Journal of Organometallic Chemistry 696 (2011) 1108e1116

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Journal of Organometallic Chemistry

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Ruthenium(II) arene complexes with oligocationic triarylphosphine ligands:

Synthesis, DNA interactions and in vitro properties
Dennis J.M. Snelders a, Angela Casini a, Fabio Edafe a, Gerard van Koten b, Robertus J.M. Klein Gebbink b,
Paul J. Dyson a, *
a
Institut des Sciences et Ingnierie Chimiques, Ecole Polytechnique Fdrale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
b
Organic Chemistry and Catalysis, Debye Institute for Nanomaterials Science, Faculty of Science, Utrecht University, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: The synthesis, DNA binding properties and cytotoxicity of a series of Ru(II)-arene complexes containing
Received 22 September 2010 oligocationic ammonium-functionalized triarylphosphines, of the type Ru(p-cymene)Cl2(L) (L oligo-
Received in revised form cationic phosphine), are reported. The complexes are highly charged (the overall charge states being 3
16 November 2010
and 6) and circular dichroism spectroscopy and gel electrophoresis studies indicate that the
Accepted 17 November 2010
compounds interact strongly with DNA via electrostatic interactions. Despite forming strong interactions
with DNA, the complexes exhibit only modest cytotoxicities on the human ovarian cancer cell lines
Keywords:
A2780 and A2780cisR.
Bioorganometallic chemistry
Ruthenium
2010 Elsevier B.V. All rights reserved.
Phosphine ligands
Metal-based drugs
DNA binding

1. Introduction complexes containing this class of ligands may be envisaged to

Classical anticancer compounds such as cisplatin and carbo-
platin primarily act by direct coordination of the metal ion to DNA
bases [1e4]. Other metal-based compounds may interact with
DNA in different ways, and this may lead to alternative types of
drug activity and selectivity [5,6]. Such interactions are often non-
covalent in nature and include hydrogen bonding, electrostatic,
hydrophobic and p-stacking interactions [7e9]. In cases where
a metal ion is coordinated to an active organic ligand, DNA binding
may occur through a combination of covalent and non-covalent
interactions [10e14].
A family of oligocationic triarylphosphine ligands termed
Dendriphos, 1ae4a (Fig. 1) that combine the triphenylphosphine
motif with three (1a) to six (2ae4a) permanent cationic substit-
uents (ammonium groups), have been reported [15e19]. Varia-
tions in the size of these ligands are achieved by altering the
ammonium substituent (R), ranging from a methyl (1a, 2a) or
benzyl (3a) group, to a Frchet dendron (4a). In view of their
bifunctional character, these compounds are interesting candi-
dates in the eld of metal-based drugs. The DNA binding of metal
* Corresponding author. Fax: 41(0)21 693 98 85.
E-mail address: paul.dyson@ep.ch (P.J. Dyson).
occur in a synergistic manner, combining electrostatic binding of
the cationic organic ligand to the negatively charged phosphate
groups of DNA, possibly with coordination of the metal ion to
DNA bases.
In this paper, we describe the synthesis and characterization of
some highly charged (cationic) ruthenium(II)-arene complexes
that can potentially interact with DNA via electrostatic and covalent
binding. Circular dichroism and gel electrophoresis were subse-
quently used to study these interactions.
2. Results
2.1. Synthesis, characterization and aquation of Ru(p-cymene)Cl2(L)
complexes
Complexes of the type Ru(p-cymene)Cl2(L) (L 1ae4a) were
prepared by reacting phosphine ligands 1ae4a with 0.5 equiv. of
[Ru(p-cymene)Cl2]2 in acetonitrile at 60  C for 1.5 h. After evapo-
ration of the solvent in vacuo, the products 1be4b were obtained
as orange powders in quantitative yield (Scheme 1). The 1H and
13
C NMR spectra of 1be4b, recorded in CD3CN, showed minor
shifts (typically Dd 0.1e0.5 ppm in 1H NMR spectra and
Dd 0.1e3.0 ppm in 13C NMR spectra) in the signals for the

0022-328X/$ e see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2010.11.025
 D.J.M. Snelders et al. / Journal of Organometallic Chemistry 696 (2011) 1108e1116 1109

