Available online at www.sciencedirect.

com
JOURNAL OF
Inorganic
Biochemistry
Journal of Inorganic Biochemistry 102 (2008) 564575
www.elsevier.com/locate/jinorgbio

Gold(III) compounds as anticancer agents: Relevance of

goldprotein interactions for their mechanism of action
Angela Casini a,*, Christian Hartinger b, Chiara Gabbiani a, Enrico Mini c, Paul J. Dyson b,
Bernard K. Keppler d, Luigi Messori a,*
a
Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy
b
Institut des Sciences et Ingenierie Chimiques, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
c
Department of Pharmacology, University of Florence, Florence, Italy
d
Institute of Inorganic Chemistry, University of Vienna, A-1090 Vienna, Austria

Received 2 July 2007; received in revised form 1 October 2007; accepted 18 October 2007
Available online 28 November 2007

Abstract

Gold(III) compounds constitute an emerging class of biologically active substances, of special interest as potential anticancer agents.
During the past decade a number of structurally diverse gold(III) complexes were reported to be acceptably stable under physiological-
like conditions and to manifest very promising cytotoxic eects against selected human tumour cell lines, making them good candidates
as anti-tumour drugs. Some representative examples will be described in detail. There is considerable interest in understanding the precise
biochemical mechanisms of these novel cytotoxic agents. Based on experimental evidence collected so far we hypothesize that these
metallodrugs, at variance with classical platinum(II) drugs, produce in most cases their growth inhibition eects through a variety of
DNA-independent mechanisms. Notably, strong inhibition of the selenoenzyme thioredoxin reductase and associated disregulation
of mitochondrial functions were clearly documented in some selected cases, thus providing a solid biochemical basis for the pronounced
proapoptotic eects. These observations led us to investigate in detail the reactions of gold(III) compounds with a few model proteins in
order to gain molecular-level information on the possible interaction modes with possible protein targets. Valuable insight on the for-
mation and the nature of goldprotein adducts was gained through ESI MS (electrospray ionization mass spectrometry) and spectropho-
tometric studies of appropriate model systems as it is exemplied here by the reactions of two representative gold(III) compounds with
cytochrome c and ubiquitin. The mechanistic relevance of gold(III)-induced oxidative protein damage and of direct gold coordination to
protein sidechains is specically assessed. Perspectives for the future of this topics are briey outlined.
2007 Elsevier Inc. All rights reserved.

Keywords: Gold(III) complexes; Anticancer agents; Proteins; ESI mass spectrometry; Mechanism of action

1. Novel gold(III) compounds as anticancer agents:
structurally diverse compounds with outstanding
antiproliferative properties
The discovery of the anticancer properties of cisplatin
during the 1960s triggered a great deal of interest in the
eld of anti-tumour metallodrugs [1]. The very favourable
*
Corresponding authors. Fax: +39 055 457 3385.
E-mail addresses: angela.casini@uni.it (A. Casini), luigi.messori@
uni.it (L. Messori).
pharmacological prole of cisplatin suggested that other
metal-based compounds might pairwise manifest impor-
tant anti-tumour eects while (ideally) exhibiting a dierent
spectrum of biological activities and a lower systemic tox-
icity [2,3]. As d8 Au(III) complexes are isoelectronic and
isostructural with Pt(II) complexes, square planar gold(III)
compounds soon appeared to be excellent candidates for
anticancer evaluation. However, at variance with plati-
num(II) compounds, gold(III) analogues were found to
manifest, on the whole, a rather poor stability prole being
kinetically more labile than the corresponding platinum(II)

0162-0134/$ - see front matter 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2007.11.003
 A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575
compounds, light-sensitive and easily reducible to metallic
gold. As a result of these diculties and, also, of detection
of important systemic toxicity in the course of the rst ani-
mal studies, gold(III) compounds were quickly abandoned.
Nonetheless, during the 1990s, renewed interest for
anticancer gold(III)-based compounds emerged place,
especially when a few novel gold(III) complexes, exhibit-
ing improved stability, lower toxicity and favourable
in vitro pharmacological properties, were made available
for pharmacological testing [4]. For instance, a series of
organogold(III) DAMP compounds (DAMP = 2-[(dimeth-
ylamino)methyl]phenyl) were developed by Buckley et al.,
and screened for anti-tumour activity with encouraging
results in vitro [5,6]. Further these compounds demon-
strated moderate activity in laboratory animals [5,6].
Later on, in the attempt of nding new biologically
active substances with an even better stability prole, some
classical square planar gold(III) complexes, based on a
variety of structurally dierent ligands, were prepared
and characterised in our laboratory [7]. To improve the sta-
bility of the gold(III) center, polydentate ligands such as
polyamines, cyclam, terpyridine and phenathroline were
preferentially employed. A few compounds, namely
[Au(en)2]Cl3 1, [Au(dien)Cl]Cl2 2, [Au(cyclam)](ClO4)2Cl
3, [Au(terpy)Cl]Cl2 4, and [Au(phen)Cl2]Cl 5, (Fig. 1), were
characterised both in the solid state and in solution [8]. On
the whole, a quite satisfactory stability prole in solution
emerged for all these gold(III) compounds that opened
the way to their in vitro pharmacological testing. This
means that the donor set comprising three or four nitrogen
atoms, produces a strong stabilisation of the gold(III) cen-
ter and a net decrease of the reduction potential, thus pre-
venting gold(III) reduction and hydrolysis.
NH2 3+
NH2
NH
Au
3Cl- Au
NH2
NH2 NH2
Cl
1 2
2+
N N
N Au Cl
2Cl
N N
4 5
Fig. 1. Schematic drawing of (1) [Au(en)2]Cl3, (2) [Au(dien)Cl]Cl2, (3) [Au(cyclam)](ClO4)2Cl, (4) [Au(terpy)Cl]Cl2, and (5) [Au(phen)Cl2]Cl.
565
The antiproliferative properties of these gold(III)poly-
amine were, then, measured by the sulforhodamine B assay
on the representative human ovarian tumour cell line
A2780, either sensitive (A2780/S) or resistant (A2780/R)
to cisplatin. In most cases, the above compounds revealed
a high cytotoxicity, with IC50 values generally falling in the
low micromolar region [9], and were also found to over-
come resistance to cisplatin in the cisplatin-resistant line.
Later on, in collaboration with the group of Minghetti
and Cinellu, we characterised and assayed other gold(III)
complexes, bearing the bipyridyl motif [10]. This family of
compounds turned out to display appreciable stability in
solution and to cause relevant tumour growth inhibition
in vitro. In particular, our investigations focused on two
members of this family, namely [Au(bipyc-H)(OH)][PF6] 6
(where bipyc = 6-(1,1-dimethylbenzyl)-2,20 -bipyridine) and
[Au(bipy)(OH)2][PF6] 7 (bipy = bipyridine) (Fig. 2) [9]. A
number of analogues were also prepared and characterised
(see Fig. 2, compounds 8 and 9) [11].
In [Au(bipy)(OH)2][PF6] 7, the gold(III) center is coordi-
nated by two nitrogens of the bidentate bipyridyl ligand and
by two hydroxide groups. At variance, 6 is an organo-
gold(III) complex in which donors to the gold(III) center
are two nitrogens from the bipyridyl moiety, the C2 carbon
of the phenyl group, and a hydroxide group. Only small
deviations from ideal square planar geometry were seen in
the classical bipyridyl complexes whereas such deviations
are quite large in the case of cyclometallated derivatives.
Both mentioned compounds exhibited sucient solubil-
ity in watery solutions. Their intense LMCT visible bands,
lying in the 300370 nm region and diagnostic of gold in
the oxidation state +3, were exploited to monitor reactions
with various kinds of biomolecular targets. Notably, these
3+
2+
NH NH 2ClO4-
Au Cl-
NH2
NH NH
3
Cl
Cl
Au
Cl
566 A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575

