Efficient oxidation of cysteine and glutathione catalyzed by a dinuclear areneruthenium trithiolato anticancer complex

Article abstract of DOI:10.1021/ic201941j

The highly cytotoxic diruthenium complex [(p-MeC₆H ₄Prⁱ)₂Ru₂(SC₆H ₄-p-Me)₃]⁺ (1), water-soluble as the chloride salt, is shown to efficiently catalyze oxidation of the thiols cysteine and glutathione to give the corresponding disulfides, which may explain its high in vitro anticancer activity.

Full text of DOI:10.1021/ic201941j

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Efficient Oxidation of Cysteine and Glutathione Catalyzed by a
Dinuclear Areneruthenium Trithiolato Anticancer Complex†
Federico Giannini,‡,§ Georg S€uss-Fink,*,§ and Julien Furrer*,‡
‡Departement f€ur Chemie und Biochemie, Universit€at Bern, CH-3012 Bern, Switzerland
§Institut de Chimie, Universitꢀe de Neuch^atel, CH-2000 Neuch^atel, Switzerland
S Supporting Information
b
we had found to be highly cytotoxic for human ovarian cancer
ABSTRACT: The highly cytotoxic diruthenium complex
[(p-MeC6H4Pri)2Ru2(SC6H4-p-Me)3]+ (1), water-soluble
as the chloride salt, is shown to efficiently catalyze oxidation
of the thiols cysteine and glutathione to give the correspond-
ing disulfides, which may explain its high in vitro anticancer
activity.
cells (with IC50 values in the nanomolar range for both the cell
line A2780 and cisplatin-resistant line A2780 CisR),13 to act as an
efficient catalyst for oxidation of the thiols cysteine (Cys) and
glutathione (GSH).
Complex 1 has been synthesized as previously described12 and
characterized by standard spectroscopic methods. The stability
1
of 1 was assessed by recording H NMR spectra over 24 h at
37 °C. The spectra show (Figure S1 in the SI) that 1 is absolutely
stable in D2O, dimethyl sulfoxide (DMSO)-d6, D2O/DMSO-d6
(95:5), with or without the addition of 100 mM NaCl (close to
extracellular [Clꢀ]).
rganometallic complexes offer aspects for medicinal chem-
O
istry that are not available with organic drugs, in particular
because of their coordination and redox properties.1 For this
reason, metal-based pharmaceuticals have found a steadily in-
creasing interest ever since the discovery of the anticancer activity
of cis-Pt(NH3)2Cl2 (cisplatin).2 The quest for alternative anti-
cancer drugs, particularly stimulated by the serious side effects of
platinum-based cancer therapies,3 resulted in a variety of cyto-
toxic organometallics, of which areneruthenium complexes oc-
cupy a prominent position because of their unique combination
of lipophilic and hydrophilic properties.4
The mode of action by which areneruthenium complexes exert
their antitumoral or antimetastatic effects is not yet fully under-
stood. By analogy with platinum complexes, it was originally
expected that DNA binding was also the main reason for the
anticancer activity of ruthenium complexes, but serum proteins
have also been discussed as possible targets.5 While the ability of
ruthenium to bind to DNA has been demonstrated,6 in particular
for areneruthenium ethylenediamine complexes,7 it was ob-
served that DNA binding of ruthenium was weaker and different
from that observed for platinum.8 These findings suggest differ-
ent modes of action depending on the type of complexes. Thus,
the RAPTA-type areneruthenium complexes (Chart S1 in the
Supporting Information, SI), originally designed to improve the
aqueous solubility,9 have been found to target thioredoxin
reductase and cathepsin B, proteins that act as enzymes in the
cells.10
The interactions between 1 and glucose, all amino acids, and
nucleotides were monitored by NMR spectroscopy, and, surpris-
1
ingly, only Cys and GSH induced changes in the H and 13C
NMR spectra compared to the free components. Figure 1 shows
1
the H NMR spectra obtained upon titration of Cys into a
solution of 1 in D2O with 50 mM NaCl. In agreement with the
1H NMR spectrum of free Cys, the addition of 1 equiv of Cys
resulted in the appearance of new Cys resonances at δ 4.17 (Hα),
3.46 (Hβ0), and 3.27 (Hβ). These new 1H signals increased over
time, relative to the signals of free Cys, and, in agreement with the
literature, were assigned as arising from cystine.14
The addition of 1 equiv of GSH resulted in the appearance of
new Cys resonances at δ 4.78 (Hα), 3.33 (Hβ0), and 3.04 (Hβ)
(Figure 2). As for Cys, these new signals increased over time,
relative to the signals of free GSH, and, in agreement with the
literature, were assigned as arising from oxidized glutathione
(GSSG).15
Interestingly, titrations with Cys and GSH revealed that only
the 1H and 13C chemical shifts of the α-CH and β-CH2 groups of
Cys were affected, whereas the chemical shifts of the complex and
the other amino acids remained unperturbed (Figures 2 and 3
and S2 in the SI). These results strongly suggest that Cys and
GSH do not form stable adducts with 1, which was further
evidenced by DOSY spectra (Figure S3 in the SI). From these 1H
NMR titration experiments, it became apparent that 1 can act as a
very efficient catalyst for oxidation of Cys to cystine and of GSH
to GSSG:
Another mode of action has been found for areneruthenium
iodoazopyridine complexes, which are surprisingly cytotoxic