Fig. 1. Oligocationic, ammonium-functionalized phosphine ligands 1ae4a.

phosphine ligand moieties compared to those for the correspond-
ing free phosphines 1ae4a. The signals for the p-cymene moieties
are observed at very similar chemical shifts to those observed in Ru
(p-cymene)Cl2(PPh3) [20]. The 31P NMR spectra of 1be4b contain
one singlet around 24e25 ppm, conrming the quantitative
formation of the desired Ru-phosphine complexes.
Extensive investigations into the water stability of Ru(II)(p-
cymene)Cl2(pta) (pta 1,3,5-triaza-7-phosphaadamantane), RAP-
TA-C (6), have been reported. It has been established that in water, 6
undergoes aquation, i.e. substitution of one or both chloride ligands
by a water molecule. Depending on the pH, the aqua ligands may be
deprotonated. At physiologically relevant pH, the dichloride I exists
in equilibrium with the mono-aquated species II and the hydroxo-
aqua species III (Scheme 2) [21e24].
The water stability of 1b, 2b and 5 was studied by 31P NMR
spectroscopy in D2O. The spectra are given in the supporting
information, Figures S1eS3. The low water solubility of 3b and
4b precluded such analysis. The 31P NMR spectrum of 1b in D2O,
recorded after 10 min, shows two major signals at 31.8 and
26.7 ppm in an approximate ratio of 1:2. After standing at RT in
solution for 16 h, no changes in the spectrum were observed.
Addition of 4 mM NaCl (the approximate chloride concentration
in cells) did not lead to a signicant change, but after addition of
100 mM NaCl (the approximate chloride concentration in plasma),
the signal at 26.7 ppm remains as the only peak in the spectrum,
indicating that this signal corresponds to the dichloride form I of
complex 1b. Addition of 1 equiv. AgBF4 led to a decrease in
intensity of the signal at 26.7 ppm in favor of the signal at

31
Fig. 2. Tendency towards aquation of 1b, 2b, 5 and 6, expressed as the percentage mono-aquated form II observed by P NMR spectroscopy in D2O at RT.
1110 D.J.M. Snelders et al. / Journal of Organometallic Chemistry 696 (2011) 1108e1116

Scheme 1. Synthesis of complexes 1be4b bearing oligocationic phosphine ligands and structures of reference compounds 5 and 6.

31.8 ppm and the appearance of a new signal at 32.3 ppm. After
addition of a second equiv. of AgBF4, the signal at 32.3 remains as
the only peak. Based on comparison with the corresponding pta
complex RAPTA-C (6) [21,24], the signal at 31.8 ppm may be
tentatively assigned to the mono-aquated form II of complex 1b
and the signal at 32.3 to the hydroxo-aqua form III. For compar-
ison, the water stability of 2b and 5 was also investigated and
Fig. 2 shows the percentage of the mono-aquated form II
observed by 31P NMR in D2O, at [NaCl] 0 or 100 mM for each of
the complexes (species of the type III are only observed following
the addition of AgBF4). The hexacationic complex 2b displayed
a remarkably different behavior from that of the tricationic
species 1b. The 31P NMR spectrum in D2O of 2b showed only one
signal, at 26.4 ppm. After standing in solution at room tempera-
ture for 24 h, no change in the spectrum was observed. Addition
of 100 mM NaCl did not lead to any change, indicating that the
observed signal corresponds to the dichoride form I of 2b. Thus,
2b does not undergo aquation in water under the employed
conditions. Titration of 2b with AgBF4 results in the formation of
two doublets centered around 15.2 ppm, with coupling constants
of 799 and 698 Hz, respectively. After addition of 3 equiv. AgBF4,
these signals were the only peaks in the spectrum. This pattern of
signals is characteristic for phosphorus nuclei coordinated to Ag(I)
[25].
In the case of 5, the 31P NMR spectrum in D2O showed two major
signals at 35.2 and 30.1 ppm, in an approximate ratio of 3.5 to 1.
Increasing the concentration of NaCl led to a shift of this ratio in
favor of the signal at 30.1 ppm, indicating that the peak corresponds
to form I of complex 5. A relatively high concentration of NaCl
was required, i.e. 300 mM, for complete disappearance of the
signal at 35.2 ppm. Addition of 1 equiv. AgBF4 led to the disap-
pearance of the signal at 30.1 ppm, leaving the signal at 35.2 as the
only peak in the spectrum, which can be assigned to the mono-
aquated form II. Addition of higher amounts of AgBF4 then led to
the appearance of a new signal at 35.5 ppm, which remained as the
only signal after addition of 3 equiv. AgBF4 and can be tentatively
assigned to the hydroxo-aqua form III.
2.2. DNA binding studies
2.2.1. CD and UV spectroscopy
In order to investigate the interaction between complexes
1be4b and DNA each compound was incubated for 24 h at room
temperature with calf-thymus DNA (ctDNA) at various ratios
(r compound:DNA base pairs 0.1, 0.2, 0.3, 0.4 and 0.5). In
addition, the Ru analogues bearing trianionic tppts (5) and pta (6),
as well as the free phosphine ligands 1ae4a were studied. After
incubation, the ctDNA-complex mixtures were analyzed by CD
spectroscopy and compared to the spectrum of ctDNA alone. Fig. 3
shows representative CD spectra obtained for 1be4b (the CD
spectra of the other compounds are provided in the Supporting
Information, Figures S4eS8). Polyvalent cations are known to

