Photolysis and thermolysis of platinum(IV) 2,2′-bipyridine complexes lead to identical platinum(II)-DNA adducts

Article abstract of DOI:10.1002/chem.201000850

Two Pt^IV and two Pt¹¹ complexes containing a 2,2′-bipyridine ligand were treated with a short DNA oligonucleotide under light irradiation at 37 °C or in the dark at 37 and 50 °C. Photolysis and thermolysis of the Pt^IV complexes led to spontaneous reduction of the Pt^IV to the corresponding Pt^II complexes and to binding of Pt^II 2,2′-bipyridine complexes to N7 of guanine. When the reduction product was [Pt(bpy)Cl₂], formation of bis-oligonucleotide adducts was observed, whereas [Pt(bpy)(MeNH ₂)Cl]⁺ gave monoad- ducts, with chloride ligands substituted in both cases. Neither in the dark nor under light irradiation was the reductive elimination process of these Pt^IV complexes accompanied by oxidative DNA damage. This work raises the question of the stability of photoacti- vatable Pt^IV complexes toward moderate heating conditions.

Full text of DOI:10.1002/chem.201000850

DOI: 10.1002/chem.201000850
Photolysis and Thermolysis of Platinum(IV) 2,2’-Bipyridine Complexes Lead
to Identical Platinum(II)–DNA Adducts
Christophe Loup,[a, b] Ana Tesouro Vallina,[a, b] Yannick Coppel,[a, b] Ulla Lꢀtinois,[a, b]
Yasuo Nakabayashi,[c, d] Bernard Meunier,[a, b] Bernhard Lippert,[c] and
Geneviꢁve Pratviel*[a, b]
Abstract: Two PtIV and two PtII com-
plexes containing 2,2’-bipyridine
nine. When the reduction product was
[Pt(bpy)Cl2], formation of bis-oligonu-
cleotide adducts was observed, whereas
[Pt(bpy)
(MeNH2)Cl]+ gave monoad-
in both cases. Neither in the dark nor
under light irradiation was the reduc-
tive elimination process of these PtIV
complexes accompanied by oxidative
DNA damage. This work raises the
question of the stability of photoacti-
vatable PtIV complexes toward moder-
ate heating conditions.
a
ACHTUNGTRENNUNG
ligand were treated with a short DNA
oligonucleotide under light irradiation
at 378C or in the dark at 37 and 508C.
Photolysis and thermolysis of the PtIV
complexes led to spontaneous reduc-
tion of the PtIV to the corresponding
PtII complexes and to binding of PtII
2,2’-bipyridine complexes to N7 of gua-
A
ACHTUNGTRENNUNG
ducts, with chloride ligands substituted
Keywords: oligonucleotides · pho-
tolysis · platinum · reduction · ther-
molysis
Introduction
the N7 atoms of two adjacent guanine (G) bases.[2,3] A seri-
ous drawback of cisplatin treatment is the intrinsic resist-
ance of some tumors or the development of resistance of
several tumors in the course of the treatment.[4] Although
no PtIV compound has been approved yet as an anticancer
drug, these six-coordinate complexes still draw attention as
alternative platinum-based anticancer drugs.[5–9] They show
anticancer activity and are toxic to tumors that are resistant
to cisplatin. PtIV complexes are considered prodrugs of their
corresponding PtII congeners. They react too slowly with tar-
gets to show any biological effect, because such reactions
cannot compete with the faster reduction of PtIV to PtII with
biological reducing agents.[10–13] However, as precursors of
PtII complexes, they offer a number of additional properties.
Variation of the axial ligand of the PtIV complexes allows
the lipophilicity of the complex to be modulated and influ-
ences its capacity to penetrate cells.[14–16] The major advan-
tages of PtIV drugs are their potential for oral administra-
tion, whereas cisplatin, carboplatin, and oxaliplatin are in-
travenously administered. Furthermore, one can take ad-
vantage of the release of two axial groups upon reduction
within cells. The two released ligands can have their own
mechanism of action. Two recent examples following this
strategy have been reported.[17,18] On the other hand, axial
positions may carry substituents that allow targeting and in-
ternalization of the platinum complex inside the appropriate
cell.[19,20]
cis-Dichloridodiammineplatinum(II), cis-[PtCl2ACHTUNGTRENNUNG
cis-tetrachloridodiammineplatinum(IV), cis-[PtCl CHTUNGTRENNUNG
(NH3)2], and
4A(NH3)2],
were identified as the Pt compounds responsible for the in-
hibition of cell division by platinum complexes discovered
by Rosenberg et al.[1] This led to cis-[PtCl
2ACTHNUTRGNEUNG(NH3)2] (cisplatin)
as one of the most widely utilized antitumor drugs. The anti-
cancer activity of cisplatin is based on its ability to form in-
trastrand covalent adducts with DNA by binding of Pt to
[a] C. Loup, Dr. A. Tesouro Vallina, Dr. Y. Coppel, Dr. U. Lꢀtinois,
Dr. B. Meunier, Dr. G. Pratviel
CNRS, Laboratoire de Chimie de Coordination
205 route de Narbonne, 31077 Toulouse (France)
Fax : (+33)561-55-3003
E-mail: genevieve.pratviel@lcc-toulouse.fr
[b] C. Loup, Dr. A. Tesouro Vallina, Dr. Y. Coppel, Dr. U. Lꢀtinois,
Dr. B. Meunier, Dr. G. Pratviel
Universitꢀ de Toulouse, Toulouse (France)
[c] Prof. Dr. Y. Nakabayashi, Prof. Dr. B. Lippert
Fakultꢁt Chemie, Technische Universitꢁt Dortmund
44221 Dortmund (Germany)
[d] Prof. Dr. Y. Nakabayashi
Kansai University, 564-8680 Suita, Osaka (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201000850.
11420
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 11420 – 11431