despite their inertness to ligand substitution: In a pioneering
study, Sadler and co-workers demonstrated these complexes to
act as catalysts for oxidation of the tripeptide glutathione,
supposed to be at the origin of their anticancer activity.11
In this paper, we report the p-cymene p-toluenethiolato
derivative [(p-CH3C6H4Pri)2Ru2(SC6H4-p-CH3)3]+ (1; Chart 1 )
of the dinuclear complex family [(arene)2Ru2(SR)3]+,12 which
1
RSH þ RSH
s
RSꢀSR þ H
ð1Þ
f
2
In order to evaluate the catalytic performance of 1, we
1
followed the H NMR spectra of mixtures of 1 with Cys and
GSH with the ratio 1:100, respectively, over time. The catalytic
Received: September 5, 2011
Published: October 07, 2011
r
2011 American Chemical Society
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Inorganic Chemistry
COMMUNICATION
Chart 1. Complex 1a
Figure 2. 1H NMR spectra of GSH (top), 1 (middle), and a mixture of
1 and GSH (ratio 1:1, bottom) recorded at 37 °C in D2O/DMSO-d6
(95:5) after 2 h of incubation. The resonances of Cys (GSH) are
indicated by * and the resonances of cystine (GSSG) by +. The residual
water signal is visible around 4.7 ppm.
aThe counteranion is Clꢀ.
Figure 1. 1H NMR spectra of Cys (top), 1 (middle), and a mixture of 1
and Cys (ratio 1:1, bottom) recorded at 37 °C in D2O/DMSO-d6 (95:5)
after 2 h of incubation. The resonances of Cys are indicated by * and the
resonances of cystine by +. The residual water signal is visible around
4.7 ppm.
Figure 3. Turnover number (TON) for mixtures of 1/Cys (2) and
1/GSH (b) (ratio 1:100) in D2O at 37 °C with 50 mM NaCl under
argon and 1/Cys (4) and 1/GSH (O) under the same reaction
conditions as those above but under O2.
conversion of GSH to GSSG may be directly related to the
anticancer activity. Cancer cells are known to have a higher GSH
pool than healthy cells, and in all living cells, more than 90% of
the total GSH pool is in the reduced form (GSH) and less than
10% exists in the disulfide form (GSSG). An increased GSSG-to-
GSH ratio is considered to be indicative of oxidative stress, which
damages all components of the cell, including proteins, lipids,
Table 1. TOFs after 50% Conversion (TOF50) for Oxidation
of Cys to Cystine and of GSH to GSSG with 1 as a Catalyst
(the Values Are Given per Ruthenium Atom)
1
and DNA and which may lead to apoptosis.16 For Cys, the H
NMR spectra showed that the reaction led to a steady oxidation
of Cys to cystine after only 14 h, which led to a turnover
frequency after 50% conversion (TOF50) of 8.1 hꢀ1 (Figure 3).
We point out here that the catalytic reaction of complex 1 could
also be performed using a ratio of 1:1000. The reaction led to a
TOF50 of 80.9 hꢀ1 (Figure S5 in the SI). Steady oxidation was
evidenced by the complete disappearance of the original reso-
nances of Cys at δ 4.03 (Hα), 3.17 (Hβ0), and 3.09 (Hβ)
(Figure 2). The formation of cystine during the reaction was
further confirmed by electrospray ionization mass spectrometry
(ESI-MS) spectra (Figure S6 in the SI).
TOF50/hꢀ1 at 37 °C
pD = 4, 50 mM pD = 7, 50 mM pD = 10, 50 mM pD = 7, 4 mM
NaCl
NaCl
NaCl
NaCl
Cys
8.1
7
8.1
7.4
7.2
6.9
6.5
6
GSH
complex 1 is about 1 order of magnitude higher than TOF
obtained for iodo-containing ruthenium(II) arene organometal-
lic derivatives (0.37 hꢀ1).11 In addition, incubation of 10 mM
GSH with these complexes led to steady oxidation of only
4.6 mM GSH to GSSG, whereas oxidation is complete for
complex 1. Moreover, complex 1 is stable during the reactions
and can be recovered unchanged as the chloride salt, as shown by
1H NMR spectra of the reaction of 1 with Cys and GSH recorded
at t = 0 and 24 h (Figures S8 and S9 in the SI). To test the stability
of the catalyst, 100 equiv of Cys was oxidized, and the reaction
was reiterated five times using the same catalyst (Figure S10 in
For GSH, the 1H NMR spectra revealed steady oxidation of
GSH to GSSG after 16 h (Figure 3), with TOF50 being 7.4 hꢀ1
(Table 1). Steady oxidation was also evidenced by the complete
disappearance of the original resonances of Cys in GSH at δ 4.61
(Hα), 3.02 (Hβ0), and 2.96 (Hβ) (Figure 2). Formation of
GSSG during the reaction was further confirmed by ESI-MS
spectra (Figure S7 in the SI). It is worth pointing out that, for an
identical complex/GSH ratio (1:100), TOF50 obtained for
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COMMUNICATION
the SI). The stability of 1 is remarkable, and TOF50 drops by only
15% after the fifth run.
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cell lines. Supramolecular enzyme inhibition, although unlikely,
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’ ASSOCIATED CONTENT
S
Supporting Information. Analytical details. This materi-
b
al is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: georg.suess-fink@unine.ch (G.S.-F.), julien.furrer@
dcb.unibe.ch (J.F.).
Author Contributions
†The manuscript was written through the contributions of all
authors. All authors have given approval to the final version of the
manuscript.
’ ACKNOWLEDGMENT
J.F. thanks the Swiss National Science Foundation (Grant
200021-131887) for financial support.
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