+ +
In the same period, Fregona and coworkers prepared
and characterised some interesting gold(III) dithiocarba-
N N CH3 PF6- N N PF6- mate compounds displaying a very encouraging biological
Au Au prole [14]. The compounds containing the N,N-di-
OH CH3
OH OH methyldithiocarbamate and ethylsarcosinedithiocarbamate
ligands (compounds 10 and 11 in Fig. 2) were the most
7
6 intensely studied ones. Early in vivo data on murine models
looked very promising and led to the rapid patenting of
+
these novel compounds. Afterwards, some additional
N N H3COOC N investigations were carried out aimed at elucidating the
CH3 CH3
Au PF6- Au main features of their mechanism of action, both at the cel-
CH3 HN CH3 H3COOC CH3 lular and biochemical level [15,16]. It is worth noting that
very recent results suggest that these gold(III) dithiocarba-
CH3 mate compounds most likely act through inhibition of the
9
8
O cancer cell proteasome [17], a quite novel and unexpected
H3C S X S X
mechanism for anticancer metallodrugs.
C
N C Au Au
Concomitantly, a series of interesting gold(III) meso-tet-
RO CH2 N C
raarylporphyrins complexes were developed by Chi Ming
H3C S X CH3 S X
X = Cl, Br Che, and coworkers at the University of Hong Kong [18].
10 R = CH3CH2 11 These compounds, of general formula [Au(III)(p-Y-
TPP)]Cl, [with Y being H (a), Me (b), OMe (c), Br (d) or
Fig. 2. Schematic drawing of [Au(bipyc-H)(OH)][PF6] (6), [Au(bipy)
Cl (e)] (see Fig. 3), were characterised by classical physio-
(OH)2][PF6] (7), [Au(bipydmb-H)(2.6-xylidine-H)][PF6] (8), [Au(pydmb-H)-
(AcO)2] (9) (where bipydmb = 6-(1,1-dimethylbenzyl)-2,20 -bipyridine); chemical methods [18]. Interestingly, coordination to the
pydmb = 2-(1,1-dimethylbenzyl)-pyridine)) and of the gold(III) dithiocar- tetrapyrrole ring causes a large stabilisation of the gold(III)
bamate complexes containing N,N-dimethyldithiocarbamate (10) and oxidation state leading to its high stability in aqueous solu-
ethylsarcosinedithiocarbamate (11) ligands. tions even in the presence of glutathione (GSH), the most
important intracellular reducing agent.
two complexes revealed a clearly dierent reactivity toward All the mentioned gold(III) porphyrins displayed excel-
ascorbate as only compound 7 was shown to be reduced. lent in vitro antiproliferative eects, with IC50 values of
This nding implies that the oxidation state +3 is far more 0.11.5 lM. The lack of cross-resistance with classical
stable in the case of the organogold(III) species compared platinum(II) compounds again suggests that gold(III) por-
to compound 7, in agreement with electrochemical results phyrins and cisplatin induce cytotoxicity through quite dis-
[12]. tinct mechanisms. Remarkably, a zinc(II) analogue [Zn(II)
The in vitro cytotoxic properties of these bipyridyl (TPP)] was also investigated and found to be at least 100-
Au(III) complexes were measured toward the human ovar- fold less cytotoxic than gold(III) porphyrins (IC50 >
ian carcinoma cell line A2780, either sensitive or resistant
to cisplatin. Both gold(III) complexes showed excellent
cytotoxic eects, with IC50 values staying in the low micro-
Y
molar range [9,10]. Compound 6 turned out to be the most
active with a twofold higher activity than cisplatin in the
A2780/R cell line. The antiproliferative properties of these
complexes were also investigated on the human ovarian cell
line SKOV3 (inherently resistant to cisplatin) and on the
+
CCRF-CEM leukemic cell line, either sensitive (CCRF-
CEM/S) or resistant (CCRF-CEM/R) to cisplatin: signi-
N N
cantly lower activities were measured on all these latter
Y Au Y
lines. In any case, an appreciable activity was retained
N N
toward the cisplatin-resistant A2780/R and CCRF-CEM/
R lines suggesting that the biochemical mechanisms of
resistance to cisplatin most likely a more ecient intracel-
lular detoxication and an increased repair of DNA dam-
age are modestly eective toward these gold(III)
complexes. In addition, independent studies pointed out
quite unambiguously that the interactions of these com-
plexes with nucleic acids are weak whereas stronger Y
adducts are formed in the reactions with model proteins Fig. 3. Gold(III) meso-tetraarylporphyrins complexes. Y = H (a), Me (b),
and serum proteins [13]. OMe (c), Br (d), Cl (e).
 A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575 567