FULL PAPER
In the above examples the release of the two axial groups
is due to chemical reduction by the intracellular reductant
pool, and the axial ligands are released as anions. However,
reduction of PtIV complexes may be independent of the
presence of reductants, and can also occur “spontaneously”
upon light irradiation (photolysis).[21–32] In this case, the two
electrons required for reduction of PtIV to PtII are provided
by intramolecular electron transfer from two ligands to the
platinum ion.[25,26,30] As with chemical reduction, a square-
planar PtII complex results, but in this case, it is associated
with two oxidized ligands. While some PtIV complexes can
be reduced either by reductants or through photoactiva-
tion,[21,27,30] some others, more interestingly, are only reduced
upon illumination.[22–24,28] It was reported that light-promot-
ed photolysis, in addition to intracellular reduction, can po-
tentiate the anticancer activity of a PtIV complex.[27] Except
for some examples,[26,27,29] the nature and fate of the two oxi-
dized ligands was not studied in detail. We reported that
photolysis of PtIV complexes with a 2,2’-bipyridine ligand
triggered the release of HOCl.25] The formation of hypochlo-
rous acid conceivably takes place through direct reductive
elimination of HOCl or reductive elimination of Cl2 and
subsequent disproportionation of Cl2 in H2O. These diffusi-
ble oxidative species may be capable of damaging biomole-
cules in addition to the action of the PtII drug generated.
Additionally, in the absence of reductant, in vitro substitu-
elimination of PtIV complexes was not associated with con-
comitant oxidative DNA damage.
Our data also support the view that the tested PtIV com-
plexes do not undergo ligand substitution with G located
either inside or at the 3’-end of a DNA strand, and are thus
unable to induce guanine oxidation by inner-sphere electron
transfer at these G residues. Spontaneous reduction upon
photolysis and thermolysis is the major reaction route.
Results
The PtIV complexes with a 2,2’-bipyridine ligand, [PtCl
bpy)] (1) and [PtCl3A(2,2’-bpy)(MeNH2)]Cl (2), tested in the
4ACHTUNGTRENNUNG(2,2’-
C
ACHTUNGTRENNUNG
present work (Scheme 1) were shown previously to be light-
tion of PtIV complex [PtCl
4ACTHNUTRGNE(NUG dach)] (dach=1,2-diaminocyclo-
hexane) with an oxidizable biological ligand such as a gua-
nine nucleobase (G) was reported to lead to inner-sphere
two-electron transfer from guanine to the platinum ion.[33–36]
Considering the redox potential of G (1.29 V vs. NHE), an
outer-sphere electron-transfer mechanism is not possible be-
tween G and the PtIV complex.[37] An inner-sphere mecha-
nism induced by coordination of PtIV to G must be consid-
ered. In this example, up to now, only guanine residues at
the 5’-end of a DNA strand were shown to be reactive.[34]
Besides, substitution reactions of PtIV complexes with G
may be catalyzed by traces of PtII.[36,38] Although such a re-
action may not be biologically relevant due to competition
with intracellular reduction of the PtIV complex, it illustrates
the potential oxidative behavior of PtIV complexes toward
DNA.
Scheme 1. Structures of the complexes applied in this study.
sensitive.[25] Light irradiation of 2 promotes its photoisomeri-
zation and photolysis.[25] Photolysis (l>300 nm) generates
the corresponding PtII complex [PtCl
A
(MeNH2)]+
ACHTUNGTRENNUNG
CTHUNGTRENNUNG
may originate from direct reductive elimination of HOCl or
from reductive elimination of Cl2 and subsequent reaction
with H2O. The reactivity of PtIV complexes 1 and 2 in the
presence of DNA models was studied under photolysis in
order to detect DNA platinum adducts due to the released
PtII complexes and oxidative DNA damage due to HOCl.
Complexes 1 and 2 were also treated with DNA models
under dark conditions in order to evidence any substitution
reactions of the PtIV complexes with guanine nucleobase
and, eventually, guanine oxidation by inner-sphere electron
transfer within adducts of PtIV complexes with guanine.
Thus, adducts of DNA with PtIV and/or PtII and oxidative
DNA damage were considered. The PtIV complexes (1, 2)
and their PtII analogues (3, 4) were treated with 5’-
CAGCTG oligonucleotide (ODN) in the dark and under
light irradiation. The time course of the reactions was moni-
tored by liquid chromatography coupled to UV/Vis detec-
tion or coupled to electrospray mass ionization (LC/ESI-
We have studied, in this work, the reactivity of two photo-
activatable (PtIV complexes with a 2,2’-bipyridine ligand,
[PtCl
4ACHTUNGTRENNUNG(2,2’-bpy)] (1), and [PtCl3AHCUTNRTGEG(NNUN 2,2’-bpy)CATHNUGTREN(NGUN MeNH2)]Cl (2).
Photolysis of 2 is known to generate the corresponding PtII
(MeNH2)]+ with concomitant re-
complex [PtClACHTUNGTRENNUNG(2,2’-bpy)HCATUNGTRENNUGN
lease of diffusible oxidative species (HOCl or Cl2).[25]
Photolysis of PtIV complexes 1 and 2 in the presence of
oligonucleotide DNA models induced reduction of the PtIV
to the corresponding PtII complexes and subsequent binding
of the PtII species to N7 of G. Mono-oligonucleotide–PtII ad-
ducts and bis-adducts stemmed from 2 and 1, respectively.
Interestingly, we found that a heating step in the dark
(508C) has the same effect as light irradiation and likewise
causes reduction of PtIV complexes to the corresponding PtII
complexes. Under the reaction conditions applied, reductive
Chem. Eur. J. 2010, 16, 11420 – 11431
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
11421

G. Pratviel et al.
MS). This method is highly sensitive and has previously al-
lowed us to detect about 1% of oxidative DNA
damage.[39,40] Under these experimental conditions the short
ODN was single-stranded.
Complex 2 is soluble in H2O. Complexes 1, 3 and 4 are
soluble in DMSO. Therefore, initial dilution of these com-
plexes in DMSO was followed by addition of water, and the
reaction medium contained 1% DMSO. The complexes
were never stored in solution to avoid ligand exchange. Buf-
fers were not used to avoid complications arising from
buffer coordination to platinum. The UV/Vis spectra of the
tested complexes are shown in Figure S1 in the Supporting
Information.
Figure 1. HPLC trace (260 nm) of the products formed during reaction of
complex 1 (100 mm) with 5’-CAGCTG (10 mm) after 24 h under light irra-
diation (378C). Pt-I, Pt-II, and Pt-III eluting at 20–25 min are platinum–
oligonucleotide adducts. Intact ODN 5’-CAGCTG was eluted at
28.5 min. Pt refers to excess platinum complex.
Cyclic voltammetry of PtIV complexes 1 and 2: The cyclic
voltammogram of PtIV complex 1 in DMSO is shown in Fig-
ure S2 in the Supporting Information. This complex displays
a first irreversible two-electron cathodic peak (Ep =0.12 V/
NHE) for the PtIV/PtII reduction process followed by a re-
excess (100 mm). The irradiated oligonucleotide showed no
DNA damage.
duction pattern identical to that exhibited by [PtCl
(2,2’-
When PtIV complex 1 (100 mm) was treated with 5’-
CAGCTG, the initial ODN (28.6 min) transformed into
three different products eluted at shorter retention times
(20-24 min, Figure 1). The ratio of the peaks corresponding
to the modified ODNs did not change during the course of
the reaction. Pt-I, Pt-III, and Pt-II accounted for approxi-
mately 45, 20 and 35%, respectively. Their increase was as-
sociated with a proportional decrease of the intact ODN.
After 24 h, the area of these peaks (HPLC trace at 260 nm)
accounted for about 60% of the starting ODN material
(Table 1, entry 2). The area of the remaining ODN corre-
bpy)].[41] The PtIV complex, upon addition of two electrons,
loses the two axial ligands, affording the corresponding plat-
inum(II) species. The second reduction process is a one-elec-
tron reversible process (E1/2 =À0.86 V/NHE), which sug-
gests that it is mainly localized on the 2,2’-bipyridine ligand.
The cyclic voltammogram of PtIV complex 2 in aqueous
media (see Figure S2 in the Supporting Information) exhib-
its a first irreversible two-electron cathodic peak (Ep =
0.40 V/NHE) for the PtIV/PtII reduction process. The fea-
tures are the same as reported for other platinum com-
plexes.[42] This reduction is irre-
versible due to loss of the axial
Table 1. Yield of adducts (%, with respect to ODN) at different reaction time for the reaction of complex 1
and 2, at 378C, in the dark (À) or under light irradiation (+). The concentration of the oligonucleotide (ODN)
was constant and the concentration of the platinum complex was 10 or 100 mm. Data are the means (Æstandard
deviation, SD) of three independent experiments.
ligands. Complex
1 was not
soluble in H2O at the typical
concentration used for cyclic
voltammetry. However, it is
possible to predict the PtIV/PtII
peak potential of 1 in aqueous
solution[43] from the known
electrochemical parameters of
the separate ligands.[44] The pre-
dicted Ep of 1 in H2O is conse-
quently Eppred =0.11 V/NHE.
Entry
Light
Complex (mm)
/ODN (mm)
Complex
1 h
3 h
6 h
24 h
1
2
3
4
5
6
7
8
+
+
+
+
À
À
À
À
10/10
100/10
10/10
100/10
10/10
100/10
10/10
1
1
2
2
1
1
2
2
29 (Æ11)
57 (Æ6)
46 (Æ9)
10[a]
23 (Æ6)
20 (Æ3)
74 (Æ9)
0
10 (Æ5)
9.8 (Æ1)
29
41 (Æ6)
29 (Æ6)
10[a]
60 (Æ10)
0
0
0
19 (Æ6)
14 (Æ2)
34 (Æ9)
22 (Æ7)
26 (Æ2)
54 (Æ6)
5 (Æ1)
100/10
15[a]
According to the PtIV/PtII
redox potentials, complex
1
2
[a] One measurement.
(Eppred =0.11 V/NHE) and
(Ep =0.12 V/NHE) should be
capable of G oxidation by inner-sphere electron transfer
within a guanine–PtIV adduct provided they can coordinate
at N7 of G.[45]
sponded to about 40% of the initial area. The new products
exhibited a dual absorbance at 260 and 320 nm compatible
with covalent binding of a platinum complex to an oligonu-
cleotide strand, as confirmed by LC-ESI/MS analysis (see
below). For the three compounds, the on-line UV/Vis spec-
tra were identical; one example is shown in Figure S3A in
the Supporting Information. Assuming that the e values of
the starting ODN and the platinum modified ODN at
260 nm are on the same order, the area of the HPLC peaks
of the adducts (Pt-I+Pt-II+Pt-III) gives the yield of the re-
Reaction of PtIV complexes 1 and 2 with 5’-CAGCTG: light-
promoted formation of Pt–DNA adducts: Platinum(IV)
complexes 1 and 2 were treated with 5’-CAGCTG (10 mm)
in H2O at 378C under light irradiation. The platinum com-
plex was present either in stoichiometric amount (10 mm)
with respect to the oligonucleotide or was added in tenfold
11422
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 11420 – 11431

