzon et al. / Inorganica Chimica Acta 393 (2012) 230–238
231
visible light the complex releases selectively the pyridine ligand
spectral width of 14 ppm. 2D COSY and NOESY spectra were re-
corded using standard pulse-pulse sequences. For NOESY spectra,
data were acquired with 72 transients into 2048 data points over
a spectral width of 14 ppm using a relaxation delay of 1.5 s and a
mixing time of 0.4–0.6 s.
generating the active aqua species [(g
6-p-cymene)Ru(bpm)
(H2O)]2+ which is capable of binding to DNA model bases such as
9-ethylguanine [5].
On the other hand, Alessio and co-workers have demonstrated
that the formal replacement of the arene ligand in half-sandwich
complexes with a neutral face-capping tridentate macrocycle
(e.g. 1,4,7-trithiacyclononane, [9]aneS3) leads to half-sandwich
coordination compounds that maintain in vitro cytotoxic activity
against cancer cells provided that they undergo aquation with sub-
stitution of the monodentate ligand at a reasonable rate and that
the chelate ligand is a hydrogen-bond donor. Thus [Ru([9]ane-
S3)(en)Cl][PF6] and [Ru([9]aneS3)Cl(dach)][PF6] (dach = 1,2-diami-
nocyclohexane), that fulfill both requirements, show cytotoxic
activity comparable to their organometallic analogs [6].
Given such premises, the purpose of this work was to investi-
gate the photochemistry of selected inert Ru(II) half-sandwich
coordination compounds, namely [Ru([9]aneS3)(bpy)(py)][PF6]2
(1), [Ru([9]aneS3)(en)(py)][PF6]2 (2), and [Ru([9]aneN3)(en)(dm-
so-S)][PF6]2 (3, [9]aneN3 = 1,4,7-triazacyclononane) (Chart 1) that
are structurally similar to the photoactivable anticancer organo-
metallic compounds developed by Sadler and co-workers. More
specifically, we wanted to assess the rate and extent of dissociation
of the monodentate ligand upon irradiation with visible light and
the capability of the resulting aqua-species to interact with se-
lected biomolecules. To obtain a deeper understanding of the pho-
tochemical processes which occur, DFT and TD-DFT calculations
were also performed.
2.1.2. Mass spectrometry
Positive ion electrospray mass spectrometry ESI(+) was per-
formed on an Esquire2000 Mass Spectrometer (Bruker, Coventry,
UK Ltd). All data were acquired and processed with Bruker Micro-
TOF control Bruker Compas DataAnalysis (version 4.0) and Origin-
Pro8.1. Mass spectrometry work with the d(ATACATGCTACATA)
oligonucleotide was carried out on a Bruker MaXis ESI-HR-MS in
the negative mode (290–5000 m/z scan range), capillary 4000 V,
end plate offset ꢀ500 V, dry gas 4.0 L/min, dry heater 180 °C, neb-
uliser 0.4 bar.
2.1.3. UV–Visible spectroscopy
UV–Vis absorption spectra were recorded on a Cary 300-spec-
trophotometer using 1-cm pathlength quartz cuvettes (600 lL)
and a PTP1 Peltier temperature controller. Spectra were recorded
at 310 K in deionized water from 200 to 800 nm and were pro-
cessed using Cary WinUV software for Windows XP and
OriginPro8.1.
2.1.4. HPLC
HPLC studies were carried out on an Agilent Technologies 1100
system with a Rheodyne 7725i manual injector fitted with a 100 lL
loop and a DAD UV–Vis detector. The mobile phase for all analyses
was solvent A: H2O 10 mM NH4OAc (pH 5.5); solvent B: CH3CN,
10 mM NH4OAc. The column used was a Hichrom ACE 5 C8
250 ꢁ 4.6 mm 300 Å pore size. At all times the flow rate was
1 mL/min, the wavelength of detection was set to 260 nm and
the column oven was between 308 and 313 K. Solvent gradient
was as follows: increasing 0–20% B over 40 min, followed by 20–
80% B over 40–41 min, and between 41 and 45 min remaining at
80% B.
2. Experimental
2.1. Materials and methods
Silver nitrate, methanol, NH4PF6, D2O, 9-ethylguanine (9-EtG)
and L-glutathione reduced (GSH) were purchased from Sigma–Al-
drich, pyridine from Fluka and NaH2PO4, Na2HPO4 and acetone
from Fisher Chemicals. The PBS buffer was prepared in D2O with
a concentration of 0.1 M and pH 7.2. The single strand oligonucleo-
tide d(ATACATGCTACATA) was purchased from DNA Technology A/
S (Denmark), purified twice by RP-HPLC and contains sodium as
the counter ion. HPLC mobile phase: HPLC grade CH3CN and H2O
were purchased from Fisher Scientific. Ammonium acetate
(99.99%) was purchased from Sigma–Aldrich. All samples were
prepared in doubly deionised water purified using Multipore Milli
Q and a USF-Elga UHQ water deioniser. Omix pipette tips for micro
extraction were purchased from Varian Inc.