Scheme 2. Aquation/hydrolysis of complexes of the type Ru(p-cymene)Cl2(L) (L phosphine ligand).

 D.J.M. Snelders et al. / Journal of Organometallic Chemistry 696 (2011) 1108e1116
Fig. 3. CD spectra of calf-thymus DNA following incubation with complexes 1be4b in 20 mM phosphate buffer (pH 7.4). r compound:DNA base pairs 0, 0.1, 0.3 and 0.5.
bind strongly to DNA and can induce DNA condensation or
precipitation in vitro as a result of phosphate charge neutralization
[26]. Therefore, to monitor possible DNA precipitation, the UV
absorption at 260 nm, which is directly related to the concentration
of DNA in solution, was also measured for each CD sample. Fig. 4
shows the percentage of ctDNA remaining in solution after incu-
bation with the various complexes and ligands in comparison to
cisplatin treated DNA.
The CD spectrum of untreated ctDNA shows a positive band
around 275 nm and a negative band at about 245 nm [26]. After
addition of the tricationic complex 1b, a signicant decrease in
intensity of the positive CD band at 275 nm was observed at r 0.5,
along with a decrease in the UV absorption at 260 nm. Increasing
r up to 1.5 did not lead to further changes in the CD spectrum.
Similar effects were observed for 4b. For the hexacationic
complexes 2b and 3b, only a small change was observed in the CD
spectrum at r 0.1, but a nearly complete loss of both the CD signal
and the UV absorption at 260 nm was observed at r 0.5 in both
Fig. 4. Percentage of ctDNA remaining in solution after incubation with compounds
1e6 or cisplatin at r 0.5, in 20 mM phosphate buffer, measured by UV absorption at
260 nm.
1111
cases. The ctDNA samples incubated with free ligands 1ae4a
afforded nearly identical CD spectra and very similar UV absorption
patterns compared with 1be4b (Fig. 4 and Figs. S4eS7). The cor-
responding Ru complex of the trianionic ligand tppts (5) and that
of pta (6) did not lead to any change in the CD spectrum of ctDNA
(Figure S8), nor did these species inuence the UV absorption at
260 nm (Fig. 4). Cisplatin does not lead to any change in the UV
absorption (Fig. 4), but it is known to signicantly alter the CD
spectrum of DNA [27]. The interaction of the hexacationic ligands
2a and 3a with ctDNA at increments of r 0.1 up to r 0.5 was
studied. For all the solutions, CD spectra and UV absorption at
260 nm were measured (Fig. 5, Figs. S5 and S6). For 2a, not much
change is observed in either the CD spectrum or the UV absorption
up to r 0.3. At r 0.4, however, an abrupt decrease in the UV
absorption is observed with concomitant collapse of the CD spec-
trum. For 3a, the CD spectrum gradually changes with an increasing
r. The UV absorption shows a similar abrupt change as seen for 2a,
but it occurs at a higher value of r, i.e. 0.5.
Fig. 5. Percentage DNA remaining in solution after incubation with compounds 2a or
3a at r 0.1e0.5 in 20 mM phosphate buffer, measured by UV absorption at 260 nm
r molar ratio of compound vs. DNA base pairs.
1112 D.J.M. Snelders et al. / Journal of Organometallic Chemistry 696 (2011) 1108e1116

Fig. 6. Gel electrophoresis of pBR322 plasmid DNA treated with 1e6 in compound to DNA base pair ratios (r) of 0.1 and 0.05. B blank (DNA only).