Platinum(II)–DNA Adducts
FULL PAPER
action. The yield of reaction depended on the irradiation
time and the concentration of the platinum complex. Plati-
num adducts Pt-I, Pt-II, and Pt-III reached about 30% after
24 h under light irradiation for a stoichiometric amount of 1
with respect to ODN (Table 1, entry 1).
The LC/ESI-MS analyses of platinum-modified oligonu-
cleotides Pt-A and Pt-B were identical. Each exhibited two
signals. One of them gave the same m/z signal, observed
before for complex 1 adducts, at 1069.2 amu for a doubly
charged ion (Figure S5, Supporting Information). As for the
The LC-ESI/MS analysis of the reaction mixture of the
photolysis of 1 in the presence of 5’-CAGCTG shows the
starting ODN 5’-CAGCTG with an m/z signal at 894.8 amu
with z=2 (not shown). The three different platinum modi-
fied oligonucleotides exhibited the same m/z signals at
1069.2 amu (z=2; see Figure S4 in the Supporting Informa-
adducts of complex 1, this ion was attributed to a [Pt
bpy)ODN]2À species. The second doubly charged ionic spe-
cies observed at 1085.5 amu was attributed to a [Pt(2,2’-
bpy)
(MeNH2)ODN]2À ion, corresponding to a platinum
adduct carrying one 2-2’-bipyrine and one methylamine
ligand. Fragmentation (loss of methylamine ligand) in the
mass spectrometer is likely the origin of the 1069.2 ion.
Complex 2 formed two well-separated monoadducts (Pt-A,
Pt-B) with identical mass spectra, each of which still carries
one methylamine ligand. As was confirmed by enzymatic
hydrolysis, they correspond to PtII adducts of the 5’-
CAGCTG sequence at the two individual G sites.
ACHTUNGTRENNUNG(2,2’-
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
tion). It was assigned to a [PtACHTNUGRTNEUNG
(2,2’-bpy)ODN]2À species com-
patible with a +2 oxidation state for platinum. As shown
thereafter by NMR spectroscopy, for each of the three ad-
ducts, the platinum center is coordinated to two bases on
the ODN (bis-adduct). However, the oxidation state of plat-
inum is not certain, since reduction of metal complexes
during ESI-MS analysis may be possible.[46]
In summary, complexes 1 and 2 are capable of platinating
5’-CAGCTG under light irradiation. The mass data are in
favor of the platinum ion for all these DNA adducts being
PtII, yet reduction of a PtIV species in the mass source may
be possible. Consequently, the oxidation state of platinum–
oligonucleotide adducts could not be determined with cer-
tainty. The formation of PtII adducts under light irradiation
can be explained by photolysis of PtIV complexes 1 and 2 to
the corresponding PtII species.[25] Oxidative damage of DNA
associated with light-induced decomposition of PtIV com-
plexes was never evidenced, even at high concentration of
PtIV complex (100 mm). The nature and/or concentration of
the diffusible oxidative species produced by the photolysis
of PtIV complexes was probably not compatible with oxida-
tive DNA damage. Indeed, DNA oxidation mediated by oxi-
dative species such as H2O2 is observed at millimolar con-
centration of reagent, whereas in the present work HOCl
(or Cl2) is produced at micromolar concentration.
As for complex 1, the reaction of complex 2 with the
short ODN 5’-CAGCTG under light irradiation led to for-
mation of platinum–DNA adducts. The reaction was also de-
pendent on light and on the concentration of the complex.
However, the nature of the products, the kinetics, and yields
were different. First, in the case of complex 2, the platinum–
DNA adducts were eluted after the starting ODN on the
HPLC chromatograms, that is, they were structurally differ-
ent from the previous ones. They appeared in the form of
two peaks of almost equal intensity Pt-A and Pt-B
(Figure 2). The UV/Vis specta of the two reaction products
To determine the mechanism of the platination reaction
mediated by the PtIV complexes under light and to unambig-
uously identify the PtII–oligonucleotide adducts, the corre-
sponding PtII complexes 3 and 4 were treated with 5’-
CAGCTG.
Reaction of PtII complexes 3, 4, and cisplatin with 5’-
CAGCTG: The concentrations of ODN and PtII complexes
3 and 4 were 10 mm. Chromatograms of the reaction mix-
tures of complexes 1 and 3 and of complexes 2 and 4
showed the same adducts. Complex 3 led to the formation
of Pt-I, Pt-II, and Pt-III (Figure S6A, Supporting Informa-
tion). Complex 4 led to the formation of Pt-A and Pt-B
(Figure S6B, Supporting Information). On-line UV/Vis spec-
tra were also similar (Figure S7, Supporting Information).
LC/ESI-MS analysis confirmed that the same adducts
formed with the PtIV and the PtII complexes in each series
(not shown). As expected, the reaction rate was much
higher with PtII compared to the corresponding PtIV com-
plexes. Under stoichiometric conditions (10 mm of ODN and
10 mm of PtII complex) adduct production reached more
than 80% within 1 h for both complexes 3 and 4 (Figure S6,
Figure 2. HPLC trace (260 nm) of the products formed during reaction of
complex 2 (10 mm) with 5’-CAGCTG (10 mm) after 24 h of light irradia-
tion (378C). Pt-A and Pt-B are platinum–oligonucleotide adducts.
were similar to that of the products from the reaction of
complex 1 (Figure S3, Supporting Information). They were
attributed also to platinum–DNA adducts. As in the case of
complex 1, the peaks corresponding to the platinated ODNs
increased simultaneously during the reaction, and their for-
mation was quantitative with the decrease of the initial
ODN. Complex 2 proved more reactive than complex 1
(Table 1, entries 3 and 4).
Chem. Eur. J. 2010, 16, 11420 – 11431
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
11423