2.1.5. Photoirradiation of 1–3 in aqueous solution and in the presence
of 9-ethylguanine and L-glutathione
Photochemistry studies were performed by irradiating aqueous
solutions of complexes 1–3 as well as buffered solutions (PBS) of
the complexes and 9-ethylguanine (9-EtG) in a 1:1 mixture. In
the case of complex 1, PBS solutions containing L-glutathione
(GSH) (1:1 mixture) were also irradiated with blue light. As light
source, a broadband visible light lamp (16 tubes, model LZC420;
Luzchem research Inc., Ottawa, Canada), operating with a maxi-
mum output at 420 nm and a maximum power of ca. 20 mW/
cm2 was used. Excitation at 467 and 517 nm was performed using
LED sources (Philips Accent Color GU10). UV–Vis absorption spec-
2.1.1. NMR spectroscopy
NMR spectra were acquired in 5 mm NMR tubes at 298 K on
either Bruker DRX-500, Bruker AV III 600 or Bruker AV II 700
NMR spectrometers. All data processing was carried out using
XWIN-NMR version 3.6 (Bruker UK Ltd.). 1H-chemical shifts were
internally referenced to TMS via 1,4-dioxane (d = 3.71). 1D spectra
were recorded using standard pulse sequences. Typically, data
were acquired with 128 transients into 16 k data points over a
tra were recorded at a 100 lM concentration using a reduced irra-
diation power of 7 mW/cm2, while 1H NMR spectra were recorded
for 3 mM solutions using the full light output. Solutions were
stored in the dark to minimize unwanted photoreactions between
measurements. Irradiation experiments in the presence of GSH
were performed in an inert nitrogen atmosphere.
2+
2.1.6. Photoirradiation of 1 in the presence of oligonucleotides
Compound 1 was dissolved in water and mixed with the oligo-
nucleotide d(ATACATGCTACATA) in a 1:1 mol ratio (final concen-
2+
2+
NH
S
Ru
N
S
Ru
N
H2
N
H2
N
NH
S
S
N
N
S
S
Ru
N
N
N
H
tration of 250 lM). The concentration of 1 was determined by
H2
H2
S
-
-
-
ICP-MS and the oligonucleotide by UV–Vis spectroscopy. The reac-
tion was monitored by HPLC and mass spectrometry. The solution
was irradiated in a Luzchem photoreactor fitted with 420 nm
bulbs, with a power of ca. 20 mW/cm2, at 310 K for a total of 2 h
with aliquots taken every 30 min starting at time 0 (non-irradiated
O
2PF6
2PF6
2PF6
(1)
(2)
(3)
Chart 1.

232
G. Ragazzon et al. / Inorganica Chimica Acta 393 (2012) 230–238
sample). Each aliquot was then further diluted in doubly-deionised
water and analysed by HPLC. For MS analysis 400 L of the solution
irradiated for a total of 2 h was de-salted using Omix micro extrac-
tion C18 pipette tips and eluted in 40 mM ammonium acetate
(60:40 CH3CN:H2O).
Yield: 23.7 mg, 71%. Anal. Calc. for C22H27F12N3P2RuS3: C, 31.27;
H, 3.12; N, 5.21. Found C, 31.19; H, 2.75; N, 4.93%. UV–Vis (H2O)/
l
nm: 240 (
e
/Mꢀ1 cmꢀ1 1948), 282 (4018), 398 (596). 1H NMR dH
3
(500 MHz; D2O, dioxane): 9.22 (d, J 5.6, 2H, H6,60), 8.65 (d, 3J
6.7, 2H, H2-py), 8.37 (d, J 8.2, 2H, H3,30), 8.14 (t, 3J 7.9, 2H,
3
H4,40), 7.75 (m, 3H, H4-py + H5,50), 7.25 (t, 3J 6.3, 2H, H3-py),
3.15 (m, 2H, CH2 [9]aneS3), 2.88 (m, 2H, CH2 [9]aneS3), 2.79 (m,
2H, CH2 [9]aneS3), 2.73 (m, 4H, CH2 [9]aneS3), 2.63 (m, 2H, CH2
2.1.7. Computational details
All calculations on complex 1 were performed with GAUSSIAN03
[7], employing the DFT method with the PBE1PBE functional [8].
The LanL2DZ effective core potential [9] was used for the Ru atom
and the 6-31G⁄⁄+ basis set [10] was used for all other atoms.
Geometry optimization of the singlet ground state and the low-
est-lying triplet state was performed in the gas phase. The nature
of the stationary points was confirmed by normal mode analysis.