2.2.2. Gel electrophoresis assays (A2780cisR), using the MTT assay (see Experimental Section and
The interaction of 1ae4a, 1be4b, 5 and 6 with DNA was further Table 1).
studied by evaluating the electrophoretic mobility of plasmid Among the cationic complexes, only the trication 1b displayed
pBR322 DNA on agarose gels after incubation for 24 h at 37  C with a moderate cytotoxic effect towards the cell line A2780. The hex-
the compounds (compound:DNA base pair ratio r 0.1 and 0.05) acationic analogue 2b and the dendritic ligand 4a, as well as co-
in comparison to cisplatin treated DNA (Fig. 6). Plasmid DNA exists mplex 4b, are somewhat cytotoxic against the cisplatin-resistant
as two main forms, a supercoiled (SC) form, which migrates fastest cell line. In both cell lines cisplatin is considerably more cytotoxic
(lowest band in Fig. 6) and an open circular form (OC) which than any of the ruthenium complexes.
migrates more slowly. Changes in the mobility of these bands are
generally interpreted as evidence for the occurrence of DNA 3. Discussion
binding [27e30].
In general, at r 0.1, most of the cationic ligands and 3.1. Water stability of the complexes
complexes led to a slight retardation of both the SC and the OC
bands along with some increase in intensity of the OC band and The extent of aquation of the complexes studied herein follows
a decrease in intensity of the SC band. For cisplatin, a more the trend 5 > 6 > 1b >> 2b. The phosphine ligand has a large
pronounced retardation of the SC band is observed. For the free inuence on the positions of the equilibria depicted in Scheme 2,
ligand 3a, the effect was somewhat more pronounced than that i.e. the tendency of the Ru-phosphine complex to undergo aqua-
observed for the corresponding complex 3b. For 2a and 2b, tion. There is no clear correlation between the extent of aquation
dramatic effects were observed, involving extensive retardation of these complexes and the basicity of the corresponding phos-
of both bands. In contrast, for the anionic complex 5 and RAPTA-C phine ligands, which have the following order of decreasing
6, no signicant effects were observed. At r 0.5, extensive DNA basicity, determined through the frequency of the CO stretching
precipitation occurred in many cases, which prevented DNA vibration in the IR spectrum of complexes of the type trans-RhCl
migration. (CO)L2 (L monodentate phosphine ligand): pta (1963 cm1) [33]
>tppts (1968 cm1) [34] >1a (1971 cm1) [18] >2a (1981 cm1)
2.3. Cytotoxicity studies [18]. With the negatively charged phosphine in 5, extensive and
rapid aquation is observed and at the other extreme, for 2b
The cytotoxicities of 1e5 were determined on the human which contains a hexacationic phosphine ligand, aquation is not
ovarian cancer (A2780) cell line and its cisplatin-resistant variant observed. It is not unreasonable to assume that the ability of
a complex to undergo aquation (which results in a change of the
overall charge of the complex by 1) is lower for positively
Table 1
charged complexes (i.e. 1b and 2b), as this leads to an increase of
IC50 values of 1e5 compared with 6 (RAPTA-C) and cisplatin against human ovarian
carcinoma cell lines sensitive (A2780) and resistant (A2780cisR) to cisplatin. the overall positive charge of the complex, which contrasts