50 lM), thus stressing the crucial role of the gold(III)
center in the biological action. Nonetheless, the porphyrin
ligand was found to be essential for anticancer activity
leading these authors to conclude that the porphyrin ligand
is crucial both in stabilising the Au(III) center and in deliv-
ering the metal to its cellular targets [18].
A quite common strategy in the eld of anticancer
metallodrugs is the design and synthesis of polymetallic
compounds, derived from the fusion of two or more
monometallic molecular fragments. It follows that the spe-
cic reactivity of each metal center in the resulting com-
pound is further controlled by its interactions with the
nearby metal center(s) and by the overall molecular scaf-
fold. Incorporation of two (or more) metal centers within
an extended molecular framework may greatly aect the
overall charge of the resulting polynuclear compound, its
redox properties, the kinetics of hydrolysis, and its specic
reactivity toward biomolecules in comparison to mononu-
clear analogues. Some interesting examples of this strategy
are available for platinum(II) and ruthenium(III) antican-
cer metallodrugs [19,20].
These observations prompted us to prepare novel dinu-
clear gold(III) species to be tested as anticancer agents,
starting from the above described mononuclear gold(III)
bipyridyl complexes. A series of six dinuclear oxo gold(III)
complexes with bipyridyl ligands (Fig. 4), of general for-
mula [Au2(N,N)2(l-O)2][PF6]2 [where N,N = 2,20 -bipyri-
dine (Auoxo1), 4,40 -di-tert-butyl- (Auoxo2), 6-methyl-
(Auoxo3), 6-neopentyl- (Auoxo4), 6-(2,6-dimethylphenyl)-
(Auoxo5), 6,60 -dimethyl-2,20 -bipyridine (Auoxo6)] was thus
prepared and characterised in our laboratory, and their
in vitro antiproliferative properties measured against the
reference A2780 human ovarian carcinoma cell line [21].
While ve compounds of this series manifested only
moderate cytotoxic properties (with IC50 typically falling
in the 1030 lM), the sixth one (Auoxo6), turned out to
be 515 times more active against both cell lines, thus
meriting further investigations. In particular, much atten-
tion was focused on the chemical and structural reasons
for the higher biological activity of Auoxo6. Remarkably,
a rather evident positive correlation was identied in a sub-
sequent study between the oxidising power of this com-
pound, its reactivity with biomolecular targets and its
antiproliferative eects.
In Table 1 we have summarised the cytotoxicity data for
the above mentioned gold(III) compounds developed in
our group. According to this data the most active com-
pounds, compared to cisplatin, are compound 4 and
Auoxo6. For these two complexes the extension of the bio-
logical screening over a wider series of cancer cell lines is
now in progress in our group. Furthermore, these most
promising molecular scaolds will be used for the drug
design of novel gold(III) complexes. For example we have
recently synthesised the gold(III)-dinuclear derivative of 4
whose cytotoxicity properties are under evaluation.
Overall, in the course of the last decade, thanks to the
eorts of a few research laboratories a signicant number
of structurally diverse gold(III) compounds were prepared
and characterised that revealed attracting antiproliferative
activities. In view of the great structural variety of the
investigated gold(III) species it is reasonable to assume that
their biological eects may depend on dierent biochemical

Me 3C CMe3

N O N N O N
Au Au [PF6]2 Au Au [PF6]2
N O N N O N

Me 3C CMe3

Auoxo1 Auoxo2

R Me Me

N O N N O N
Au Au [PF6]2 Au Au [PF6]2
N O N N O N

R Me Me

Auoxo3: R = Me Auoxo6
Auoxo4: R = CH2CMe 3
Auoxo5: R = C6H3Me2-2,6

Fig. 4. Schematic drawings of the dinuclear gold(III) complexes Auoxo. Auoxo3 is a ca. 1:1 mixture of the cis and trans isomer while Auoxo4 and Auoxo5
are, as depicted, exclusively trans isomers.
568 A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575

Table 1
Cytotoxicity (IC50-lM) of the gold compounds studied in Florence during the last years towards dierent tumour cell lines
Compounds A2780/S A2780/R CCRF-CEM/S CCRF-CEM/R SK-OV-3 MCF7 HT29 A549
Cisplatin 1.2 0.43 14 2.72 0.7 0.1 20.1 7.2 5.2 5.30 0.87 6.30 0.23
1 [Au(en)2]Cl3 8.36 0.77 17.0 4.24
2 [Au(dien)Cl]Cl2 8.2 0.93 18.7 2.16 12.6 2.0 32.7 6.6
3 [Au(cyclam)]ClO4)2Cl 99.0 >120.0
4 [Au(Terpy)Cl]Cl2 0.2 0.37 0.032
5 [Au(Phen)Cl2]Cl 3.8 1.1 3.49 0.91 2.3 6
6 [Au(bipyc-H)(OH)][PF6] 3.3 1.4 8.2 1.5 11.9 2.1 51.2 5.6 13.3 1.6 35.30 8.8 24.60 >50
7 [Au(bipy)(OH)2][PF6] 8.8 3.9 24.1 8.7 52.9 11.6 58.6 0.9 34.4 4.7
8 [Au(bipydmb-H)(2.6-xylidine- 2.50 0.43 5.7 0.3 5.20 0.40 25 35
H)][PF6]
9 [Au(pydmb-H)(AcO)2] 2.90 0.34 6.40 1.0 7.70 0.44 8.60 49
Auoxo1 22.8 1.53 23.3 0.35
Auoxo2 12.1 1.5 13.5 1.8
Auoxo3 25.4 2.47 29.8 3.1
Auoxo4 12.7 1.06 19.8 1.8
Auoxo5 11.0 1.5 13.2 1.2
Auoxo6 1.79 0.17 4.81 0.5
Cisplatin is reported as reference compound. Data were collected after 72 h exposure to drug.

