Genotoxic potential of N-(benzothiazolyl)sulfonamide copper(II) complexes on yeast cells transformed with YEGFP expression constructs containing the RAD54 or RNR2 promoter

Article abstract of DOI:10.1002/ejic.200600305

Four ternary complexes [Cu(L)₂(phen)] where L is an N-(benzothiazol-2-yl)sulfonamide derivative have been prepared and their ability to cleave DNA has been studied. The complexes were structurally characterized with the aid of single-crystal X-ray crystallography. Whereas the molecular structure of the [Cu(L1)₂(phen)] (1) [HL1 = N-(6-chlorobenzothiazol- 2-yl)benzenesulfonamide] and [Cu(L3)₂(phen)] (3) [HL3 = N-(benzothiazol-2-yl)benzenesulfonamide] complexes can best be described as having a distorted square-planar geometry, that of the [Cu(L4) ₂(phen)] (4) [HL4 = N-(benzothiazol-2-yl)toluenesulfonamide] complex shows a strictly square-planar geometry. The [Cu(L2)₂(phen)MeOH] (2) [HL2 = N-(6-chlorobenzothiazol-2-yl)toluenesulfonamide] complex displays an axially elongated square-pyramidal coordination geometry in which the phen ligand binds at the basal plane. Viscosity and fluorescence measurements indicated that [Cu(L4)₂(phen)] (4) has a propensity for binding calf thymus DNA. The four complexes were found to be efficient chemical nucleases, with ascorbate/H₂O₂ activation giving rise to hydroxyl and superoxide radicals as active cleaving species. The nuclease activity of 4 is not only the highest of the four complexes, but also much higher than that of the copper-phenanthroline complex. The ability of the complexes to cleave DNA within cells has been tested by monitoring the expression of the yEGFP gene containing reporter plasmid. The significant induction of fluorescence by complex 4 indicates that it is able to cleave DNA inside the cell. Wiley-VCH Verlag GmbH & Co. KGaA, 2006.

Full text of DOI:10.1002/ejic.200600305

FULL PAPER
DOI: 10.1002/ejic.200600305
Genotoxic Potential of N-(Benzothiazolyl)sulfonamide Copper(II) Complexes
on Yeast Cells Transformed with YEGFP Expression Constructs Containing
the RAD54 or RNR2 Promoter
Marta González-Álvarez,[a] Gloria Alzuet,[a] Lucas del Castillo,[b] Joaquín Borrás,*[a] and
Malva Liu-González[c]
Keywords: Copper complexes / DNA damage / Genotoxicity / yEGFP expression
Four ternary complexes [Cu(L)2(phen)] where L is an N-
(benzothiazol-2-yl)sulfonamide derivative have been pre-
pared and their ability to cleave DNA has been studied. The
complexes were structurally characterized with the aid of
single-crystal X-ray crystallography. Whereas the molecular
structure of the [Cu(L1)2(phen)] (1) [HL1 = N-(6-chlorobenzo-
thiazol-2-yl)benzenesulfonamide] and [Cu(L3)2(phen)] (3)
basal plane. Viscosity and fluorescence measurements indi-
cated that [Cu(L4)2(phen)] (4) has a propensity for binding
calf thymus DNA. The four complexes were found to be ef-
ficient chemical nucleases, with ascorbate/H2O2 activation
giving rise to hydroxyl and superoxide radicals as active
cleaving species. The nuclease activity of 4 is not only the
highest of the four complexes, but also much higher than that
of the copper–phenanthroline complex. The ability of the
complexes to cleave DNA within cells has been tested by
monitoring the expression of the yEGFP gene containing re-
porter plasmid. The significant induction of fluorescence by
complex 4 indicates that it is able to cleave DNA inside the
cell.