with the anionic complex, i.e. 5, which leads to a decrease in the
IC50 (mM)
overall charge.
Compound A2780 A2780cisR
1a >1000 >1000 3.2. DNA interactions of oligocationic phosphines and their Ru
1b 241 >1000
2a >1000 >1000
complexes
2b >1000 247
3a >1000 >1000 Circular dichroism spectroscopy is widely used to study the
3b >1000 >1000 solution structure of DNA and the conformational changes that
4a >1000 211
occur upon ligand binding [11,26,27,35e37]. The CD spectrum of
4b >1000 169
5 >1000 >1000 the normal form of DNA (B-DNA) is characterized by a positive
6a 353 n.d. band around 275 nm and a negative band around 245 nm [26].
Cisplatinb 4.3 18.2 After incubation of ctDNA with the tricationic complex 1b, or the
a
Ref. [31]. corresponding free ligand 1a, a signicant decrease in intensity of
b
Ref. [32]. the positive CD band at 275 nm was observed at r 0.5. This
 D.J.M. Snelders et al. / Journal of Organometallic Chemistry 696 (2011) 1108e1116
suggests that a signicant interaction occurs between the DNA
and 1a and 1b and that the DNA undergoes some conformational
changes due to this interaction. The UV absorption at 260 nm is
slightly decreased indicating that DNA precipitation has taken
place. In the case of the hexacationic species 2a, 2b, 3a and 3b,
the UV absorption at 260 nm was almost reduced to zero at
r 0.5, indicating that nearly all of the DNA has precipitated,
explaining the loss of the CD signal in these cases. Similar ob-
servations have been reported upon titration of DNA with cationic
liposomes [37].
In the case of 3a and 3b, a distinct CD spectrum was observed
at r 0.3e0.4, characterized by a collapse of the positive band at
275 nm and a strong increase of the negative band. Such CD char-
acteristics have been observed under a variety of conditions and
correspond to a more highly wound form of B-DNA [35]. It thus
seems that 3a and 3b induce strong conformational changes up to
r 0.3e0.4, while leading to DNA precipitation at r 0.5. For
complexes 1be4b, the observed changes in the CD spectra were
very similar to those observed for the corresponding free ligands
1ae4a. This indicates that the observed conformational changes
are ligand-based, rather than metal-based, and that the interaction
of the cationic complexes and ligands with DNA occurs via elec-
trostatic interactions between the positively charged ammonium
groups in the ligands and the negatively charged phosphate groups
of the DNA.
In contrast, the trianion 5 did not lead to any changes in the
CD spectrum, presumably due to repulsive electrostatic interac-
tions between the anionic sulfonate groups in the tppts ligand
and the phosphate groups of DNA. Addition of RAPTA-C (6) to
DNA did not lead to any spectral change either. Although it is
known that various ruthenium compounds, including 6 [38,39],
bind to DNA, for example via coordinative binding of the Ru ion
to N atoms of the DNA bases [40], these interactions do not alter
the CD spectrum of ctDNA under the employed conditions. This is
in line with previous investigations that showed no signicant CD
spectral change upon addition of several ruthenium compounds
[27].
The DNA precipitation observed upon addition of 2a, 2b, 3a
and 3b most likely occurs due to charge neutralization through