G. Pratviel et al.
Supporting Information). Thus, reduction of the PtIV com-
plexes 1 and 2 to PtII complexes 3 and 4, respectively, is
likely to account for the formation of the observed platinum
adducts upon incubation of the PtIV complexes with ODN.
Furthermore, since the reactivity of the PtII complexes is
high, the formation of DNA adducts is probably not the
rate-determining step in the reaction of PtIV complexes with
DNA under light irradiation.
Complexes 3 and 4 were compared with cisplatin. The
yield of the cisplatin–ODN adduct was only 20% after 2 h
of incubation of 5’-CAGCTG (10 mm) with cisplatin (10 mm)
at 378C, as opposed to almost total reaction with 3 or 4 (Fig-
ure S8, Supporting Information). However, when cisplatin
was allowed to exchange the chloride ligands with water
molecules before incubation in the presence of ODN, the
yield reached 80% within 1 h, which is comparable to those
of 3 and 4. Under these two reaction conditions cisplatin led
to only one peak of a PtII–DNA adduct on the HPLC trace,
which was eluted before intact ODN (Figure S8, Supporting
Information).
rying one abasic site due to loss of one guanine nucleobase
on the basis of its on-line mass spectrum showing a signal at
828.4 amu.
The yields of reactions in the dark can be calculated in
the same way as those under light irradiation because the
formation of the platinum–DNA adducts was quantitative
on the basis of the decrease of the initial ODN. The forma-
tion of PtII adducts under light irradiation can be explained
by photolysis of PtIV complexes 1 and 2 to the corresponding
PtII species.[25] The origin of PtII adducts in the dark reac-
tions of the PtIV complexes is puzzling. One hypothesis is
traces of PtII in the PtIV complex. However, this is not com-
pletely consistent with the data. Indeed, 2% of 3 may justify
the observed 2.2 mm concentration of adducts when
1
(100 mm) reacts with ODN in the dark for 24 h (Table 1
entry 6 and Figure S10A in the Supporting Information).
Similarly, 5% of 4 may be responsible for the formation of
adducts (5.4 mm) when 2 (100 mm) was incubated with ODN
in the dark (Table 1 entry 8 and Figure S10B in the Support-
ing Information). However, the reaction of 2 (10 mm) in the
dark for 24 h led to 2.6 mm concentration of adducts (Table 1
entry 7 and Figure S10B in the Supporting Information),
which is too high to be accounted for by a concentration of
0.5 mm (5%) of 4. Additionally, the kinetics of the control
reactions involving PtII complex 3 (2 mm) or 4 (5 mm) were
more rapid than the corresponding reactions of 100 mm of
PtIV complex 1 or 2, respectively, in the dark (Figure S11,
Supporting Information).
As previously reported, the 2,2’-bipyridine ligand decreas-
es the electron density and increases the electrophilicity of
the PtII center, which results in increased reaction rates for
nucleophilic substitution reactions.[47]
In summary, the platinum-oligonucleotide adducts formed
during photolysis of PtIV complexes 1 and 2 are adducts of
the PtII species 3 and 4.
Reaction of PtIV complexes 1 and 2 with 5’-CAGCTG in the
dark: heat-promoted formation of Pt-DNA adducts: Control
reactions in the absence of light were performed. Platinu-
m(IV) complexes 1 and 2 were treated with 5’-CAGCTG
(10 mm) in H2O, at 378C in the dark. As under light irradia-
tion, the platinum complex was present either in stoichio-
metric amount (10 mm) with respect to the oligonucleotide
or was added in a tenfold excess (100 mm). Complex 1
(100 mm) led to the formation of the three adducts, Pt-I, Pt-
II, and Pt-III, that were previously observed under light irra-
diation and with PtII complex 3 (Figure S9A, Supporting In-
formation). They formed in the same ratio (45/20/35) but
with lower yield (Table 1, entries 5 and 6 and Figure S10A,
Supporting Information). LC/ESI-MS of the dark reaction
exhibited the usual 1069.2 amu (z=2) signal for Pt-I, Pt-II,
and Pt-III (not shown). An excess of complex 1 with respect
to the ODN was necessary to observe some reaction in the
dark, and no reaction occurred between 1 (10 mm) and 5’-
CAGCTG after 24 h of incubation in the dark.
The dark reactions of 2 with 5’-CAGCTG gave rise to Pt-
A and Pt-B adducts (Figure S9B, Supporting Information).
LC/ESI-MS analysis of the reaction showed the two signals
at 1085.5 and 1069.2 amu (z=2) for Pt-A and Pt-B (not
shown). As for complex 1, the yields of the reactions were
lower in the dark (Table 1, entries 7 and 8 and Figure S10B,
Supporting Information). A minor product (ca. 2% of the
initial ODN after 24 h) eluted before the ODN was evi-
denced in the HPLC trace of the reaction of 2 (100 mm) in
the dark. It was attributed to an oligonucleotide strand car-
Micromolar concentration of reductant in the reaction
medium might account for the generation of PtII species in a
light-independent process. Indeed, ascorbate was able to
reduce PtIV complexes 1 and 2 to the corresponding PtII
complexes. However, the source of electrons in the dark re-
actions of the PtIV complexes is not obvious. Although DNA
might be considered as a possible source of electrons
through guanine oxidation, oxidation of 5’-CAGCTG was
never observed. The abasic site seen with 2 in the dark (Fig-
ure S9B, Supporting Information) is not an oxidation prod-
uct of DNA. Besides, the yield of its formation cannot com-
pare with that of adducts Pt-A and Pt-B.
Alternatively, decomposition of PtIV to PtII complexes in
the dark and at 378C may be heat-promoted instead of
light-promoted (thermolysis instead of photolysis). The PtIV
complexes 1 and 2 were treated with 5’-CAGCTG in the
dark at 508C. Figure 3 and Table S1 in the Supporting Infor-
mation show unambiguously that heating the reaction
medium at 508C in the dark leads to the same kinetics of
platination as light irradiation whatever the concentration of
PtIV complex.
In conclusion, moderate heating (378C, 508C) as well as
light promote decomposition of PtIV complexes 1 and 2 to
PtII complexes 3 and 4, respectively.
Isolation and stability of the oligonucleotide–platinum ad-
ducts of 1 and 2 formed under light irradiation: Oligonu-
cleotide–platinum adducts formed under light irradiation
were isolated by semipreparative HPLC. In the case of ad-
11424
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 11420 – 11431

Platinum(II)–DNA Adducts
FULL PAPER
as Pt-III with some material from Pt-I. From the photolysis
reaction of complex 2, Pt-A and Pt-B were isolated sepa-
rately.
The stability of the ODN–platinum adducts was studied
further on the isolated products. Adducts Pt-I and Pt-II
were incubated separately in 10 mm TEAA buffer pH 6.5 or
10 mm phosphate buffer pH 7.6 at 378C. After 4 h, each iso-
lated adduct was found to be a mixture of the previous
three forms of adducts of the platination reaction and with
the same ratio (45, 20, and 35% for adducts Pt-I, Pt-III and
Pt-II, respectively). These three forms of adducts are clearly
in equilibrium. The same stability test was performed in
H2O containing 100 mm NaCl at 378C. Again, isomerization
took place following the same kinetics, with one pure
adduct at the beginning of the incubation time giving rise to
three products. Equilibrium is reached after 4 h and the mix-
ture was then stable for 24 h. The three adducts were found
to be conformers of platinum bisadducts on purine groups
of 5’-CAGCTG (see NMR data below). Rotation about the
Pt–purine bond is hindered by the 2,2’-bipyridine ligand,
and this leads to slowly interconvertible rotamers. The same
phenomenon was reported before for PtII bisadducts of com-
plexes bearing bulky ligands designed to reduce the dynamic
À
motion by destabilizing the transition state for Pt G rota-
tion.[48]
Similarly, adducts Pt-A and Pt-B were incubated separate-
ly in either 10 mm TEAA buffer pH 6.5 or 10 mm phosphate
buffer pH 7.6 at 378C. They were relatively stable; only
10% of intact 5’-CAGCTG was observed after 24 h of incu-
bation. The two adducts did not interconvert with each
other. No other product, except the unmodified oligonucleo-
tide, was observed. It was identified by its on-line UV/Vis
spectrum and by co-injection with a standard. Contrary to
Pt-I, Pt-II, and Pt-III, adducts Pt-A and Pt-B were sensitive
to chloride ions, with their lability increasing by a factor of
2–3 upon incubation in 100 mm NaCl at 378C. After 24 h
30% 5’-CAGCTG formed. Long-term storage at À208C
also led to 20% labilization of the dried isolated adducts Pt-
A and Pt-B.
Figure 3. Kinetics of the formation of platinum adducts upon photolysis
(empty symbols) compared to thermolysis (filled symbols) for PtIV com-
plexes 1 (A) and 2 (B). Complex 1 (100 mm) and 2 (10 mm) were incubat-
ed with 5’-CAGCTG (10 mm) under light irradiation at 378C (&, *) or
in the dark at 378C (&, *) and 508C (~).
ducts from photolysis of PtIV complex 1, although the HPLC
peaks corresponding to the two major adducts Pt-I and Pt-II
were well separated, neither product was recovered in pure
form after lyophilization. Each product was contaminated
by the other. These ODN–platinum adducts interconvert
slowly. Adducts formed with PtII complex 3 behaved in the
same way. This conversion was avoided by keeping the col-
lected fractions at low temperature (48C).
In summary, as confirmed below by NMR data, bisad-
ducts Pt-I, Pt-II, and Pt-III (from 3 or from photolysis or
thermolysis of 1) are interconvertible rotamers in slow ex-
change. Monoadducts Pt-A and Pt-B (from 4 or from pho-
tolysis or thermolysis of 2) are relatively labile.
The ODN–platinum adducts from photolysis of PtIV com-
plex 2, Pt-A and Pt-B, proved to be labile during the lyophi-
lization step. Lyophilization of the chromatography solvent,
which contained 10 mm triethylammonium acetate (TEAA)
buffer, induced decoordination of platinum and led to 25–
50% of intact 5’-CAGCTG in the dried fractions. This prob-
lem was solved by introducing a desalting step (C18 Sep-
Pak cartridge) before lyophilization. Finally, all adducts
could be isolated in pure form and in suitable amount for
characterization. Three isolated fractions of platinum–oligo-
nucleotide adducts from photolysis of complex 1 were sepa-
rated: one corresponded to the peak referred to as Pt-I, an-
other to Pt-II, and third to a mixture of the peak referred to
NMR characterization of the platinum adducts formed by
photolysis of complex 1: The self-complementary 5’-
CAGCTG oligonucleotide is single-stranded at 310 K in
D2O, as deduced from the temperature dependence of the
1
chemical shifts. Assignments of the H NMR resonances of
the 5’-CAGCTG oligonucleotide were made on the basis of
the established procedure developed for right-handed DNA
duplexes[49] and the chemical shifts are listed in Table S2 in
1
the Supporting Information. The H NMR spectrum of 5’-
CAGCTG is shown in Figure 4A.
1
The H NMR spectrum shown in Figure 4B corresponds
to the mixture of oligonucleotide–platinum adducts obtained
Chem. Eur. J. 2010, 16, 11420 – 11431
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
11425