The conductor-like polarizable continuum model method (CPCM)
[11] with water as solvent was used to calculate the electronic
structure of the excited states of the complexes in solution
(H2O). Fifty singlet excited states and eight triplet excited states,
as well as the corresponding oscillator strength, were determined
with TD-DFT calculation [12] using the singlet ground state and
the lowest-lying triplet state geometries, respectively. The elec-
tronic distribution and the localization of the singlet and triplet ex-
cited states were visualized using the electron density difference
maps (EDDMs) [13]. GaussSum 1.05 [14] was used for EDDMs cal-
culations and for the electronic spectrum simulation. A summary
of the calculated electronic transitions is reported in the Support-
ing Information. Molecular graphics images were produced using
the UCSF Chimera package from the Resource for Biocomputing,
Visualization, and Informatics at the University of California, San
Francisco (supported by NIH P41 RR001081) [15].
[9]aneS3). ESI-MS m/z: 258.5 [Ru([9]aneS3)(en)(py)]2+
.
2.2.2. Synthesis and characterization of [Ru([9]aneS3)(en)(py)][PF6]2
(2)
In a light-protected round bottomed flask at ambient tempera-
ture [Ru([9]aneS3)(en)Cl][PF6] (35.2 mg, 0.067 mmol) and AgNO3
(11.2 mg, 0.066 mmol) were dissolved in a methanol/water 4:1
mixture (10 mL). The yellowish solution was stirred for about
24 h and afterwards the cloudy mixture was centrifuged. The gray
AgCl precipitate was discarded. Pyridine (0.109 mL, 1.348 mmol)
was added to the filtrate and the reaction mixture was stirred
again for ca. 24 h. The yellow solution obtained was filtered and
an excess of NH4PF6 (109.4 mg, 0.674 mmol) and acetone (1 mL)
were added. Slow evaporation of the solvent afforded a light yel-
low solid. Yield: 15.0 mg, 31%. Anal. Calc. for C13H25F12N3P2RuS3:
C, 21.97; H, 3.55; N, 5.91. Found C, 21.69; H, 3.38; N, 5.96%. UV–
Vis (H2O)/nm: 237 (e
/Mꢀ1 cmꢀ1 4615), 316 (4280). 1H NMR d
(400 MHz; D2O, dioxane): 8.79 (d, 3J 5.3, 2H, H2-py), 7.96 (t, 3J
7.8, 1H, H4-py), 7.53 (t, 3J 6.40, 2H, H3-py), 3.79 (br, 2H, NH en),
a
2.83–2.64 (m, 10H, CH2 en and[9aneS3]), 2.43 (m, 2H, CH2
[9]aneS3), 2.29 (m, 2H, CH2 [9]aneS3), 2.22 (m, 2H, CH2 [9]aneS3).
Ethylenediamine diasterotopic NHb 1H NMR signals overlap with
the residual water peak.
2.2. Synthesis and characterization of compounds
2.3. X-ray crystallography
The procedure for obtaining the precursor complex [Ru([9]
aneS3)(bpy)Cl][PF6] is similar to that previously described for the
corresponding CF3SO3 compound [16]. To [Ru([9]aneS3)(dmso-
S)2Cl][PF6] (100 mg, 0.16 mmol) suspended in methanol (10 mL),
2,20-bipyridine (25.6 mg, 0.16 mmol) was added and the mixture
was heated under reflux for 1 h. During this time the color of the
solution changed from yellow to orange and the formation of the
product as deep yellow solid was observed. After cooling, the solid
was collected by filtration, washed with ethanol and diethyl ether
Single crystals of 1 suitable for X-ray analysis were obtained
from slow evaporation of the reaction mixture as described above.
The data were collected using an Oxford Diffraction Gemini four-
circle system with Ruby CCD area detector and the structure was
solved by direct methods using SHELXS [17] (TREF) with additional
light atoms found by Fourier methods. X-ray crystallographic data
for complex 1 are available as Supporting Information and have
been deposited in the Cambridge Crystallographic Data Centre un-
der the accession number CCDC 880474. X-ray crystallographic
data in CIF format are available from the Cambridge Crystallo-
graphic Data Centre (http://www.ccdc.cam.ac.uk/).
and
vacuum
dried
(69.4 mg,
70%).
Anal.
Calc.
for
C16H20ClF6N2PRuS3: C, 31.09; H, 3.26; N, 4.53. Found: C, 30.98; H,
3.07; N, 4.38%. The spectral patterns of this compound are coinci-
dent with those of the corresponding CF3SO3 salt [16]. The precur-
sors [Ru([9]aneS3)(en)Cl][PF6] and [Ru([9]aneN3)(en)(dmso-
S)][Cl]2 (3) were prepared as reported elsewhere [6].