strong electrostatic interactions between the ammonium groups of
these hexacationic species and the phosphate groups of DNA [26].
It is known that multivalent cation-induced DNA condensation
occurs when 90% of the DNA phosphate charge is neutralized by
cation binding [41]. Thus, the ability of 2a and 3a to neutralize the
phosphate charges even at a considerable ionic strength indicates
a strong interaction with DNA. The slightly higher ratio needed
for 3a compared with 2a might be due to an increased steric
hindrance of 3a due to the added benzyl substituents. Similarly,
the relatively weak interaction of 4a and 4b with DNA can be
rationalized by their large size and the buried position of the
ammonium groups within the dendritic structure. In the case of 1a
and 1b the weaker DNA binding, compared with 2a and 2b, may
be attributed to the presence of only three positive charges in
these species.
The plasmid DNA gel electrophoresis mobility studies are
generally in accordance with the CD spectroscopy studies and
showed that the cationic complexes, as well as their correspond-
ing ligands, interact with DNA. Most of the compounds promoted
the conversion of supercoiled (SC) DNA into its relaxed open
circular form (OC), along with a slight decrease in mobility for
both forms, although in most cases the effects are less pronounced
than for cisplatin. In general the ruthenium complexes were
equally or slightly less effective than the corresponding free
ligands. This is not unexpected due to the low ability of the
cationic complexes to undergo aquation. For the hexacationic
1113
compounds 2a and 2b, a dramatic effect of the DNA mobility was
observed, indicating a strong interaction with the DNA. In line
with the CD studies, the tricationic 1aeb and the dendritic
systems 4aeb showed much smaller effects, while anionic 5 and
neutral 6 did not interact with the DNA under the conditions. The
interaction of 6 is known to depend on the pH of the solution and
occurs only at pH < 7 [29].
3.3. Cytotoxicity studies
In general all the ligands and complexes are only slightly
cytotoxic or are not cytotoxic to the human ovarian cancer cell
lines A2780 and A2780cisR. Among the class of compounds of the
type Ru(p-cymene)Cl2(L) (L 1e5), only the smallest, tricationic
complex 1b is moderately active against the A2780 cell line,
while only the hexacationic complexes 2b and 4b and the ligand
4a are active against the A2780cisR cell line. The cytotoxicity
values for these compounds fall in the range commonly observed
for various Ru(h6-arene)-type complexes [31,42,43]. Since the
compounds interact strongly with DNA it seems likely that they
do not reach the DNA in vitro. This may be due to reduced
cellular uptake although various studies show that highly charged
(cationic) ruthenium compounds, albeit based on multiple
ruthenium centers, can enter cells and are cytotoxic [44e46]. It
should be noted that despite having modest cytotoxicities
a number of ruthenium complexes are highly efcient in vivo
[22,47,48].
4. Conclusions
A series of ruthenium(II)-arene complexes with charged
monodentate phosphine ligands were prepared and studied. The
complexes with charged cationic ligands are quite resistant to
aquation and they appear to bind strongly with DNA, mainly via
electrostatic interactions. Depending on the number of charges as