[HL3
=
N-(benzothiazol-2-yl)benzenesulfonamide] com-
plexes can best be described as having a distorted square-
planar geometry, that of the [Cu(L4)2(phen)] (4) [HL4 = N-
(benzothiazol-2-yl)toluenesulfonamide] complex shows
a
strictly square-planar geometry. The [Cu(L2)2(phen)MeOH]
(2) [HL2 = N-(6-chlorobenzothiazol-2-yl)toluenesulfonamide]
complex displays an axially elongated square-pyramidal co-
ordination geometry in which the phen ligand binds at the
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
leads to DNA damage. It has been shown, for example, that
in phosphate buffer CuII/H2O2 induces DNA damage at the
thymine and guanine positions by generating ROS.[6] More-
over, copper has a dual role in oxidative DNA damage: it
generates ROS through autoxidation of carcinogens and
also activates H2O2 to form a reactive CuI–hydroperoxo
complex.
Introduction
The mechanism of metal carcinogenesis has been widely
studied since a number of metals are known to have carcin-
ogenic potential. In 1986, for example, Kawanishi et al.[1]
proposed the hypothesis that metal carcinogenesis involves
the generation of endogenous reactive oxygen species
(ROS). These same researchers subsequently reported that
various carcinogenic metal compounds are capable of caus-
ing oxidative DNA damage in the presence of H2O2.[2–5]
Indeed, metals are considered to act not only as carcino-
gens, but also as cocarcinogens that activate carcinogenic
chemicals, since a large number of weak, nonmutagenic car-
cinogens have been found to act through metal-mediated
oxidative DNA damage. Endogenous metals ions such as
copper and iron seem to play a particularly important role
in ROS generation from various carcinogens, which in turn
In recent years the interaction of transition metal com-
plexes with nucleic acids has attracted attention because of
their relevance in the development of new reagents for bio-
technology and medical research. Interest in the rational
design of novel transition metal complexes that bind and
cleave duplex DNA with high sequence or structure selec-
tivity has been especially keen. Indeed, there is already a
considerable amount of literature that deals with the practi-
cal use of transition metal complexes as chemical nucleases.
The redox activity of copper complexes of 1,10-phenan-
throline as artificial nucleases, for instance, is well
known.[7,8] Meunier et al.[9] even developed the Clip-Phen
series containing two phen moieties linked through tethers
in order to favor the stoichiometry of two phen units per
copper. These authors showed that the DNA cleavage ac-
tivity of copper Clip-Phen complexes increased dramati-
cally in relation to that of the phen parent compound. They
then went on to determine the cytostatic activity of these
[a] Departament de Química Inorgànica, Facultat de Farmàcia,
Universitat de València,
Avda. Vicente Andrés Estellés s/n, 46100 Burjassot, Spain
Fax: +34-963544960
E-mail: Joaquin.Borras@uv.es
[b] Departament de Microbiologia i Ecologia, Facultat de Farmà-
cia, Universitat de València,
Avda. Vicente Andrés Estellés s/n, 46100 Burjassot, Spain
[c] SCSIE, RX. Universitat de València,
Dr. Moliner 50, 46100 Burjassot, Spain
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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M. González-Álvarez, G. Alzuet, L. del Castillo, J. Borrás, M. Liu-González
FULL PAPER
complexes.[10] Our group has previously reported[11] several gene in cells containing the reporter plasmid in response to
copper ternary complexes of N-substituted sulfonamides the exposure to DNA-damaging agents is thus monitored
and phen with the ability to damage DNA in the presence by noting the presence of its characteristic green fluores-
of various reducing agents.
cence.