3. Results and discussion
2.2.1. Synthesis and characterization of [Ru([9]aneS3)(bpy)(py)][PF6]2
(1)
3.1. Synthesis of complexes and molecular structure of 1
In a light-protected round bottomed flask at ambient tempera-
ture [Ru([9]aneS3)(bpy)Cl][PF6] (25.5 mg, 0.041 mmol) and AgNO3
(6.8 mg, 0.040 mmol) were dissolved in a methanol/water 4:1 mix-
ture (10 mL) giving an almost colorless solution. The solution was
stirred for ca. 24 h and precipitation of AgCl was observed. The so-
lid was removed by centrifugation followed by filtration. A large
excess of pyridine (0.066 mL, 0.824 mmol) was added to the
remaining solution and the reaction mixture stirred again for ca.
18 h. The yellow solution obtained was filtered to eliminate any
residual AgCl or any other particulates and NH4PF6 (66.9 mg,
0.412 mmol) and acetone (1 mL) were added. After two days yel-
low crystals suitable for X-ray analysis were obtained by slow
evaporation of the solvent. The crystals were collected by filtration,
washed with methanol and dried in air.
Complex [Ru([9]aneS3)(bpy)(py)][PF6]2 (1) was prepared in a
similar manner as reported for [(g6-arene)Ru(N-N0)(L)]2+ com-
pounds [5]: the parent chloride complex [Ru([9]aneS3)(b-
py)Cl][PF6] reacted with AgNO3 to afford the aqua adduct
[Ru([9]aneS3)(bpy)(H2O)]2+ which was then treated with an excess
of pyridine to give 1 after precipitation with NH4PF6.
The same procedure was adopted for complex 2, while 3 was
prepared as described elsewhere [16]. All complexes were fully
characterized by 1H NMR spectroscopy, mass spectrometry and
elemental analysis. Moreover, the molecular structure of 1 was
determined by X-ray crystallography. A perspective view of the
cation is given in Fig. 1, while selected bond distances and angles
are reported in Table 1 and compared with the DFT-optimized
structural parameters.

G. Ragazzon et al. / Inorganica Chimica Acta 393 (2012) 230–238
233
3.2. Dark stability and photochemical behavior of 1–3 in aqueous
solution
Complex 1 is very stable in aqueous solution in the dark. In-
deed, no changes were observed in the UV–Vis and 1H NMR spec-
tra of 1 even after six days at 298 K or overnight at 310 K.
However upon light irradiation (kex = 420 nm) of 1 in H2O, rapid
changes occurred in the UV–Vis spectrum. Three isosbestic points
were observed when the photoreaction was monitored by UV–Vis
spectroscopy (Fig. 2A). The band at 282 nm decreased in intensity
while the bands at 240 and 398 nm were slightly red-shifted.
After ca. 10 min of irradiation no further significant changes were
observed at the irradiation power (7 mW/cm2) and complex con-
centration (100 lM) used. These changes correspond to the re-
lease of the coordinated pyridine and formation of the aqua
adduct (Scheme 1). Light activation was also achieved using
467 nm light, while excitation at 517 nm did not induce formation
of photoproducts.
Analogously, complex [Ru([9]aneS3)(en)(py)][PF6]2 (2) was sta-
ble in aqueous solution (up to 6 days), but readily reacted upon
irradiation. Light excitation caused a decrease in the intensity of
the two main bands of its UV–Vis spectrum (237 and 316 nm),
and the appearance of new bands at 247 and 375 nm. Three isos-
bestic points were observed at 246, 266 and 377 nm, suggesting
that the photoreaction produces a single product. After 40 min of
irradiation there was no further change in the spectrum (Fig. S2).
Complex [Ru([9]aneN3)(en)(dmso-S)][Cl]2 (3) also appeared to
be stable in the dark (overnight at 310 K), however its photoactiv-
ity was lower. In the UV–Vis spectrum of 3, the band at 214 nm de-
creased in intensity while the band at 306 nm was red shifted and
more intense. Similarly to 1 and 2, an isosbestic point is present at
Fig. 1. Solid state structure of the cation of complex [Ru([9]aneS3)(bpy)(py)][PF6]2
(1) with atom numbering scheme. Thermal ellipsoids show 50% probability.
Table 1
Selected X-ray bond lengths (Å) and angles (°) for 1 together with calculated (DFT)
values.
254 nm (Fig. S3), confirming the formation of
photoproduct.
a
single
X-ray
DFT
GSa
LL-Tb
The photoreactions of complexes 1–3 were also followed by 1H
NMR spectroscopy in D2O. After 20 min of irradiation at 420 nm
(Fig. 2B), complex 1 completely released the pyridine ligand form-
ing the aqua adduct [Ru([9]aneS3)(bpy)(H2O)]2+ (1a, see Scheme 1),
as clearly indicated by the chemical shift of the bpy resonances
[16] and by the appearance of free pyridine peaks in the 1H spec-
trum. These results are in agreement with the reported observa-
tions for analogous piano-stool ruthenium complexes [5]. ESI-MS
further confirmed the nature of the photoproduct giving the ex-
pected peak for the aqua complex at m/z 227.9962 (predicted
227.9964 m/z) with the expected isotopic pattern. 1H NMR shows
that in the photolyzed solution the reverse binding of pyridine to
1a is very slow and not quantitative (see below).