well as on the steric effects of the substituents at the ammonium
groups in the phosphine ligands, the DNA binding afnity of the
complexes follows the order 2b > 3b > 1b > 4b. Despite forming
strong interactions with DNA, the compounds display only
modest cytotoxic effects on ovarian cancer cells. These studies
highlight that no single mechanism of action can be attributed to
ruthenium(II)-arene compounds [49] and each compound must
be considered separately. The cationic phosphine ligands studied
herein may be regarded as effective agents for linking metal ions
to DNA and as such they might nd application in other elds.
For example, these systems might be of interest in the nascent
eld of DNA-based hybrid catalysts for asymmetric synthesis
[50,51].
5. Experimental section
[RuCl2(p-cymene)]2 [52], 1ae4a [18], 5 [53] and 6 [29] were
synthesized according to published methods. NMR spectra were
measured on a Bruker Avance II 400 spectrometer at 25  C unless
stated otherwise. 1H and 13C{1H} NMR spectra were referenced to
residual solvent signals. 31P NMR chemical shifts are given relative
to 85% H3PO4. UV/Vis spectra were recorded on a Jasco V-550
spectrophotometer. Elemental analyses were obtained in-house
using a Thermo Fischer Scientic Flash 2000 Organic Elemental
Analyzer. ElectroSpray-Ionisation MS data were acquired on a Q-Tof
Ultima mass spectrometer (Waters) tted with the LockSprayTM
dual electrospray ion source and operated in positive and ionization
mode.
1114 D.J.M. Snelders et al. / Journal of Organometallic Chemistry 696 (2011) 1108e1116
5.1. General procedure for the synthesis of complexes Ru(p-cymene)
Cl2(L) (1be4b)
Phosphine ligands 1ae4a and 0.50 equiv. of [Ru(p-cymene)Cl2]2
were dissolved in deoxygenated CH3CN (3e5 mL) and stirred at
60  C for 1.5 h under an inert atmosphere. The solvent was evap-
orated in vacuo affording the products 1be4b as orange powders.
5.2. 1b
Starting materials: 1a (84.2 mg, 0.114 mmol) and [Ru(p-
cymene)Cl2]2 (33.7 mg, 55.0 mmol, i.e. 0.110 mmol Ru). Yield: 118 mg
(quant.). 1H NMR (400.1 MHz, CD3CN): d (ppm) 7.95 (dd,
3
JH,H 9.0 Hz, 3JP,H 9.0 Hz, 6H, o-Ar), 7.55 (d, 3JH,H 7.0 Hz, 6H, m-
Ar), 5.31 (d, 3JH,H 6.2 Hz, 2H, 2-Ar), 5.25 (d, 3JH,H 5.8 Hz, 2H, 3-Ar),
4.46 (s, 6H, CH2), 3.04 (s, 27H, N(CH3)3), 2.60 (sept, 3JH,H 6.9 Hz,1H),
1.85 (s, 3H), 0.99 (d, 3JH,H 6.9 Hz, 6H, C(CH3)2). 13C{1H} NMR
(100.6 MHz, CD3CN): d (ppm) 137.2 (d, 1JP,C 43.5 Hz, i-Ar), 136.0
(d, JP,C 9.8 Hz, Ar), 133.2 (d, JP,C 9.9 Hz, Ar), 130.9 (d, 4JP,C 2.4 Hz,
p-Ar),111.0 (s,1-Ar), 97.5 (s, 4-Ar), 90.9 (s, 2-Ar), 88.3 (s, 3-Ar), 69.2 (s,
CH2), 53.4 (s, N(CH3)3), 31.2 (s, C(CH3)2), 21.9 (s, C(CH3)2), 17.9 (s, Me).
31 1
P{ H} NMR (162.0 MHz, CD3CN): d (ppm) 24.7 (s). Elem. anal. for
C40H58B3Cl2F12N3PRu (1044.27): calc. (%) C 46.01, H 5.60, N 4.02;
found C 46.01, H 5.25, N 4.31.
5.3. 2b
Starting materials: 2a (56.5 mg, 46.5 mmol) and [Ru(p-cym-
ene)Cl2]2 (14.2 mg, 23.2 mmol, i.e. 46.4 mmol Ru). Yield: 71 mg
(quant.). 1H NMR (400.1 MHz, CD3CN): d (ppm) 8.29 (d,
3
JP,H 9.6 Hz, 6H, o-Ar), 7.79 (s, 3H, p-Ar), 5.37 (d, 3JH,H 5.1 Hz, 2H,
2-Ar), 5.28 (d, 3JH,H 5.2 Hz, 2H, 3-Ar), 4.40 (s, 12H, CH2), 3.02
(s, 54H, N(CH3)3), 2.43 (sept, 3JH,H 6.8 Hz, 1H), 1.84 (s, 3H), 1.03
(d, 3JH,H 7.7 Hz, 6H, C(CH3)2). 13C{1H} NMR (100.6 MHz, CD3CN):