In this paper we describe the synthesis, characterization,
A great number of chemical substances are known to
possess in vitro biocatalytic activity, emulating different en- and biological activity assays of four copper ternary com-
zymatic processes. Among these chemicals, copper-coordi- plexes with N-substituted sulfonamides and o-phenan-
nated compounds have shown superoxide dismutase and/or throline. The sulfonamide ligands selected for this study
DNAsa activities, depending on the type of ligand present. (Scheme 1) possess two main features that facilitate the syn-
However, in vitro activity occurs under conditions that are thesis of copper(II) complexes with nuclease activity; both
usually not equivalent to the physiological environment in a benzothiazole ring and an aromatic ring (benzene or tolu-
the cell. This is an important consideration in evaluating ene) able to intercalate between the DNA bases, as well as
research in this field, as, in order to have any effect, the a sulfonamido group that can interact with DNA through
chemical product in question needs to penetrate within the hydrogen bonds. In addition, the benzenesulfonamido
cell itself. Once the substance is in the cytoplasm, the dif- group has been suggested to be a functional element that
ferent mechanisms of cell detoxification can reduce or even facilitates various biological activities such as the inhibition
prevent its activity, either through degradation or extrusion of tumor cell growth.[19] The biological activity studies in-
out of the cell or to intracellular compartments.
clude evaluations of DNA binding ability, nuclease activity,
In the past, yeast has been used as a model for under- and the ability to damage DNA in yeast cells transformed
standing physiological processes in superior eukaryotes and with yEGFP expression constructs containing either the
it is still considered to be an ideal organism for in vivo RAD54 or the RNR2 promoter.
studies on the activity of biological compounds. Our group
has used an S. cerevisiae strain deficient in superoxide dis-
Results and Discussion
mutase for in vivo evaluation of the SOD-like activity of
various copper complexes.[12,13] Induction of different yeast
genes in response to exposure to DNA-damaging agents
represents an important and promising approach for de-
Spectroscopic and Structural Aspects
Ternary copper(II) complexes [Cu(L)2phen] (L = L1, L2,
veloping genotoxicity tests. Induction of RAD54 and L3, and L4) were prepared and characterized with the aid
RNR2, two promoters that control the expression of dam- of analytical, spectroscopic, and X-ray diffraction data.
age-inducible genes, has already been used successfully to
All the complexes had the same infrared spectrum
evaluate DNA damage.[14–17] We have gone one step further pattern, and the most significant feature was a shift of the
and developed reporter plasmids that express the green flu- characteristic band of the benzothiazole ring from
orescent protein yEGFP gene.[18] The expression of this 1550 cm–1 in the free ligands to 1460 cm–1 in the complexes.
As for the sulfonamidato group, the bands assigned to
ν(SO2)as and ν(SO2)s are shifted to slightly lower frequencies
while the ν(S–N) vibration appears at slightly higher fre-
quencies than those of the uncoordinated ligands. In gene-
ral, the IR spectra are similar to those observed for other
copper N-sulfonamide derivatives[20,21] with the modifica-
tions noted above being mainly because of the ligand depro-
tonation at the sulfonamide group.
The diffuse reflectance spectra of complexes 1, 3, and 4
exhibit a d–d shoulder in the range of 652 to 671 nm while
that of complex 2 shows two well-defined d–d bands at 543
and 702 nm. The room temperature magnetic moments of
complexes 1 (µeff = 1.73 BM), 2 (µeff = 1.75 BM), 3 (µeff
=
1.82 BM), and 4 (µeff = 1.77 BM) are consistent with the
presence of a single unpaired electron. The EPR spectra of
the complexes are axial with gʈ ranging from 2.22 to 2.30
and gЌ around 2.05.
The complexes have been structurally characterized with
the aid of single-crystal X-ray diffraction techniques. OR-
TEP drawings of the complexes, including the atomic num-
bering schemes, are shown in Figure 1, Figure 2, Figure 3,
and Figure 4. Selected bond lengths and angles are listed in
Table 1.
The crystal structures of compounds 1, 3, and 4 consist
of a discrete monomeric copper(II) species surrounded by
Scheme 1. N-(Benzothiazolyl)sulfonamide ligands.