Bond length (Å)
Ru2–N101
Ru2–N112
Ru2–N201
Ru2–S301
Ru2–S304
Ru2–S307
2.0903(12)
2.0898(11)
2.1295(12)
2.3117(3)
2.3151(4)
2.3021(4)
2.100
2.072
1.995
2.118
2.403
2.425
2.416
2.099
2.136
2.361
2.367
2.365
Bond angles (°)
N101–Ru2–N112
N101–Ru2–N201
N112–Ru2–N201
S301–Ru2–S304
S301–Ru2–S307
S304–Ru2–S307
78.25 (5)
88.83 (5)
87.92 (5)
85.392 (12)
88.845 (13)
88.449 (13)
77.996
89.430
88.370
86.485
87.678
87.316
80.817
89.637
91.898
85.551
87.476
86.063
a
Similar behavior was observed when photolysis experiments
were performed in PBS buffer. However, a new minor species (ca.
7%) (bpy resonances at 9.14 (d), 8.41 (d), 8.09 (t) 7.59 (t)) also
formed in PBS buffer under prolonged light irradiation (150 min),
probably due to the formation of the phosphate adduct, since com-
parison with the 1H NMR spectrum of complex 1 rules out the for-
mation of the chlorido derivative (Fig. S4).
GS, ground state geometry.
LL-T, lowest lying triplet geometry.
b
Complex 1 displays a Ru–N(py) distance of 2.1295(12) Å,
slightly longer than the Ru–N(bpy) distances of 2.090 Å (see Ta-
ble 1). The sulfur atoms of the [9]aneS3 ligand are at 2.302–
2.315 Å from the ruthenium center and binding angles of the li-
gand approach 90°. Only the S301–Ru2–S304 angle is significantly
smaller (85.391°), causing a distortion in the [9]aneS3 ligand struc-
ture. DFT-optimized ground state geometry is in very good agree-
ment with the X-ray data. Bond distances are overestimated by less
than 0.06 Å. The lowest-lying triplet geometry was calculated as
well, by virtue of the key role that such a state plays in the photo-
chemistry of transition metal complexes [18]. It is observed that in
the triplet state all the Ru–N bonds are slightly contracted while
the Ru–S bonds are elongated by ca. 0.1 Å compared to the ground
state structure. Crystallographic details and crystal packing infor-
mation for complex 1 are reported in the Supporting Information
(Table 1 and Fig. S1).
1H NMR monitoring of the photolysis of complex 2 gave compa-
rable results (Fig. S5). Upon light-induced formation of the aqua
2+
2+
S
Ru
N
S
N
N
N
hν, H O
N
N
S
S
2
S
S
+
Ru
OH2
1a
1
Scheme 1.

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G. Ragazzon et al. / Inorganica Chimica Acta 393 (2012) 230–238
Fig. 2. (A) UV–Vis spectra of [Ru([9]aneS3)(bpy)(py)][PF6]2 (1) in aqueous solution recorded at different irradiation times (kex = 420 nm, 7 mW/cm2); (B) 1H NMR spectra of 1
in D2O at t = 0 min and after 20 min of irradiation (kex = 420 nm, 20 mW/cm2); 1a is the aqua species [Ru([9]aneS3)(bpy)(H2O)]2+, py(f) indicates free pyridine.
complex [Ru([9]aneS3)(en)(H2O)]2+ (2a) the signals of the diaste-
reotopic NH 1,2-diaminoethane protons became observable as
they were no longer overlapped by the solvent and reference sig-
nals. The formation of the aqua species 2a was also confirmed by
comparison with the spectrum of the labile [Ru([9]ane-
S3)(en)Cl][PF6], which gave the same aqua species 2a upon release
of the Cl ligand [6,16]. Interestingly, resonances for coordinated py
in 2 could still be seen after 60 min of irradiation, indicating that
the photo-induced ligand release was not complete (92% from
NMR signal integration).
and 9-ethylguanine (9-EtG) in a 1:1 mol ratio was monitored by
1H NMR spectroscopy. In the dark at 310 K no dissociation of pyr-
idine and consequently no interaction with 9-EtG was observed. In
contrast, light excitation in the presence of 9-EtG resulted in the
release of the py ligand and formation of the aqua complex 1a,
which readily reacted with 9-EtG to give the [Ru([9]aneS3)(b-
py)(9-EtG)]2+ adduct (1-G). The progress of the reaction is shown
in Fig. 3, where the formation of 1-G is demonstrated by the
appearance of a singlet at about 8 ppm, corresponding to the H8
proton of the coordinated 9-EtG. The resonances of coordinated
bpy in 1-G are slightly shifted compared to both 1 and 1a, and
the relative integration of the new bpy and 9-EtG signals is consis-
tent with the formation of 1-G. A 1H–1H NOESY experiment, per-
formed after 20 min of irradiation, showed a clear correlation
peak between the H8 singlet of bound 9-EtG and the H6,60 doublet
of bpy, confirming the formation of the nucleobase adduct 1–G
(Fig. S7). After six further hours in the dark, the reaction reached
equilibrium, and compound 1-G was the most abundant (50%)
compared to 1 (15%) and its aqua derivative 1a (35%).