d (ppm) 140.9 (d, 3JP,C 10.4 Hz, o-Ar), 140.3 (s, p-Ar), 137.3
(d, 1JP,C 43.7 Hz, i-Ar), 130.0 (d, 2JP,C 10.0 Hz, m-Ar), 112.4 (s, 1-
Ar), 99.0 (s, 4-Ar), 90.3 (s, 2-Ar), 88.0 (s, 3-Ar), 68.8 (s, CH2), 53.4
(s, N(CH3)3), 31.3 (s, C(CH3)2), 21.9 (s, C(CH3)2), 18.5 (s, Me). 31P{1H}
NMR (162.0 MHz, CD3CN): d (ppm) 24.9 (s). Elem. anal. for
C52H89B6Cl2F24N6PRu (1522.08): calc. (%) C 41.03, H 5.89, N 5.52;
found C 41.21, H 5.99, N 5.52.
5.4. 3b
Starting materials: 3a (33.0 mg, 19.7 mmol) and [Ru(p-cym-
ene)Cl2]2 (6.0 mg, 9.8 mmol, i.e. 19.6 mmol Ru). Yield: 39 mg (quant.).
1
H NMR (400.1 MHz, CD3CN): d (ppm) 8.39 (d, 3JP,H 10.0 Hz, 6H,
o-Ar), 7.76 (s, 3H, p-Ar), 7.53 (m, 30H, Ph), 5.40 (d, 3JH,H 5.8 Hz, 2H,
2-Ar), 5.15 (d, 3JH,H 5.9 Hz, 2H, 3-Ar), 4.43 (12H, NCH2), 4.38 (12H,
NCH2), 2.81 (s, 36H, N(CH3)2), 2.25 (sept, 3JH,H 7.0 Hz, 1H), 1.76
(s, 3H, CH3), 0.90 (d, 3JH,H 6.9 Hz, 6H, C(CH3)2). 13C{1H} NMR
(100.6 MHz, CD3CN): d (ppm) 141.3 (d, 2JP,C 8.6 Hz, o-Ar),
140.8 (s, p-Ar), 134.2 (s, Ph), 131.8 (s, Ph), 130.2 (s, Ph), 129.8
(d, 3JP,C 11.1 Hz, m-Ar), 128.0 (s, Ph), 111.6 (s, 1-Ar), 98.5 (s, 4-Ar),
91.3 (s, 2-Ar), 87.3 (s, 3-Ar), 69.0 (s, NCH2), 68.4 (s, NCH2), 49.4 (s, N
(CH3)2), 31.2 (s, C(CH3)2), 21.9 (s, C(CH3)2), 18.4 (s, Me). 31P{1H}
NMR (162.0 MHz, CD3CN): d (ppm) 24.2 (s). HR-ESI MS: (m/z)
902.3638 {[M-2BF4]2, calc. 902.3677}.
5.5. 4b
Starting materials: 4a (40.0 mg, 7.28 mmol) and a stock solu-
tion of [Ru(p-cymene)Cl2]2 (0.92 mL; 2.36 mg/mL in deoxygenated
CH3CN; 3.54 mmol, i.e. 7.09 mmol Ru). Yield: 42 mg (quant.). 1H NMR
(400.1 MHz, CD3CN): d (ppm) 8.36 (br. s, 6H, o-Ar), 7.72 (br. s, 3H, p-
Ar), 7.4e7.2 (br. m, 120H, Ph), 6.7e6.5 (br. m, 42H, overlapping Ar0,
Ar00 ), 6.48 (s, 12H, o-Ar0 ), 5.34 (br. s, 2H, 2-Ar), 4.90 (br. s, 74H, over-
lapping OCH2, 3-Ar), 4.31 (br. s, 12H, NCH2Ar0 ), 4.20 (br. s, 12H,
NCH2Ar), 2.71 (br. s, 36H, N(CH3)2), 1.76 (s, 3H, CH3), 0.81 (br. s, 6H, C
(CH3)2) (signal for CH(CH3)2 not resolved). 13C{1H} NMR (100.6 MHz,
CD3CN): d (ppm) 161.0, (s, m-Ar00 ), 161.0 (s, m-Ar0 ), 140.1 (s, o-Ar0 ),
138.0 (s, o-Ar00 ), 129.8 (s, Ph), 129.4 (s, Ph), 128.9 (s, Ph), 128.6 (s, Ph),
113.0 (s, p-Ar0 ), 107.5 (s, p-Ar00 ), 105.2 (s, i-Ar0 ), 102.2 (s, i-Ar00 ), 70.6 (s,
OCH2), 69.2 (br. s., NCH2), 68.4 (br. s, NCH2), 49.6 (s, N(CH3)2), 30.8 (s,
C(CH3)2), 21.8 (br. s, C(CH3)2), 18.3 (s, Me) (signals for Ar not
resolved). 31P{1H} NMR (162.0 MHz, CD3CN): d (ppm) 23.8 (s).
Elem. anal. for C340H329B6Cl2F24N6O36PRu (5799.04): calc. (%) C
70.42, H 5.72, N 1.45; found C 70.15, H 6.03, N 1.44.
5.6. Aquation studies
Solutions of 1b, 2b and 5 in D2O (10e20 mM) were analyzed
by 31P NMR spectroscopy. Subsequently, aliquots of stock solutions
 D.J.M. Snelders et al. / Journal of Organometallic Chemistry 696 (2011) 1108e1116 1115

of NaCl or AgBF4 in D2O were added and the resulting mixtures Appendix. Supplementary material
were shaken at room temperature for 1 min, followed by analysis
by 31P NMR spectroscopy. The concentrations of NaCl amounted to Supplementary data related to this article can be found online
4, 100, 200 or 300 mM and the concentrations of AgBF4 amounted at doi:10.1016/j.jorganchem.2010.11.025.
to 1, 2 or 3 equiv with respect to the Ru complex.

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