G. Pratviel et al.
Figure 4. 1H NMR spectra of the aromatic region A) of the 5’-CAGCTG oligonucleotide and B) of the mixture of adducts of 1 and 5’-CAGCTG at 310 K
in D2O. Assignments of H8/H6 base protons of the free 5’-CAGCTG oligonucleotide in (A) and of H8 protons of the platinated guanines of the four ad-
ducts in (B) are indicated.
from isolated Pt-II adduct that was equilibrated in D2O at
room temperature for several days. The binding of the metal
complex to the 5’-CAGCTG oligonucleotide results in large
chemical shift changes of selected oligonucleotide resonan-
ces. Although three different peaks of adducts were seen in
the HPLC trace, four different adducts are evidenced in the
NMR spectrum of the equilibrium mixture (Figure 4B) with
a distribution of 44 (Pt-I), 34 (Pt-II), 12 (Pt-III), and 10%
(referred to as Pt-IV), respectively. The Pt–oligonucleotide
adducts are deduced to be single-stranded at 310 K from the
temperature dependence of the chemical shifts and the
broadening of resonances when lowering the temperature,
due to equilibrium between single-stranded and double-
stranded oligonucleotide.
Owing to the single-stranded nature of adducts and the
elevated temperature, the oligonucleotides have a random
structure and most of the internucleotide NOEs could not
be observed. Furthermore, only very few NOEs between
protons of the oligonucleotide and of the Pt 2,2’-bipyridine
moiety were detected. Chemical-shift changes were only ob-
served for protons in close proximity to the metal complex.
Important chemical shift changes (>0.2 ppm) were only de-
tected for H8 base proton and H1’, H2’, and H2’’ deoxyri-
bose protons of the nucleotides bound to the Pt complex
and for H1’, H2’, and H2’’ deoxyribose protons of their 5’
neighbor residues (Tables S3–S6, Supporting Information).
Base protons of the residues not linked to the metal com-
plex and H3’, H4’, H5’, and H5’’ sugar protons, close to the
phosphodiester backbone, were only slightly shifted by bind-
ing of the Pt complex.
Each set of deoxyribose proton resonances could be as-
signed with 2D TOCSY and 2D ROESY spectra and related
to the corresponding base H8 or H6 protons by means of in-
tranucleotide NOEs. Assignment of cytosine resonances was
made possible by the characteristic correlation between H6
and H5 base protons observed in 2D TOCSY spectra. Dis-
tinction between C(1) and C(4) resonances was based on
the fact that sugar protons of C(1) resonate at lower fre-
quency than those of C(4) due to their position at the 5’-
edge of the nucleotide and on the absence of 31P couplings
for H5’ and H5’’ C(1) protons. Assignments of thymine T(5)
were confirmed by means of correlations between H6 and
methyl base protons detected in 2D TOCSY and 2D
ROESY spectra. Purine resonances were assigned primarily
by comparison with the free 5’-CAGCTG oligonucleotide.
Guanine G(6) resonances could be identified with the aid of
H3’ and H4’ sugar protons that resonate at lower frequency
for guanine G(6) than for guanine G(3) and adenine A(2)
due to their positions at the 3’-edge of the nucleotide. Reso-
nances of adenine A(2) and guanine G(3) were distinguish-
ed on the basis of the H8 proton chemical shift of the purine
base that is not linked to the metal complex (d=8.27 , 8.26,
8.12, and 8.21 ppm for adducts Pt-I, Pt-II, Pt-III, and Pt-IV,
respectively). These chemical shifts are close to the adenine
A(2) H8 resonance (d=8.20 ppm) but seem too shifted
compared to the guanine G(3) H8 resonance (d=7.91 ppm)
11426
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 11420 – 11431

Platinum(II)–DNA Adducts
FULL PAPER
of free 5’-CAGCTG. The assignments of guanine G(3) and
G(6) and adenine A(2) were confirmed in adduct Pt-I by a
weak NOE between H6-C(4) and H2’’-G(3) protons, in
adduct Pt-II by NOEs between H8-G(3) and H1’-A(2) (Fig-
ure S12, Supporting Information) and between H8-G(3) and
H2’’-A(2) protons, and in adduct Pt-IV by NOEs between
H8-G(3) and H1’-A(2) and between H8-G(6) and H1’-T(5)
protons (Figure S12, Supporting Information). Distinction
between resonances of the four different adducts was based
on integration and confirmed by the few internucleotide
NOEs detected. Furthermore, Pt-I and Pt-II and a mixture
of Pt-I and Pt-III adducts were analyzed separately at 279 K
(Figure 5). Assignment of Pt-III versus Pt-IV adducts, which
Figure 6. 1H NMR spectra (guanine-H8 resonances only) of the HPLC
fraction of adduct Pt-II A) at 279 K, B) after 15 h at 279 K, C) 15 min,
D) 30 min, E) 45 min after increasing the temperature to 310 K, and
F) after several days at room temperature (spectrum acquired at 310 K).
NOEs between H8 protons of G(3) and G(6) residues and
H6 and H6’ protons of the 2,2’-bipyridine moiety (Figur-
es S12–S14 and Table S7, Supporting Information).
Although determination of the fine structure of the four
different adducts is beyond the scope of the present study,
their presence could be explained by the possibility of
obtain multiple Pt–oligonucleotide conformers (head-to-
head and head-to-tail), as has been reported for other Pt–
DNA adducts.[50,51] NOEs between H8-G(3) and H8-G(6)
for Pt-IV indicate that Pt-IV is a head-to-head conformer,
while the absence of NOEs between H8-G(3) and H8-G(6)
for Pt-I and Pt-II may imply a head-to-tail conformation for
the two major forms of adducts (Figure S15, Supporting In-
formation). The conformation of Pt-III could not be as-
signed by NOE measurements because the two H8 signals
are too close. No satellite signal for platinum was detected.
Figure 5. 1H NMR spectra (guanine-H8 resonances only) of: HPLC frac-
tion (A) of Pt-I adduct, (B) of Pt-II adduct, and (C) of a mixture of Pt-I
and Pt-III adducts at 279 K in D2O.
are in close ratio at 310 K, could be validated (Tables S8–
S10, Supporting Information). Increasing the temperature
from 279 to 310 K restored the distribution of the four Pt-5’-
CAGCTG adducts after several minutes (Figure 6), support-
ing the hypothesis that adducts interconvert slowly to reach
equilibrium.
1
The absence of coupling between H and 195Pt suggests a +2
In summary, we were able to detect and assign four differ-
ent oligonucleotide platinum adducts isolated from the pho-
tolysis of complex 1 with 5’-CAGCTG that interconvert
slowly at 310 K in D2O. The four adducts are bis-adducts
and show platination of G(3) and G(6) nucleotides of 5’-
CAGCTG. No binding to the A(2) residue was detected, no-
tably confirmed by the fact that only very slight shifts of the
C(1) proton resonances were detected for the four Pt–ODN
adducts (Tables S3–S6, Supporting Information). Pt binding
through the N7 position of guanine, as expected, is validated
by 1) the high-frequency shifts of H8 resonances of G(3)
and G(6) residues (between 0.5 and 1.0 ppm) and 2) by
oxidation state for platinum adducts, although an unambigu-
ous assignment is not possible.
Enzymatic digestion of the platinum adducts from photolysis
of complex 2: Under light irradiation, the reaction of com-
plex 2 with 5’-CAGCTG gives rise to two platinum adducts,
Pt-A and Pt-B. The two adducts were isolated as pure sam-
ples and subjected separately to enzymatic digestion by
phosphodiesterase I (followed by alkaline phosphate). Phos-
phodiesterase I from snake venom removes nucleotide resi-
dues from the 3’-OH terminus of oligonucleotides in the
form of nucleotides. Alkaline phosphatase removes the 5’-
Chem. Eur. J. 2010, 16, 11420 – 11431
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
11427