In the case of 3, 1H NMR spectra recorded at different times of
irradiation showed the release of the dmso ligand (Fig. S6). A sin-
glet at d = 2.72 corresponding to free dmso progressively increased
in intensity, while the peak of the coordinated dmso-S at d = 3.33
decreased in intensity, indicating the formation of [Ru([9]ane-
N3)(en)(H2O)]2+ (3a). The concentration of free dmso increased
during the first two hours of irradiation, eventually reaching a pla-
teau corresponding to the formation of ca. 23% 3a.
A similar procedure was employed for complexes 2 and 3,
although in unbuffered D2O. In the presence of 9-EtG both com-
plexes were stable in the dark at 37 °C (overnight), and underwent
photoreaction to give the corresponding aqua derivatives and
3.3. Irradiation of 1–3 in the presence of model biomolecules
To study the ability of 1 to bind to DNA bases, photolysis
(kex = 420 nm) of a PBS solution (pH 7.2) containing the complex

G. Ragazzon et al. / Inorganica Chimica Acta 393 (2012) 230–238
235
Fig. 3. Aromatic region of the 1H NMR spectrum of [Ru([9]aneS3)(bpy)(py)][PF6]2 (1) and 9-EtG in D2O (1:1 mixture, PBS buffer, pH 7.2) recorded after various irradiation
times (kex = 420 nm, 20 mW/cm2); py (f) indicates free pyridine and H8(1-G) is the resonance of the H8 proton of the coordinated 9-EtG in [Ru([9]aneS3)(bpy)(9-EtG)]2+
.
subsequent coordination of the nucleobase. New H8 peaks of the 2-
G and 3-G adducts were observed at lower fields (0.5 and 0.2 ppm,
respectively) in the 1H NMR spectra of the irradiated samples
(Figs. S8 and S9). After 180 min under light excitation (when
100% py dissociation had occurred), 2-G accounted for ca. 56% of
the total complex in solution, while with 135 min of irradiation
and incubation at 37 °C overnight only a small fraction of 3-G
(ca. 7%) was formed.
but a full assignment was not possible due to the presence of
extensive overlap. Nevertheless, a new signal (dd) at 3.60 ppm
was tentatively assigned to one of the CH2 protons adjacent to
the S atom of GSH that are diastereotopic. The signals of 1-GS in-
creased with time until they reached a plateau after 140 min of
irradiation. At that point the composition of the solution was
(according to bpy integration) ca. 43% 1-GS and 57% 1a.
Given the relevance of glutathione (c-L-Glu-L-Cys-Gly, GSH) –
3.4. Oligonucleotide binding of 1 under light irradiation
the abundant intracellular tripeptide – as metal detoxification
agent [19], its interaction with 1 was studied by 1H NMR spectros-
copy (PBS, pH 7.2) both in the dark and under light irradiation
(Fig. 4). When left in the dark overnight no reaction was observed
between 1 and GSH (1:1 mol ratio). On the contrary, light excita-
tion clearly caused the formation of Ru-GSH adducts. In the aro-
matic region of the 1H NMR spectrum a new set of signals for
coordinated bpy appeared, in addition to those expected for 1a.
The new resonances were tentatively attributed to the adduct
[Ru([9]aneS3)(bpy)(GS)]+ (1-GS), in which GSH is presumably
bound through the deprotonated S atom. In the aliphatic part of
the spectrum new signals were observed for GSH and [9]aneS3,
The interaction of 1 with the single strand oligonucleotide
d(ATACATGCTACATA) (1:1 mol ratio, 250 lM) under irradiation
with visible light was investigated by HPLC and mass spectrome-
try. After just 30 min of irradiation (kex = 420 nm) complex 1a
was found to bind significantly to the oligonucleotide. The integra-
tion of the total peak area of the chromatograms indicated that the
extent of binding by 1a to the oligonucleotide was ca. 50% after
30 min of light exposure (Fig. S10), increasing to 65% ( 3%) after
90 min. MS analysis of the irradiated solution detected the pres-
ence of the unreacted oligonucleotide, the oligonucleotide with
one {Ru([9]aneS3)(bpy)}2+ fragment bound (Fig. S11, predicted
Fig. 4. Aromatic region of the 1H NMR spectrum of 1 and GSH in PBS (1:1 mixture, pH 7.2) recorded at different irradiation times; py (f) indicates free pyridine, 1-GS indicates
[Ru([9]aneS3)(bpy)(GS)]2+
.