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Genotoxic Potential of N-(Benzothiazolyl)sulfonamide Copper(II) Complexes
FULL PAPER
Figure 1. ORTEP drawing of the [Cu(L1)2(phen)] (1) complex.
Figure 3. ORTEP drawing of the [Cu(L3)2(phen)] (3) complex.
Figure 2. ORTEP drawing of the [Cu(L2)2(phen)MeOH] (2) com-
plex.
Figure 4. ORTEP drawing of the [Cu(L4)2(phen)] (4) complex.
Table 1. Selected bond lengths [Å] and angles [°] for [Cu(L1)2(phen)] (1), [Cu(L2)2(phen)MeOH] (2), [Cu(L3)2(phen)] (3), and [Cu(L4)2-
(phen)] (4).
[Cu(L1)2(phen)]
[Cu(L2)2(phen)MeOH]
[Cu(L3)2(phen)]
[Cu(L4)2(phen)]
Cu(1)–N(2A)
Cu(1)–N(2B)
Cu(1)–N(1C)
Cu(1)–N(1D)
1.989(18)
1.989(16)
2.027(12)
1.957(12)
Cu(1)–N(2B)
Cu(1)–N(2A)
Cu(1)–N(1C)
Cu(1)–N(2C)
Cu(1)–O(1)
1.983(2)
2.018(2)
2.020(2)
2.036(2)
2.353(3)
Cu(1)–N(2B)
Cu(1)–N(2A)
Cu(1)–N(2C)
Cu(1)–N(1C)
1.949(4)
1.964(4)
2.006(4)
2.013(4)
Cu(1)–N(2)#1
Cu(1)–N(2)
Cu(1)–N(3)
1.954(15)
1.954(15)
1.989(14)
1.989(13)
Cu(1)–N(3)#1
N(1D)–Cu(1)–N(2B) 96.9(7)
N(1D)–Cu(1)–N(2A) 165.7(9)
N(2B)–Cu(1)–N(2A) 94.32(9)
N(2B)–Cu(1)–N(1C) 159.55(10)
N(2A)–Cu(1)–N(1C) 91.05(10)
N(2B)–Cu(1)–N(2C) 93.98(10)
N(2A)–Cu(1)–N(2C) 171.67(10)
N(1C)–Cu(1)–N(2C) 81.35(10)
N(2B)–Cu(1)–N(2A)
N(2B)–Cu(1)–N(2C)
N(2A)–Cu(1)–N(2C) 162.05(19)
N(2B)–Cu(1)–N(1C) 167.69(17)
N(2A)–Cu(1)–N(1C) 92.05(16)
N(2C)–Cu(1)–N(1C) 80.98(18)
96.93(15)
92.69(16)
N(2)#1–Cu(1)–N(2) 94.2(6)
N(2)#1–Cu(1)–N(3) 162.7(7)
N(2B)–Cu(1)–N(2A)
N(1D)–Cu(1)–N(1C) 79.9(8)
N(2B)–Cu(1)–N(1C) 167.8(8)
92.6(6)
N(2)–Cu(1)–N(3)
95.8(10)
N(3)–Cu(1)–N(3)#1 78.(2)
N(1)–Cu(1)–N(2)
N(1)–Cu(1)–N(3)
58.57(3)
91.39(3)
N(2A)–Cu(1)–N(1C) 92.9(7)
N(2B)–Cu(1)–O(1)
N(2A)–Cu(1)–O(1)
N(1C)–Cu(1)–O(1)
N(2C)–Cu(1)–O(1)
107.03(11)
98.82(9)
91.57(11)
78.09(9)
N(1)–Cu(1)–N(1)#1 159.03(5)
N(1)–Cu(1)–N(2)#1 105.88(3)
N(1)–Cu(1)–N(3)#1 104.96(2)
Eur. J. Inorg. Chem. 2006, 3823–3834
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FULL PAPER
four N atoms belonging to a phenanthroline molecule and
two deprotonated N-sulfonamide ligands in a distorted
square-planar geometry. In complexes 1 and 3 the tetrahe-
drality value of 15.51° and 18.79°, respectively, indicates
that the copper ion adopts a distorted square-planar geom-
etry.[22] The metal ion in complex 4 is located at an inver-
sion symmetry center, hence its coordination polyhedron is
strictly square planar. The Cu–N bond lengths lie in the
range 1.949(4) to 2.027(12) Å, which are normal lengths for
Cu–Nphen
and
Cu–N-benzothiazole
coordination
bonds.[11,13,23]
The copper(II) ion in complex 2 is five-coordinate with
a distorted square-pyramidal geometry. The basal plane is
defined by the benzothiazole N atoms of two deprotonated
sulfonamide ligands and the two N atoms of a phenan-
throline molecule. The Cu–N bond lengths are similar to
those found in complexes 1, 3, and 4. The oxygen atom of
a coordinated methanol molecule occupies the axial posi-
tion with a bond length of 2.353 Å. The changes in bond
length are described by tetragonality (T5); in this case, the