mechanisms. Current eorts to identify those mechanisms
are described in the next section. In any case sucient
evidence has been collected so far that the presence of a
gold(III) species is usually associated with outstanding
cytotoxic properties so that the metal itself seems to be
one of the major determinants of the biological actions.
However careful selection and chemical modication of
the ligands might allow the respective complexes proper-
ties to be eciently tuned with regard to inertness, rate
of binding to biomolecules and cytotoxic activity.
2. Current views concerning the biochemical actions of
cytotoxic gold(III) compounds: a variety of molecular
mechanisms
As outlined above, the renaissance of interest for
gold(III) compounds as potential anticancer metallodrugs
has resulted, in the course of the last decade, in the synthe-
sis of a number of structurally diverse gold(III) species,
endowed with sucient chemical stability and with relevant
antiproliferative activities. Detection of in vitro cytotoxicity
has represented, for these metal-based agents, the primary
screening criterion in order to assess their potential anti-
cancer properties. IC50 values of 105 M or lower were
used to indicate a promising, or at least acceptable, anti-
tumour ecacy.
Notably, for a few of these compounds, the pharmaco-
logical studies could be extended well beyond assessment
of mere in vitro cytotoxicity by analysing some of the eects
they produce at the cellular level such as direct DNA dam-
age, modication of the cell cycle, alterations of mitochon-
drial functions, induction of apoptosis, and so on.
Moreover, the reactions of some cytotoxic gold(III)
complexes with some specic biomolecular targets, e.g.
calf-thymus DNA and a few representative proteins, were
analysed at the molecular level by taking advantage of a
vast array of biochemical, biophysical and physicochemical
methods [13,22].
A representative example is the case of gold(III) porphy-
rins whose reactions with nucleic acids were investigated.
In particular, Sun and coworkers examined the interactions
of representative gold(III) porphyrins with duplex DNA by
UVvis absorption titrations [18]. Isosbestic changes and
signicant hypochromicity of the Soret band were noticed
in the electronic spectra of gold(III) porphyrins after addi-
tion of calf-thymus DNA supporting a direct and strong
interaction with the DNA double helix. Accordingly, in a
subsequent study the same authors reported that treatment
with gold(III) porphyrins induces signicant and character-
istic changes in protein expression proles [23].
In general, these cellular and biochemical studies have
provided further valuable insight into the reactivity and
the mode of action of novel gold(III)-based metallodrugs.
Although the ultimate molecular mechanisms of these
new anticancer agents remain largely unknown and elusive,
some considerations and suggestions may be anticipated
based on the available information.
Most of the mechanistic studies carried out on cytotoxic
gold(III) compounds have been generally referred and
compared to the behaviour of cisplatin, for which DNA
is one of the major putative targets [24]. However, it
emerges from the experimental results collected so far that
the respective molecular mechanisms are rather distinct.
Also for cisplatin the major resistance mechanisms fall
into the following four categories: limitation of drug levels
by reduced uptake and/or increased eux; increased cellu-
lar thiol levels; enhanced DNA repair and/or increased
damage tolerance; and failure of cell death pathways [25].
If we consider the cytotoxic eects reported for some of
the above mentioned gold(III) complexes (Table 1) it is
 A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575 569

worth noting that most of them have similar activity Table 2

toward the cisplatin sensitive and resistant cell lines, and Inhibition (IC50-lM) of thioredoxin reductase by gold compounds
therefore may overcome the resistance problems seen with Compound IC50 (lM)
cisplatin itself. However further biological screening is Rat Rat Human
needed to demonstrate these peculiar pharmacological mitochondrial cytosolic cytosolic
properties. TrxR2 TrxR1 TrxR1
Overall, the few gold(III) compounds on which Cisplatin 36.0
advanced pharmacological testing was performed oer a Auranon 0.020
Au triethylphosphine chloride 0.065
rather variegate mechanistic scenario that drastically con- Aurothiomalate 0.280 0.005
trasts with the rather homogeneous biological pattern pro- 2 [Au(2,20 - 0.420 0.20
vided by classical platinum(II) compounds. Therefore, even diethylendiamine)Cl]Cl2
if in a few cases e.g. gold(III) porphyrins some evidence 9 [(Au(2-(1,1-dimethylbenzyl)- 1.420
for a direct DNA damage has been obtained, for most pyridine) (CH3COO)2]
6 [Au(6-(1,1-dimethylbenzyl)- 0.280
other cases, the eects on nucleic acids and on the cell cycle 2,20 - bipyridine)(OH)](PF6)
appear to be very modest so that it is very unlikely that 8 [Au(6-(1,1-dimethylbenzyl)- 0.210
DNA may be the primary target. 2,20 -bipyridine)-H)(2,6-
For instance, we have shown that the large cytotoxic xylidine)](PF6)
and proapoptotic eects induced by the gold(III) com- Tetrachloroaurate 0.0058
12 0.012
plexes 2 and 5 involve very marginal DNA damage [26]; 13 0.030
accordingly only weak cell cycle eects were detected, at 14 0.036
variance with cisplatin (that is known to produce an evi- 15 0.180
dent G2/M arrest), further suggesting a modest impact on 16 0.030
DNA related functions. A similar situation was previously 17 0.0022
18 1.80
reported for gold(III) DAMP compounds pairwise show- 19 0.68
ing very weak DNA interaction properties [5]. Thus, Au triphenylphosphine 0.001
DNA is likely not to be the primary target for several of chloride
the novel gold(III) compounds. Even in the cases where Au(III) chloride trihydrate 0.00075
some evidence of a direct interaction with DNA has been Au acetate 0.040
Au thiosulfate Na+ 0.0005
achieved, the mechanisms of DNA damage and cell death
appear to be profoundly distinct from those induced by Cisplatin is reported as reference compound.
platinum drugs.
All these arguments suggest that alternative biochemical
mechanisms must be operative, most likely associated to pounds are known to target, rather strongly and selectively,
selective modication of some crucial proteins. In this thiol and imidazole groups of proteins (as well as selenol
respect it is worth noting that gold(I) and gold(III) com- groups) [27]; this type of reactivity might reasonably repre-
sent the true molecular basis for their biological actions.
In this context, a recent hypothesis is that gold com-
pounds may exert their relevant cytotoxic and proapoptot-
ic eects through a direct antimitochondrial action: this
idea originally developed for classical antiarthritic and
cytotoxic gold(I) compounds like auranon and aurothi-
omalate (nonetheless showing a relevant cytotoxicity
in vitro) [28] has later been extended to novel anticancer
gold(III) complexes. The key event for the biological mech-
anism of gold compounds would be the direct, strong inhi-
bition of thioredoxin reductase (TrxR), a selenoenzyme
critically involved in the regulation of the intracellular
redox state and of mitochondrial functions [29]. As a con-
sequence of large TrxR inhibition the opening of the mito-
chondrial pore is induced, eventually leading to cyt c
release and apoptosis.
Recent studies have shed light into the molecular and
structural aspects of thioredoxin reductases. The thiore-
doxin reductases are enzymes belonging to the avoprotein
family of pyridine nucleotidedisulphide oxidoreductases
Fig. 5. Schematic representation of the molecular structure of mammalian that also includes lipoamide dehydrogenase, glutathione
TrxR1 visualised with the Swiss-Pdb Viewer software. reductase and mercuric ion reductase [30]. Members of this
570 A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575