G. Pratviel et al.
phosphate of nucleotides. Pt-A proved completely resistant
to digestion. The presence of intact Pt-A adduct at the end
of the digestion time was checked by negative LC/ESI-MS
This process is accompanied by loss of the axial ligands and
release of HOCl due to reductive elimination of HOCl or
reductive elimination of Cl2 and further reaction of Cl2 with
H2O.[25] The release of two axial ligands from PtIV complexes
is likely to be quantitative with respect to the generated PtII
species upon light irradiation. In this work, photolysis of
PtIV complexes 1 and 2 was carried out in the presence of
the short oligonucleotide 5’-CAGCTG. Upon photolysis,
PtIV complexes gave rise to PtII–ODN adducts identical to
those identified with their respective PtII complexes. Com-
plex 1 was at the origin of complex 3, which produced a
mixture of four conformers of bis-adducts with N7 of gua-
nine bases G(3) and G(6) of 5’-CAGCTG. Complex 2 trans-
formed into complex 4, which formed two mono-adducts at
G(3) or G(6) of 5’-CAGCTG. For all these adducts, the sub-
stitution reactions involved the chlorido ligands. The 2,2’-bi-
pyridine ligand was still present in the bis-adducts PtII-ODN
adducts of 3. In the case of 4, the PtII–ODN adducts carried
one methylamine ligand and one 2,2’-bipyridine ligand. In
the HPLC traces the folded bis-adducts were eluted first,
and the linear mono-adducts after intact ODN 5’-CAGCTG.
Photoisomerization of complex 2 involving migration of
the methylamine ligand to an axial position was evidenced
in previous work.[25] In the present work we did not observe
the formation of complex 3 upon light irradiation of 2. Pho-
tolysis of 2 in the presence of 5’-CAGCTG never produced
Pt-I, Pt-II, and Pt-III DNA bis-adducts. It may be concluded
that PtIV complex with an axial methylamine ligand do not
undergo photolysis.
analysis. The expected signals at m/z 1085.5 for an [Pt
bpy)
(MeNH2)ODN]2À ion associated with a signal at m/z
1069.5 for [Pt
(2,2’-bpy)ODN]2À ion were observed. These
ACHTUNGTRNE(NUNG 2,2’-
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
data allowed us to propose that Pt-A consists of an oligonu-
cleotide with a platinum ion covalently bound at N7 of gua-
nine G(6) at the 3’-end of 5’-CAGCTG.
Figure 7 displays the HPLC trace of the products of enzy-
matic digestion of Pt-B (Figure 7B, C) compared to those of
5’-CAGCTG (Figure 7A). Digestion of Pt-B shows three
We observed that the PtIV complexes were not stable at
378C and transformed spontaneously into their correspond-
ing PtII complexes in the dark. The dark decomposition of
PtIV complexes increased with increasing temperature. Thus,
reduction of the PtIV bipyridine complexes can be promoted
either by light irradiation or heat. Heating at 508C had the
same efficiency as light irradiation under the tested experi-
mental conditions. Complex 2 was more susceptible to spon-
taneous reduction than complex 1.
Figure 7. HPLC trace of the enzymatic digestion of enzymatic digestion
of 5’-CAGCTG (A) and adduct Pt-B (B,C).
We found that the reaction of the PtII complexes 3 and 4
(reduction products of 1 and 2, respectively) with 5’-
CAGCTG was faster than that of cisplatin. The rapid and
quantitative reaction of PtII complexes with DNA allowed
us to estimate the yield of spontaneous reduction (photolysis
and/or thermolysis) of the PtIV complex. Indeed, the quanti-
ty of PtII complex generated upon photolysis of the PtIV
complexes at 378C can be measured by means of the quanti-
ty of PtII–ODN adducts. Light irradiation of complexes 1
and 2 (10 mm) for 24 h yielded 29 and 46% of PtII complex
with respect to the starting PtIV complex. At higher concen-
tration of PtIV, the oligonucleotide became a limiting factor
under the present conditions.
nucleosides dC, dG, and dT associated with a peak at 28 min
whose mass spectrum in positive LC/ESI-MS analysis is con-
sistent with a platinated trinucleotide 5’-CAG. Two main sig-
nals at m/z 1219.3 and 1250.1 account for [Pt
ACHTUNGTRENUN(NG 2,2’-bpy)-
A
E
N
ACHTUNGTRENNUNG
ions, respectively (Figure S16, Supporting Information). The
unmodified trinucleotide 5’-CAG was also detected in the
on-line mass spectrum of the trinucleotide adduct at m/z
870.5 due to adduct lability during mass analysis. Adduct Pt-
B is thus a mono-adduct at G(3) in the middle of 5-
CAGCTG with the platinum ion coordinated to a 2,2’-bipyr-
idine ligand, a methylamine ligand, and N7 of guanine.
Oxidative DNA damage was never evidenced when PtIV
complexes 1 and 2 reacted with 5’-CAGCTG. The retention
times and the mass data of oxidized ODN were known from
previous work on 5’-CAGCTG.[39,40] Formation of an oxi-
dized oligonucleotide strand (even 1% yield, 0.1 mm) would
have been detected by the method of analysis. The yield of
Discussion
It was previously reported that PtIV complexes 1 and 2 are
reduced to the corresponding PtII species upon photolysis.
11428
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 11420 – 11431