236
G. Ragazzon et al. / Inorganica Chimica Acta 393 (2012) 230–238
Fig. 5. (A) Selected molecular orbitals for 1 in the singlet ground state; (B) SOMOs and spin density surface for 1 in the lowest lying triplet state.
1554.2438 m/z, observed 1554.2254 m/z), and a very small amount
of the oligonucleotide bearing two {Ru([9]aneS3)(bpy)}2+ frag-
ments (predicted 1699.8950 m/z, observed 1699.8774 m/z). All
species were detected in the 3ꢀ charge state. As demonstrated by
the 9-EtG binding experiment, it is likely that 1a binds first at
the single guanine residue, as the N7 of guanine is usually the pre-
ferred DNA site for transition metal ions [20]. A minor adenine ad-
duct may also be present, giving rise to the species observed at
1699.8774 m/z that however eluded HPLC detection.
3.5. Molecular orbitals and spin surface analysis for complex 1 in the
singlet ground state and in the lowest-lying triplet state geometry
Complex 1 was selected for a more detailed computational
study aimed at characterizing its electronic properties. The elec-
tronic structure of 1 was calculated in solution (H2O) for both
the singlet ground state and the lowest-lying triplet state geome-
tries using the CPCM method [10]. Selected molecular orbitals
and the spin density surface for the triplet geometry are described
in Fig. 5. In the ground state configuration, the frontier occupied
orbitals – HOMO, HOMOꢀ1, and HOMOꢀ2 – have all a marked me-
tal-centered character, although contributions from pyridine, bpy,
and [9]aneS3 ligands are present. The four lowest unoccupied orbi-
tals are all prevalently bpy-based while the LUMO+4 and LUMO+5
have a significant metal contribution. Moreover, these two latter
Fig. 6. Experimental absorption spectrum (black line) and calculated singlet excited
state transitions (magenta vertical bars) of 1 in H2O. The vertical bar height is the
oscillator strength. Selected electron density difference maps (EDDMs) of singlet
excited state transitions of 1 are reported on the top of the box. Yellow indicates a
decrease in electron density, while orange indicates an increase. (For interpretation
of the references to color in this figure legend, the reader is referred to the web
version of this article.)
orbitals have a strong
r-antibonding character towards the bpy
and pyridine ligands. Such orbitals are likely to play a role in the
photochemistry of complex 1 [21].
In the lowest-lying triplet geometry, the unpaired electrons are
localized on two bpy-based SOMOs (singly occupied molecular
orbitals). The lowest-SOMO (l-SOMO) has also a noteworthy ruthe-
nium contribution. This can also be appreciated in the spin density
surface where the Ru-bpy character is particularly evident and it is
in agreement with the TDDFT results on the lowest energy triplet
excited state (vide infra).

G. Ragazzon et al. / Inorganica Chimica Acta 393 (2012) 230–238
237
Fig. 7. Selected electron density difference maps (EDDMs) of the four lowest energy triplet excited states of 1. Magenta indicates a decrease in electron density, while purple
indicates an increase.
3.6. Absorption properties of complex 1
and [Ru([9]aneS3)(en)(py)][PF6]2 (2), were prepared and investi-
gated as well as the [9]aneN3 compound [Ru([9]aneN3)(en)(dm-
so-S)][Cl]2 (3). They were found to be completely inert in
solution in the absence of light, which is a desirable pre-requisite
for photoactivatable pro-drugs.
The photochemical studies showed that the three half-sand-
wich Ru(II) complexes 1–3 dissociate to some extent the monoden-
tate ligand upon irradiation with visible light (kex = 420 nm) of
moderate power (ca. 20 mW/cm2) and give selectively the corre-
sponding aqua species. When the photoactivation is performed in
the presence of an equimolar amount of the model DNA base 9-
EtG, coordination through N7 occurs. Data summarizing the results
for the three complexes are given in Table 2.
As discussed in Section 3.2, the absorption spectrum of 1 shows
three major bands and a shoulder (Fig. 6). Singlet transitions were
calculated employing TDDFT to assign the character of the bands in
the UV–Vis spectrum. All the details on the calculated transitions
are reported in the Supporting Information (Table S2).