N N N
Cl
Au AuCl4 AuCl3 AuCl2

Cl
N

12 13 14

NMe2 NMe2 NMe2

Au Au Au
Cl2 Ph
(OAc)2
Cl
15 16
17

Me PPh3
NMe2
Au
Me Me
Au
Me

Me
18 19

Fig. 6. Schematic drawings of gold(III) complexes screened for the inhibition of human TrxR1.

family are homodimeric proteins in which each monomer
includes an FAD prosthetic group, an NADPH binding
site and an active site containing a redox-active selenol
group. Electrons are transferred from NADPH via FAD
to the active-site selenol of TrxR, which then reduces the
substrate [31]. TrxRs are named for their ability to reduce
oxidised thioredoxins (Trxs), a group of small
(10 12 kDa) ubiquitous redox-active peptides which have
a conserved -Trp-Cys-Gly-Pro-Cys-Lys- catalytic site that
undergoes reversible oxidation/reduction of the two Cys
residues (see Fig. 5).
The crystal structure of thioredoxin reductase has been
recently solved [32]. Notably, these structural studies evi-
denced the presence of a selenocysteine group at its active
site that, in the reduced form, displays a high reactivity
toward soft metal ions. There is today sucient evidence
that this selenol group is the primary anchoring site for a
vast array of metals and metal complexes that are known
to inhibit thioredoxin reductase [33,34].
Thus, one can propose quite reasonably that gold(III)
compounds exert their cytotoxic eects by causing direct
mitochondrial damage through selective modication of
the selenol active site in thioredoxin reductase as previously
suggested for auranon and some related gold(I) com-
pounds [3537]. As a matter of fact, in a recent study con-
ducted in collaboration with the group of Bindoli and
Rigobello in Padova, we have observed that a few gold(III)
complexes developed in our laboratory behave as eective
inhibitors of the cytosolic form of the selenoenzyme thiore-
doxin reductase (TrxR) (see Table 2), accordingly, these
compounds were also found to perturb greatly the mito-
chondrial functions [38].
This view is now reinforced by a recent paper by Powis
et al. reporting the inhibitory properties of a series of
Au(III) complexes (Fig. 6), against human thioredoxin
reductase 1 (TrxR1), including some gold(III) DAMP com-
pounds [39]. Table 2 contains a compilation of representa-
tive literature data for the inhibition of TrxR by various
gold compounds.
Thus, according to the data described above, it is
reasonable to propose that direct antimitochondrial
eects are the determinant for the large proapoptotic
and cytotoxic eects produced by anticancer gold(III)
compounds.
Nonetheless, other recent studies identied the thiol-
dependent cathepsin enzymes as possible, alternative tar-
gets for gold-based anticancer agents [40]. These lysosomal
enzymes are mainly cysteine proteases responsible for
extracellular matrix degradation, bone resorption and joint
destruction. Their marked inhibition arising from coordi-
nation of gold to the active-site cysteine has been reported
for cathespin L and B [41,42].
 A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575 571

1.2

1.1

1.0

0.9

0.8

0.7

A 0.6 b
0.5

0.4

0.3

0.2
a
0.1

0.0
200 250 300 350 400 450 500 550 600 650 700

nm

0.50

0.45

0.40

0.35 b

0.30

A 0.25

0.20

0.15

0.10

0.05
a
0
200 250 300 350 400 450 500 550 600 650 70 0

nm

Fig. 7. Time dependent spectral proles of Auoxo1cyt c (A) and Auoxo1ubiquitin (B) adducts. Spectra correspond to proteins 105 M before (a) and
after the addition (b) of Auoxo1 in a 1:1 ratio. The further evolution of the various systems over time is reported up to 24 h incubation at 37 C. The buer
was 10 mM phosphate, 20 mM NaCl, pH 7.4.

Very recently a paper on the ecient inhibition of
cathepsin K and S by two gold(I) derivatives, auranon
and gold thiomalate, has been published [43]. In this paper
the authors present an X-ray analysis of the gold thioma-
late/cathepsin K complex, showing the selective binding
of gold to the active-site cysteine residue of the protease.
On the ground of the above arguments and of the large
structural variety of the tested compounds, it can be pro-
posed that gold(III) complexes produce their biological
eect through a number of distinct biochemical mechanisms
ranging from classical DNA damage to oxidative membrane
damage, from proteasome inhibition to mitochondrial dam-
age. However, the latter mechanism, i.e. mitochondrial
damage induced through inhibition of thioredoxin reduc-
tase, seems to be a rather peculiar and recurrent mechanistic
feature for gold compounds. The focus of current research is
now on the search of the most important protein targets for
gold(III) compounds.
3. Modelling the interactions of gold(III) compounds with
protein targets: ESI MS and spectroscopic studies of the
reaction of two representative gold(III) complexes with
cytochrome c and ubiquitin
In recent years, ESI MS has emerged as an extremely
powerful tool to monitor, at the molecular level, the forma-
tion of protein adducts of classical anticancer platinum(II)
drugs, and to identify the precise nature of the metallic
fragments coordinated to protein side chains in these
adducts [4447]. However, only very few ESI MS studies
572 A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575

953.067
100 885.333

95

90

85

80
826.800
75

70 1032.133

65
Relative Abundance

60

55

50 774.867

45
1125.600
40

35

30
1237.533
25
729.400
1374.467
20

15 1149.733
1545.733
1265.133
10 1274.267
689.200 1409.200
1554.800
5
651.200 1604.333 1772.333
403.133 586.000
0
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
m/z