Platinum(II)–DNA Adducts
FULL PAPER
Platinum complexes: [PtCl
G
3ACHUTGTNREN(NUG 2,2’-bpy)ACHTUNGTNER(NUGN MeNH2)]Cl
spontaneous reduction determines the subsequent concen-
tration of diffusible oxidative species from the released axial
ligands (HOCl or Cl2). When the photolysis reaction was
performed at 10 mm of PtIV complexes, about 3 and 5 mm of
PtII complex formed with 1 and 2, respectively. Consequent-
ly, no more than 3–5 mm of diffusible oxidative species is ex-
pected. Increasing the concentration of PtIV complex did not
lead to detectable oxidative DNA damage either. The PtIV
complexes were incubated at a concentration of 100 mm with
5’-CAGCTG (10 mm). The concentrations of PtII–oligonu-
cleotide adducts were 5.7 mm for 1 after 24 h and 7.4 mm for
2 after 3 h, that is, at least 6–8 mm of PtII complex and associ-
ated potential oxidant were produced during photolysis. In
no case was the concentration of diffusible oxidative species
(HOCl or Cl2) high enough to allow us to detect oxidative
DNA damage.
(2), [PtCl2A(2,2’-bpy)] (3), and [PtCl
E
pared according to published procedures.[25] They were characterized by
NMR spectroscopy before use. They were never stored in solution to
avoid ligand exchange. From an initial solution of complex in acetonitrile
(3, 4) or in H2O (2), aliquots containing 100 nmol or 200 nmol were pre-
pared in Eppendorf tubes and immediately dried under vacuum. Dry ali-
quots were stored in the absence of light at À208C. New solutions of
complex 1, dissolved in DMSO, were prepared before each experiment.
UV/Vis [lmax/nm (e/mÀ1 cmÀ1)] of 1 in acetonitrile: 307 (10500), 319
(9500); 2 in H2O: 308 (11000), 320 (11400); 3 in acetonitrile: 276
(18000), 312 (7000), 323 (8600); 4 in acetonitrile: 250 (15500), 308
(9700), 321 (13200).
Electrochemical experiments: Voltammetric measurements were carried
out with a potentiostat Autolab PGSTAT100. Experiments were per-
formed at room temperature in a homemade airtight three-electrode cell
connected to a vacuum/argon line. The reference electrode consisted of a
saturated calomel electrode (SCE) separated from the solution by a
bridge compartment. The counterelectrode was a platinum wire of ca
1 cm2 apparent surface area. The working electrode was a glassy carbon
microdisk (0.5 mm diameter). In aqueous media, the platinum complex
was dissolved to a final concentration of 1 mm in 0.1m KCl, and the pH
was adjusted to 7.0. In aprotic media, the supporting electrolyte tetrabu-
tylammonium tetrafluoroborate (Fluka, 99% puriss electrochemical
grade) and the solvent (DMSO) were used as received. Acetonitrile was
freshly distilled over Na/benzophenone prior to use. The solutions used
during the electrochemical studies were typically 1 mm in complex and
0.1m in supporting electrolyte. Before each measurement, the solutions
were degassed by bubbling Ar, and the working electrode was polished
with a polishing machine (Presi P230). Experimental peak potentials
were measured versus SCE and converted to the NHE scale by adding
0.242 V.
On the other hand, the PtIV 2,2’-bipyridine complexes
were inert toward substitution reactions with 5’-CAGCTG.
Thus, despite the fact that the 2,2’-bipyridine ligand confers
to the PtIV complexes a high redox potential, no guanine ox-
idation by inner-sphere electron transfer was possible, since
neither the intrastrand G nor 3’-G of 5’-CAGCTG coordi-
nated to the PtIV center. Rather, reductive elimination to
PtII species was the preferred pathway.
Conclusion
HPLC and HPLC coupled to electrospray mass analysis (LC/ESI-MS):
The HPLC apparatus was a Hewlett Packard 1050 equipped with a
diode-array detector. The oligonucleotide reactions were analyzed on a
reverse-phase Nucleosil column (C18, 10 mm, 250ꢃ4.6 mm from Inter-
chim, France) eluted with two different gradients: gradient 1: linear gra-
dient from 5 to 12% of acetonitrile in 10 mm triethylammonium acetate
buffer (TEAA), pH 6.5 for 20 min, followed by a linear gradient from 12
to 30% acetonitrile for 5 min; gradient 2: linear gradient from 5 to 10%
of acetonitrile in 10 mm TEAA, pH 6.5 for 50 min, followed by a linear
gradient from 10 to 30% acetonitrile for 5 min. Gradient 1 was used for
analysis of the products of the reactions of complexes 2 and 4, and gradi-
ent 2 for the products formed by complex 1, complex 3, and cisplatin.
The enzymatic digestion media were analyzed on a reverse-phase Nucle-
osil column (C18, 5 mm, 250ꢃ4.6 mm from Interchim, France) with gradi-
ent 1. The flow rate was 1 mLminÀ1 and detection was at l=260 and
320 nm. LC/ESI-MS analysis was carried out with the same column,
which was coupled to a Perkin-Elmer SCIEX-API 365 spectrometer
equipped with a turbo ion spray source (negative mode for oligonucleo-
tides, positive mode for nucleosides or trinucleotides). The temperature
of the gas (N2) stream was set at 4508C. The chromatography conditions
of LC/ESI-MS remained the same as above, but the flow was reduced to
0.5 mLminÀ1. All the samples were directly injected into the HPLC
system. However, in the case of enzymatic digestions a pre-column (re-
verse-phase) was added ahead of the column.
Photolysis (l>300 nm) and thermolysis in the dark (37–
508C) of PtIV 2,2’-bipyridine complexes 1 and 2 led to reduc-
tive formation of the corresponding PtII complexes. In the
presence of oligonucleotide 5’-CAGCTG, PtII complexes
readily formed typical DNA–platinum(II) adducts. All ad-
ducts carried the 2,2’-bipyridine ligand and involved guanine
N7 as binding site. When the PtII species had two chlorido li-
gands, bisadducts at G(3) and G(6) of 5-CAGCTG were ob-
served. When the PtII species had only a single chlorido
ligand, in addition to a methylamine and a 2,2’-bipyridine
ligand, only monoadducts formed.
The extent of spontaneous reduction under light irradia-
tion at 378C or in the dark at 508C after 24 h was identical
and reached about 30 and 50% with respect to PtIV com-
plexes 1 and 2, respectively. The PtIV 2,2’-bipyridine com-
plexes 1 and 2 proved inert toward substitution. The ques-
tion of the stability of other photoactivatable PtIV complexes
under conditions of moderate warming deserves further
studies.
Reaction of the Pt complexes with oligonucleotide 5’-CAGCTG: The oli-
gonucleotide (ODN) was stored as a 2 mm solution in H2O at À208C.
For the platinum complexes 1, 3, and 4, a 1 or 10 mm fresh solution of
the platinum complex was prepared before each reaction by dissolving
100 nmol of the complex in 100 or 10 mL of DMSO, respectively. In the
case of complex 2 the same dilutions were done in H2O before each reac-
tion. The reaction medium was prepared by mixing 50 mL of a 100 mm
ODN solution, 5 mL of the 1 mm (or 10 mm) platinum complex solution,
and 445 mL of H2O. The final concentrations of the ODN and the plati-
num complexes were 10 and 10 or 100 mM for 1:1 or 1:10 reactions, re-
spectively. Except in the case of complex 2, the reaction contained 1%
DMSO. The reactions samples (in glass tubes) were incubated at 378C in
Experimental Section
Chemicals: H2O was of milliQ grade (Millipore). Oligodeoxyribonucleo-
tide (ODN) 5’-CAGCTG was from Eurogentec SA (Belgium) and puri-
fied by HPLC if necessary. Concentration was determined by UV at
[52]
260 nm, e=55 000mÀ1 cmÀ1
.
Cisplatin was purchased from Merck.
Phosphodiesterase I from Crotalus adamanteus venom was purchased
from USB. Phosphatase, alkaline from Escherichia coli was obtainedfrom
Sigma-Aldrich.
Chem. Eur. J. 2010, 16, 11420 – 11431
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
11429