Despite a blue shift of ca. 20–25 nm, calculations predict well
the shape of the spectrum. In the experimental spectrum, the low-
est-energy band is centered at 399 nm (e
= 3286 Mꢀ1 cmꢀ1) and
has a MLCT character as shown by the electron density difference
map (EDDM) S1. At higher energies the most intense peak
(e
= 19672 Mꢀ1 cmꢀ1) and the shoulder fall at 281 and 309 nm,
respectively. The corresponding EDDMs highlight a Ru ? bpy
MLCT (S13 and S17) character also for this band, although the py
is partially involved as well. The third peak is at 239 nm
These data clearly show that both the nature of the face-cap-
ping ligand and of the leaving group have a remarkable influence
on the photoactivation process: whereas both the [9]aneS3 com-
plexes 1 and 2 have excellent photoactivation parameters (rate
and extent of pyridine dissociation, extent of reaction with 9-
EtG) that compare very well with most of the organometallic ana-
logs investigated so far, the strong Ru–S(dmso) bond in the
[9]aneN3 complex 3 is not easily broken even by the action of light
and the extent of photoactivation is modest. Also the nature of the
chelating ligand has a strong impact on the photoactivation rate
(cf. 1 with 2), possibly because there is a marked increase in the
absorbance at the irradiation wavelength when the bpy aromatic
system replaces 1,2-diaminoethane. Nevertheless, complex
[Ru([9]aneS3)(py)3][PF6]2, that was extensively investigated by
Thomas and co-workers for synthetic purposes and is structurally
very similar to 1, has been described as photo-inactive [23]. In
the case of 1, DFT calculations show that dissociative 3MC (d–d li-
gand field) states are accessible and can promote specific photodis-
sociation of the pyridine ligand.
These interesting properties of 1 prompted us to extend our
studies on this compound. We found that upon photoactivation
the complex is capable of binding efficiently, not only to 9-EtG,
but also to the single strand oligonucleotide d(ATACATGCTACATA)
and to glutathione. Moreover, photoactivation occurs also with the
less energetic light at 463 nm.
In the future we plan to perform in vitro antiproliferative tests
on compounds 1 and 2, and their analogs, both in the dark and
upon photo-irradiation, in order to assess if the photoactivation
and reactivity observed in the test-tube leads to cytotoxicity
towards cancer cells. The in vitro results will be compared with
(e
= 10658 Mꢀ1 cmꢀ1) and has major intraligand character (S36:
py ? bpy). Interestingly, there are weak transitions in the
350 nm region (calculated) that have a dissociative MC character
because of significant contributions from the
tals LUMO+4 and LUMO+5 (e.g. S4, Table S1).
r-antibonding orbi-
3.7. Triplet excited states and general remarks on the photochemistry
of 1
Dissociative singlet excited states (e.g. S4, Table S1) may be
responsible for pyridine release from 1 upon light excitation. How-
ever, triplet states are generally involved in the photochemistry of
ruthenium complexes since they become efficiently populated
after intersystem crossing (ISC). For this reason we have investi-
gated the nature of the lowest-energy triplet states by TDDFT
(Fig. 7). Consistent with the UKS (unrestricted Kohn–Sham) calcu-
lations, the lowest-lying triplet has a 3MLCT character (T1). Similar
character is observed for a state lying at higher energy (T2, 0.4 eV),
while two highly dissociative 3MC states (T3 and T4) are at 0.73–
0.83 eV from T1. These 3MC states have dominant contributions
from orbitals such as the LUMO+5 represented in Fig. 5A. More-
over, according to the calculations, they have lower energy than
the lowest singlet S1 and could therefore be easily populated by
ISC and then promote the release of the py ligand [22]. This sce-
nario is in agreement with the lack of luminescence in solution
of 1 and with its high reactivity.
4. Conclusions
Table 2
The aim of this work was to study the photochemistry of some
Ru(II) half-sandwich coordination compounds, structurally similar
Extent (%) of light-induced (kex = 420 nm, 20 mW/cm2) aquation and 9-EtG binding
for complexes 1–3.
to the inert organometallic compounds of general formula [(g6
-
Complex
Irr. time (min)
% of aqua species
% binding to 9-EtG
arene)Ru(N–N’)(L)]2+ developed in recent years by Sadler and co-
workers, that can be activated by photo-induced dissociation of
the monodentate ligand upon irradiation with visible light. Thus
two new [9]aneS3 complexes, [Ru([9]aneS3)(bpy)(py)][PF6]2 (1)
1
2
3
2
40
360
100
92
23
50
56
7

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G. Ragazzon et al. / Inorganica Chimica Acta 393 (2012) 230–238
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21.
those previously obtained for the chlorido compounds [Ru([9]a-
neS3)(bpy)Cl][PF6] and [Ru([9]aneS3)(en)Cl][PF6] that in aqueous
solution undergo substitution of the Cl ligand and generate the
same aqua species as complexes 1 and 2, namely, 1a and 2a,
respectively [6,16,24].
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Note added in proof
Current interest in the photo-activation of ruthenium com-
plexes for anticancer activity is demonstrated by two recent publi-
cations that appeared after submission of the manuscript [24]
Acknowledgements
G.R. thanks COST action D39 ‘‘Metallo-Drug design and action’’
for funding. L.S. was supported by ERC BIOINCMED (Grant No.
247450 to P.J.S.). E.A. thanks Fondazione Beneficentia Stiftung for
financial support and BASF Italia Srl for a generous donation of hy-
drated ruthenium trichloride.
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