953.467
100
885.133
95
1032.400
90

85

80

75

70 1125.867
825.667
65
Relative Abundance

60

55
1238.133
1374.733
50

45

40 1143.867

35
773.800
30 1261.400

25
1390.867 1545.800
1275.733
20 1400.333

15 1416.467
728.400 1423.933
10
1448.333
1564.000
5 716.933 1588.667
681.200 1664.800 1766.333
408.800 519.200
0
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
m/z

Fig. 8. Multicharged mass spectra of cyt c (A) and Auoxo1cyt c conjugate (B) after 24 h reaction at 37 C.

have dealt so far with protein adducts formed by non-plat- niques, to analyse the reactions of two selected gold(III)
inum anticancer drugs [4850]. compounds, namely compound 7 and its dinuclear ana-
We show here that ESI MS may be advantageously logue Auoxo1, with the model proteins ubiquitin and cyto-
employed, in association with other spectroscopic tech- chrome c. Independent information on these systems was
 A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575
gained from ICP OES determinations. Overall, the results
reported here give us the chance to draw a quite detailed
description of the interactions that take place.
First of all, both 7 and Auoxo1 were reacted with either
ubiquitin or cyt c under identical experimental conditions
and the resulting samples monitored spectrophotometri-
cally over 24 h at 37 C [21].
Ubiquitin does not exhibit any absorption band above
280 nm so that the bands characteristic of the two Au(III)
compounds may be easily followed. In both cases, the pro-
gressive decrease of the CT bands typical of these gold(III)
complexes is observed, accompanied by the growth of
intense bands in the 270300 nm region, characteristic of
the free ligand. However, the process is slow, at room tem-
perature, and reaches only 30% progression within 24 h.
On the other hand, horse heart cyt c, in its typical oxi-
dised form, manifests intense bands in the visible, namely
the Soret band at about 410 nm and broad Q bands
between 500 and 550 nm. Nonetheless, the overlap with
the bands characteristic of the two tested gold(III) com-
plexes is not dramatic so that the spectral evolution of
the two distinct components of our system may be moni-
tored rather easily. The reaction of 7 and Auoxo1 with
cyt c is faster than in the case of ubiquitin. At 24 h the reac-
tion has progressed to about 70%. Spectral patterns similar
to those of ubiquitin are observed showing a continuous
decrease of the bands of the gold(III) complex and the
growth of an UV band typical of the free ligand. Typical
spectral proles obtained for Auoxo1 are shown in Fig. 7.
Ultraltration experiments accompanied by ICP OES
determinations showed that a signicant amount of gold
remains associated to cyt c (up to 60% after 24 h). In con-
trast, gold binding to ubiquitin is poor, being only around
15% even after several days of incubation.
Afterward, ESI MS studies were performed on the
adducts of 7 or Auoxo1 with either Ub or cyt c. For com-
parison purposes, all samples were prepared and analysed
under the identical conditions.1 The proteins, alone,
showed a charge distribution of +7 to +13 and +8 to
+18 for Ub and cyt c, respectively. ESI MS analyses of
1
Auoxo1 and 7 were dissolved in DMSO and the solution was diluted
1:100 with H2O to obtain a nal concentration of DMSO of 1%. The
complexes were incubated at molar ratios of 1:1 with ubiquitin and
cytochrome c in aqueous solution at 37 C yielding nal protein
concentrations of 250 lM. The samples were analysed immediately after
start of the incubation, 3 h, 24 h, 48 h, 6 days and 12 days. Before ESI IT
MS analysis, the incubation solutions were diluted (99:1) with
ACN:H2O:formic acid (30:70:1) and analysed immediately after mixing.
Analyses were performed on a Thermo Finnigan LCQ Deca XP Plus
quadrupole ion-trap instrument in positive ion mode. The capillary
temperature was set at 180 C and the source voltage to 1.50 kV, with a
mass range from 300 to 2000. The acquisition was done with Tune Plus 1.3
SR1 program (Thermo Finnigan) and the deconvolution of data were
performed with Bioworks Browser 3.0 (Thermo Finnigan) on a Windows
2000 Prof. SR4 PC system using Biomass Calculation and Deconvolution
software. The molecular weight was deconvoluted from the multiply
charged protein and proteinAu adduct peaks with charges ranging from
+7 to +13 and +8 to +18 for ubiquitin and cytochrome c, respectively.
573
the free complexes revealed for Auoxo1 peaks at m/z
369.8 and 883.0 which were assigned to [Au(bipy)OH]+
(100%) and [Au2(bipy)2O2PF6] (14%). For compound 7
the main peak at m/z 387.4 was assigned to [Au(bi-
py)(OH)2]+ and a very weak one at m/z 499.4 to [Au(bi-
py)PF6]+ (3%). It is worth noting that mass spectra of
proteinmetallodrug samples immediately after mixing
did not show any adduct formation. This observation indi-
cated that gas phase reactions do not take place under the
applied experimental conditions.
After 24 h incubation, samples of Auoxo1 and 7 with cyt
c showed, beyond the signal of the native protein, a charac-
teristic peak assigned to a 1:1 Aucyt c derivative. The mul-
ticharged spectra of free cyt c and of its adduct with
Auoxo1 are reported in Fig. 8. The ESI MS spectra,
together with spectrophotometric data, strongly suggest
that upon reaction with the protein the organic ligand is
lost whereas gold, most likely in the form of a Au+ ion,
remains tightly attached to the protein. A similar behav-