G. Pratviel et al.
a glass, thermostated water bath in the dark or under light irradiation. A
halogen lamp (40 W, Philips krypton) was mounted 10 cm to the side of
samples. Light illumination through glass dishes removes light of wave-
lengths below about 300 nm.
[15] L. R. Kelland, P. Mistry, G. Abel, S. Y. Loh, C. F. OꢄNeill, B. A.
Murrer, K. R. Harrap, Cancer Res. 1992, 52, 3857–3864.
[16] S. Ishida, J. Lee, D. J. Thiele, I. Herskowitz, Proc. Natl. Acad. Sci.
USA 2002, 99, 14298–14302.
[17] K. R. Barnes, A. Kutikov, S. J. Lippard, Chem. Biol. 2004, 11, 557–
564.
[18] W. H. Ang, I. Khalaila, C. S. Allardyce, L. Juillerat-Jeanneret, P. J.
Dyson, J. Am. Chem. Soc. 2005, 127, 1382–1383.
[19] S. Dhar, Z. Liu, J. Thomale, H. Dai, S. J. Lippard, J. Am. Chem. Soc.
2008, 130, 11467–11476.
[20] S. Dhar, F. X. Gu, R. Langer, O. C. Farokhzad, S. J. Lippard, Proc.
Natl. Acad. Sci. USA 2008, 105, 17356–17361.
[21] N. A. Kratochwil, J. A. Parkinson, P. J. Bednarski, P. J. Sadler,
Angew. Chem. 1999, 111, 1566–1569; Angew. Chem. Int. Ed. 1999,
38, 1460–1463.
[22] P. Mꢅller, B. Schrçder, J. A. Parkinson, N. A. Kratochwil, R. A.
Coxall, A. Parkins, S. Parsons, P. J. Sadler, Angew. Chem. 2003, 115,
349–353; Angew. Chem. Int. Ed. 2003, 42, 335–339.
[23] P. J. Bednarski, R. Grꢅnert, M. Zielzki, A. Wellner, F. S. Mackay,
P. J. Sadler, Chem. Biol. 2006, 13, 61–67.
[24] F. S. Mackay, J. A. Woods, P. Heringova, J. Kasparkova, A. M. Pizar-
ro, S. A. Moggach, S. Parsons, V. Brabec, P. J. Sadler, Proc. Natl.
Acad. Sci. USA 2007, 104, 20743–20748.
[25] Y. Nakabayashi, A. Erxleben, U. Lꢀtinois, G. Pratviel, B. Meunier,
L. Holland, B. Lippert, Chem. Eur. J. 2007, 13, 3980–3988.
[26] L. Ronconi, P. J. Sadler, Chem. Commun. 2008, 235–237.
[27] N. A. Kratochwil, M. Zabel, K.-J. Range, P. J. Bednarski, J. Med.
Chem. 1996, 39, 2499–2507.
Isolation of individual platinum adducts: The different adducts were col-
lected when they were eluted from the chromatography column and im-
mediately cooled in ice. The salts of the HPLC buffer were removed by a
desalting step (C18 Sep-Pak cartridge from Waters). The desalting proce-
dure consisted of dilution of the fraction with 10 mL H2O, loading of the
sample on the cartridge, washing with 15 mL H2O, and elution of oligo-
nucleotide with 5 mL H2O/CH3CN (95:5). After lyophilization, the dried
material was dissolved in H2O and quantified by UV/Vis spectroscopy,
and the adducts were stored dry, as 1 nmol aliquots after a second lyophi-
lization step. For NMR characterization, several reactions were carried
out on a 10-fold scale, adducts were collected from HPLC, and fractions
were pooled together until about 100 nmol of each adduct was isolated.
NMR spectra: NMR spectra were recorded on a Bruker Avance 600
spectrometer equipped with a 5 mm triple resonance inverse Z-gradient
cryoprobe. All chemical shifts for 1H are relative to the residual HOD
1
resonance (relative to TMS). H NMR spectra were collected at 310 K in
D2O. Presaturation was used to suppress the residual water signal. The
2D T-ROESY spectra were acquired with a mixing time of 250 ms and
TOCSY spectra were recorded with a spin-lock time of 60 ms and 90 ms.
Typically, 2048 t2 data points were collected for 512 t1 increments. Spectra
processing was performed using Bruker Topspin software.
Enzymatic digestion: Each isolated, pure, platinum adduct Pt-A, Pt-B,
and 5’-CAGCTG (1 nmol) was incubated with phosphodiesterase I (0.03
unit) in 0.18 mL (final volume) 100 mm Tris-HCl buffer pH 8.6, 100 mm
NaCl, 14 mm MgCl2 for 1 h at 378C. The reaction was quenched at 658C
for 5 min, then 0.7 units of phosphatase alkaline was added and the
sample was kept at 378C for 1 h. The analysis of the digestion products
was performed by HPLC.
[28] F. S. Mackay, J. A. Woods, H. Moseley, J. Ferguson, A. Dawson, S.
Parsons, P. J. Sadler, Chem. Eur. J. 2006, 12, 3155–3161.
[29] H. I. A. Phillips, L. Ronconi, P. J. Sadler, Chem. Eur. J. 2009, 15,
1588–1596.
[30] N. J. Farrer, J. A. Woods, V. P. Munk, F. S. Mackay, P. J. Sadler,
Chem. Res. Toxicol. 2010, 23, 413–421.
[31] F. S. Mackay, N. J. Farrer, L. Salassa, H.-C. Tai, R. J. Deeth, S. A.
Moggach, P. A. Wood, S. Parsons, P. J. Sadler, Dalton Trans. 2009,
2315–2325.
[32] N. Gomez Blanco, C. R. Maldonado, J. C. Mareque-Rivas, Chem.
Commun. 2009, 5257–5259.
[33] S. Choi, R. B. Cooley, A. S. Hakemian, Y. C. Larrabee, R. C. Bunt,
S. D. Maupas, J. G. Muller, C. J. Burrows, J. Am. Chem. Soc. 2004,
126, 591–598.
[34] S. Choi, R. B. Cooley, A. Voutchkova, C. H. Leung, L. Vastag, D. E.
Knowles, J. Am. Chem. Soc. 2005, 127, 1773–1781.
Acknowledgements
This work was supported by a joint CNRS/DFG grant. Dr. Pablo Sanz
Miguel is acknowledged for the preparation of complex 1 and Laurianne
Benoist for technical assistance. HPLC/ESI-MS data were obtained from
the Service de Spectromꢀtrie de Masse de l’Universitꢀ Paul Sabatier
FR14-LCC-CNRS Toulouse, operated by Catherine Claparols. Cyclic vol-
tammetry was carried out by Alix Sournia-Saquet (LCC).
[35] S. Choi, L. Vastag, C. H. Leung, A. M. Beard, D. E. Knowles, J. A.
Larrabee, Inorg. Chem. 2006, 45, 10108–10114.
[36] S. Choi, L. Vastag, Y. C. Larrabee, M. L. Personick, K. B. Schaberg,
B. J. Fowler, R. K. Sandwick, G. Rawji, Inorg. Chem. 2008, 47,
1352–1360.
[1] B. Rosenberg, L. VanCamp, T. Krigas, Nature 1965, 205, 698.
[2] E. R. Jamieson, S. J. Lippard, Chem. Rev. 1999, 99, 2467–2498.
[3] J. Reedijk, Chem. Rev. 1999, 99, 2499–2510.
[37] S. Steenken, S. Jovanovic, J. Am. Chem. Soc. 1997, 119, 617–618.
[38] R. M. Roat, M. J. Jerardi, C. B. Kopay, D. C. Heath, J. A. Clark,
J. A. DeMars, J. M. Weaver, E. Bezemer, J. Reedijk, J. Chem. Soc.
Dalton Trans. 1997, 3115–3621.
[39] C. Vialas, C. Claparols, G. Pratviel, B. Meunier, J. Am. Chem. Soc.
2000, 122, 2157–2167.
[40] A. Kupan, A. Sauliꢆre, S. Broussy, C. Seguy, G. Pratviel, B. Meunier,
ChemBioChem 2006, 7, 125–133.
[41] J. A. Weinstein, N. N. Zheligovskaya, M. Y. Mel’nikov, F. Hartl, J.
Chem. Soc. Dalton Trans. 1998, 2459–2466.
[42] S. Choi, C. Filotto, M. Bisanzo, S. Delaney, D. Lagasee, J. L. Whit-
worth, A. Jusko, C. Li, N. A. Wood, J. Willingham, A. Schwenker,
K. Spaulding, Inorg. Chem. 1998, 37, 2500–2504.
[43] E. Reisner, V. B. Arion, B. K. Keppler, A. J. L. Pombeiro, Inorg.
Chim. Acta 2008, 361, 1569–1583.
[4] P. J. OꢄDwyer in Cisplatin-Chemistry and Biochemistry of a Leading
Anticancer Drug (Ed.: B. Lippert), Wiley-VCH, Weinheim, 1999,
pp. 31–72.
[5] E. Wong, C. M. Giandomenico, Chem. Rev. 1999, 99, 2451–2466.
[6] L. R. Kelland in Cisplatin-Chemistry and Biochemistry of a Leading
Anticancer Drug (Ed.: B. Lippert), Wiley-VCH, Weinheim, 1999,
pp. 497–521.
[7] M. D. Hall, H. R. Mellor, R. Callaghan, T. W. Hambley, J. Med.
Chem. 2007, 50, 3403–3411.
[8] S. H. van Rijt, P. J. Sadler, Drug Discovery Today 2009, 14, 1089–
1097.
[9] A. V. Klein, T. W. Hambley, Chem. Rev. 2009, 109, 4911–4920.
[10] T. Shi, J. Berglund, L. I. Elding, Inorg. Chem. 1996, 35, 3498–3503.
[11] R. N. Bose, E. L. Weaver, J. Chem. Soc. Dalton Trans. 1997, 1797–
1799.
[44] A. B. P. Lever, Inorg. Chem. 1990, 29, 1271–1285.
[45] S. Choi, S. Mahalingaiah, S. Delaney, N. R. Neale, S. Masood, Inorg.
Chem. 1999, 38, 1800–1805.
[46] P. Bigey, S. Frau, C. Loup, C. Claparols, J. Bernadou, B. Meunier,
Bull. Soc. Chim. Fr. 1996, 133, 679–689.
[12] K. Lemma, T. Shi, L. I. Elding, Inorg. Chem. 2000, 39, 1728–1734.
[13] M. D. Hall, C. T. Dillon, M. Zhang, P. Beale, Z. Cai, B. Lai, A. P. J.
Stampfl, T. W. Hambley, J. Biol. Inorg. Chem. 2003, 8, 726–732.
[14] L. R. Kelland, B. A. Murrer, G. Abel, C. M. Giandomenico, P.
Mistry, K. R. Harrap, Cancer Res. 1992, 52, 822–828.
11430
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 11420 – 11431

Platinum(II)–DNA Adducts
FULL PAPER
[47] N. Summa, W. Schiessl, R. Puchta, N. van Eikema Hommes, R. va-
n Eldik, Inorg. Chem. 2006, 45, 2948–2959.
[51] V. Beljanski, J. M. Villanueva, P. W. Doetsch, G. Natile, L. G. Mar-
zilli, J. Am. Chem. Soc. 2005, 127, 15833–15842.
[48] G. Natile, L. G. Marzilli, Coord. Chem. Rev. 2006, 250, 1315–1331.
[49] R. M. Scheek, N. Russo, R. Boelens, R. Kaptein, J. H. Van Boom, J.
Am. Chem. Soc. 1983, 105, 2914–2916.
[52] G. D. Fasman, Handbook of Biochemistry and Molecular Biology-
Nucleic Acids, 3rd ed, CRC Press, Boca Raton, 1975.
[50] D. Bhattacharyya, P. A. Marzilli, L. G. Marzilli, Inorg. Chem. 2005,
44, 7644–7651.
Received: April 5, 2010
Published online: August 16, 2010
Chem. Eur. J. 2010, 16, 11420 – 11431
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
11431