iour was previously reported by Sadler and coworkers in
the case of the adducts of gold(I) triethylphosphine chlo-
ride with cyclophilin [51].
Conversely, ESI MS analysis of the incubation mixtures
of both complexes with Ub revealed no appreciable adduct
formation even after 12 days of reaction.
In conclusion, the above results point out quite clearly
that independently of the nature of the gold(III) complexes,
a similar reactivity with cyt c takes place, comprising a
redox process with gold(III) reduction followed by release
of the organic ligand and eventual coordination of a
gold(I) ion to a protein side chain.
Both the in situ redox process and gold(I) coordination
to a specic protein residue may be crucial events for direct
and irreversible protein damage. Further studies are in pro-
gress to identify the nature of the binding site for gold(I) in
cyt c, good candidates being Met 65 and two accessible his-
tidines (His 33 and His 26).
Recent advances in the cellular pharmacology of Au(III)
compounds are leading to the identication of a few spe-
cic proteins as probable biomolecular targets. In this
frame it is important to develop appropriate physicochem-
ical methods to monitor reactions of gold(III) complexes
with protein targets. Modelling work carried out in our
laboratory on simple proteins, primarily relying on mass
spectrometry and some other ancillary techniques, holds
much promise for a rapid and detailed characterisation of
goldprotein adducts. Some representative examples have
been reported concerning ubiquitin and cytochrome c. Fur-
ther eorts are needed to extend the investigation to the
protein targets that were mentioned in the previous section,
for which, up to now, ESI MS has been scarcely used for
the characterisation of their interactions with metallodrugs.
4. Concluding remarks
The renewed interest toward gold(III) compounds as
potential anti-tumour agents, that started at the beginning
574 A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575
of 1990s, has resulted, until now, in the identication of
several gold(III) and organogold(III) compounds charac-
terised by a signicant structural variety and by encourag-
ing in vitro pharmacological properties. Notably, these
gold(III) compounds constitute today an interesting family
of new cytotoxic agents that undoubtedly deserve more
extensive pharmacological testing and a careful analysis
of their mechanism of action. We have reported above that
these structural diverse gold(III) complexes exert outstand-
ing antiproliferative eects when tested in vitro against var-
ious tumour cell lines. The observed biological eects are
most likely the consequence of innovative and distinct
molecular mechanisms in dependence of the peculiar coor-
dination and redox chemistry of the gold(III) center and of
the nature of its ligands. DNA seems not to be a primary
target for most novel cytotoxic gold(III) compounds.
In contrast, the important reactivity detected toward
model proteins favours the idea that the cytotoxic
eects of gold(III) complexes are primarily mediated by
interactions with proteins targets. For instance, relevant
antimitochondrial eects, probably arising from direct
inhibition of thioredoxin reductase were highlighted, that
activate the apoptotic cascade after triggering mitochon-
drial cyt c release. Additionally, various Au(III) complexes
were reported to inhibit strongly the disease-specic, thiol-
containing, cysteine-protease cathepsins.
We have shown here that the reactivity of gold(III) com-
pounds with proteins may be accurately monitored, at the
molecular level, through physicochemical studies of appro-
priate model systems. In this respect, ESI MS has turned
out to be a very powerful method to follow formation of
protein adducts and identify the nature of protein bound
metal fragments. In the reported examples, concomitant
redox and metal binding processes seem to be crucial in
producing specic protein damage as documented by the
case of cyt c. However, in this eld, the development of
proteomic or metalloproteomic techniques, as well as
other combined biochemical and genetic methods, will be
fundamental to provide us with powerful tools for the dis-
covery of new proteins or pathways involved in the cellular
response to gold-based drugs. Overall, in view of their
peculiar chemical and biological properties, it seems to us
that these novel cytotoxic gold(III) complexes have the
potential of being further developed and exploited as
experimental anticancer drugs.
5. Abbreviations
bipy bipyridyl
bipyc 6-(1,1-dimethylbenzyl)-2,20 -bipyridine
cisplatin cis-diammine-dichloro platinum(II)
cyt c horse heart cytochrome c
CT charge transfer
DAMP 2-[(dimethylamino)methylphenyl]
dien diethylendiamine
en ethylendiamine
ESI MS electrospray ionization mass spectrometry
GSH glutathione
ICP OES inductively coupled plasma optical emission
spectroscopy
LMCT ligand to metal charge transfer
phen phenathroline
py pyridine
terpy terpyridine
TPP meso-tetraarylporphyrin
TrxR thioredoxin reductase
TrxR1 human cytosolic thioredoxin reductase
TrxR2 human mitochondrial thioredoxin reductase
Trxs thioredoxins
Ub ubiquitin
Acknowledgements
The consortium C.I.R.C.M.S.B and Ente Cassa di
Risparmio di Firenze are gratefully acknowledged for
nancial support. Dr. Angela Casini is grateful to AIRC
for providing her with a two years fellowship.
References
[1] B. Rosenberg, L. VanCamp, J.E. Trosko, V.H. Mansour, Nature 222
(1969) 385386.
[2] B. Lippert, Cisplatin: Chemistry and Biochemistry of a Leading
Anticancer Drug, John Wiley & Sons, Inc., New York, 1999.
[3] E. Wong, C.M. Giandomenico, Chem. Rev. 99 (1999) 24512466.
[4] E.R. Tiekink, Crit. Rev. Oncol. Hematol. 42 (2002) 225248.
[5] R.V. Parish, B.P. Howe, J.P. Wright, J. Mack, R.G. Pritchard, R.G.
Buckley, A.M. Elsome, S.P. Fricker, Inorg. Chem. 35 (1996) 1659
1666.
[6] R.G. Buckley, A.M. Elsome, S.P. Fricker, G.R. Henderson, B.R.
Theobald, R.V. Parish, B.P. Howe, L.R. Kelland, J. Med. Chem. 39
(1996) 52085214.
[7] S. Carotti, A. Guerri, T. Mazzei, L. Messori, E. Mini, P. Orioli,
Inorg. Chim. Acta 281 (1998) 9094.
[8] L. Messori, F. Abbate, G. Marcon, P. Orioli, M. Fontani, E. Mini, T.
Mazzei, S. Carotti, T. OConnell, P. Zanello, J. Med. Chem. 43 (2000)
35413548.
[9] F. Abbate, P. Orioli, B. Bruni, G. Marcon, L. Messori, Inorg. Chim.
Acta 311 (2000) 15.
[10] G. Marcon, S. Carotti, M. Coronnello, L. Messori, E. Mini, P. Orioli,
T. Mazzei, M.A. Cinellu, G. Minghetti, J. Med. Chem. 45 (2002)
16721677.
[11] M. Coronnello, E. Mini, B. Caciagli, M.A. Cinellu, A. Bindoli, C.
Gabbiani, L. Messori, J. Med. Chem. 48 (2005) 67616765.
[12] A. Vaccini, Ph.D. thesis 2002, University of Florence.
[13] L. Messori, P. Orioli, C. Tempi, G. Marcon, Biochem. Biophys. Res.
Commun. 281 (2001) 352360.
[14] L. Ronconi, L. Giovagnini, C. Marzano, F. Bettio, R. Graziani, G.
Pilloni, D. Fregona, Inorg. Chem. 44 (2005) 18671881.
[15] L. Ronconi, C. Marzano, P. Zanello, M. Corsini, G. Miolo, C.
Macca, A. Trevisan, D. Fregona, J. Med. Chem. 49 (2006) 16481657.
[16] D. Aldinucci, D. Lorenzon, L. Stefani, L. Giovagnini, A. Colombatti,
D. Fregona, Anticanc. Drugs 18 (2007) 323332.
[17] V. Milacic, D. Chen, L. Ronconi, K.R. Landis-Piwowar, D. Fregona,
Q.P. Dou, Cancer Res. 66 (2006) 1047810486.
[18] C.M. Che, R.W.-Y. Sun, W.-Y. Yu, C.-B. Ko, N. Zhu, H. Sun,
Chem. Commun. 14 (2003) 17181719.
[19] N. Farrell, Met. Ions Biol. Syst. 42 (2004) 251296.
[20] A. Bergamo, G. Stocco, B. Gava, M. Cocchietto, E. Alessio, B. Serli,
E. Iengo, G. Sava, J. Pharmacol. Exp. Ther. 305 (2003) 725732.
 A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575
[21] A. Casini, M.A. Cinellu, G. Minghetti, C. Gabbiani, M. Coronnello,
E. Mini, L. Messori, J. Med. Chem. 49 (2006) 55245531.
[22] L. Messori, G. Marcon, P. Orioli, M.A. Cinellu, G. Minghetti, Eur. J.
Biochem. 270 (2003) 46554661.
[23] Y. Wang, Q.Y. He, C.M. Che, J.F. Chiu, Proteomics 6 (2006) 131
142.
[24] L.H. Hurley, Nat. Rev. Cancer 2 (2002) 188200.
[25] D. Wang, S.J. Lippard, Nat. Rev. Drug. Discov. 4 (2005) 307320.
[26] M. Coronnello, G. Marcon, S. Carotti, B. Caciagli, E. Mini, T.
Mazzei, P. Orioli, L. Messori, Oncol. Res. 12 (2000) 361370.
[27] M.T. Coer, C.F. Shaw 3rd, A.L. Hormann, C.K. Mirabelli, S.T.
Crooke, J. Inorg. Biochem. 30 (1987) 177187.
[28] G.F. Rush, P.F. Smith, G.D. Hoke, D.W. Alberts, R.M. Snyder,
C.K. Mirabelli, Toxicol. Appl. Pharmacol. 90 (1987) 391400.
[29] C.H. Williams Jr., Eur. J. Biochem. 267 (2000) 6101.
[30] E.S.J. Arner, A. Holmgren, Eur. J. Biochem. 267 (2000) 61026109.
[31] H. Nakamura, Antioxid. Redox. Signal. 7 (2005) 823828.
[32] K. Fritz-Wolf, S. Urig, K. Becker, J. Mol. Biol. 370 (2007) 116127.
[33] A.B. Witte, K. Anestal, E. Jerremalm, H. Ehrsson, E.S. Arner, Free
Radic. Biol. Med. 39 (2005) 696703.
[34] P.J. Barnard, S.J. Berners-Price, Coord. Chem. Rev. 251 (2007) 1889
1902.
[35] M.P. Rigobello, G. Scutari, A. Folda, A. Bindoli, Biochem.
Pharmacol. 67 (2004) 689696.
[36] Y. Omata, M. Folan, M. Shaw, R.L. Messer, P.E. Lockwood, D.
Hobbs, S. Bouillaguet, H. Sano, J.B. Lewis, J.C. Wataha, Toxicol. In
Vitro 20 (2006) 882890.
[37] O. Rackham, S.J. Nichols, P.J. Leedman, S.J. Berners-Price, A.
Filipovska, Biochem. Pharmacol. 74 (2007) 9921002.
[38] M.P. Rigobello, L. Messori, G. Marcon, M. Bragadin, A. Folda, G.
Scutari, A. Bindoli, J. Inorg. Biochem. 98 (2004) 16341641.
575
[39] L. Engman, M. McNaughton, M. Gajewska, S. Kumar, A. Birming-
ham, G. Powis, Anticanc. Drugs 17 (2006) 539544.
[40] V. Turk, B. Turk, D. Turk, EMBO J. 20 (2001) 46294633.
[41] A. Chircorian, A.M. Barrios, Bioorg. Med. Chem. Lett. 14 (2004)
51135116.
[42] S.P. Fricker, R. Skerjl, B.R. Cameron, R. Mosi, Y. Zhu, Recent
developments in gold drugs in: Contribution to the Gold 2003
Conference: New Industrial Applications for Gold.
[43] E. Weidauer, Y. Yasuda, B.K. Biswal, M. Cherny, M.N. James, D.
Bromme, J. Biol. Chem. 388 (2007) 331336.
[44] I. Khalaila, C.S. Allardyce, C. Verma, P.J. Dyson, ChemBioChem 6
(2005) 17881795.
[45] T. Peleg-Shulman, Y. Najajreh, D. Gibson, J. Inorg. Biochem. 91
(2002) 306311.
[46] A. Casini, G. Mastrobuoni, C. Temperini, C. Gabbiani, S. Francese,
G. Moneti, C.T. Supuran, A. Scozzafava, L. Messori, Chem.
Commun. (2007) 156158.
[47] C.G. Hartinger, W.H. Ang, A. Casini, L. Messori, B.K. Keppler, P.J.
Dyson, JAAS 22 (2007) 960967.
[48] A.R. Timerbaev, C.G. Hartinger, S.S. Aleksenko, B.K. Keppler,
Chem. Rev. 106 (2006) 22242248.
[49] A. Casini, G. Mastrobuoni, W.H. Ang, C. Gabbiani, G. Pieraccini,
G. Moneti, P.J. Dyson, L. Messori, Chem. Med. Chem. 2 (2007) 631
635.
[50] A. Casini, G. Mastrobuoni, M. Terenghi, C. Gabbiani, E. Monzani,
G. Moneti, L. Casella, L. Messori, J. Biol. Inorg. Chem. 12 (2007)
11071117.
[51] J. Zou, P. Taylor, J. Dornan, S.P. Robinson, M.D. Walkinshaw, P.J.
Sadler, Angew. Chem. Int. Ed. Engl. 39 (2000) 29312934.