tetragonality value of 0.85 fits into the range expected for
square-pyramidal complexes with mixed nitrogen-oxygen li-
gands.[24] The in-plane angular distortion described by the
ratio τ represents the percentage of trigonal distortion in a
square-pyramidal geometry.[25] The τ value of 0.20 found
here thus indicates the presence of a slight distortion.
The coordination mode of the sulfonamide as a mono-
dentate ligand that binds through the benzothiazole N atom
in complexes 1, 2, 3, and 4 is the same as that usually found
in other CuII complexes with N-substituted sulfonamide an-
alogs.[12,13,23]
Figure 5. Effect of increasing amounts of the [Cu(L4)2(phen)] (4)
complex on the relative viscosity of CT-DNA.
The different behavior exhibited by complex 4 may be
attributable to its strictly square-planar geometry; in the
other complexes, the distortion of the coordination polyhe-
dron may prevent the intercalation between the bases of the
DNA.
Effect of the Complexes on the Emission Spectra of the
DNA-EB System
To corroborate the findings from the viscometry results
on the mode of DNA interaction, we carried out competi-
tive ethidium bromide (EB) binding studies. Since EB shows
a reduced emission intensity in buffers because of the
quenching by the solvent molecules and a significantly en-
hanced intensity when bound to DNA, the emission inten-
sity of this compound is often used as a spectroscopic
probe.[27] Binding of the complexes to DNA decreases the
emission intensity of EB and the extent of the emission in-
tensity reduction actually gives a measure of the DNA
binding propensity of the complexes in question. The re-
sults in Figure 6 (a) show that the fluorescence intensity
of CT-DNA–EB decreases remarkably with the addition of
complex 4, which indicates that the complex binds to DNA,
replacing the EB from the CT-DNA. Such a result is com-
mon in intercalative DNA interactions and is consistent
with the results obtained from the viscosity measurements.
The fluorescence quenching of EB bound to DNA by
complex 4 is in agreement with the classic linear Stern–
Volmer equation[27]
DNA Binding Properties
The mode of and propensity for binding of the com-
plexes to calf thymus DNA (CT-DNA) has been studied
with the aid of different techniques.
Viscosity Measurements
Viscosity measurements were carried out on CT-DNA by
varying the concentration of the complexes. Hydrodynamic
measurements that are sensitive to length changes are re-
garded as the least ambiguous and the most critical tests of
binding in solution. One classic intercalation model results
in the lengthening of the DNA helix as the base pairs are
separated to accommodate the binding ligand, thus leading
IF0/IF = 1 + kq [Q],
to the increase of DNA viscosity. In contrast, complexes where IF0 is the emission intensity in the absence of
that bind exclusively in DNA grooves by means of partial quencher, IF is the emission intensity in the presence of
and/or nonclassic intercalation under the same conditions quencher, kq is the quenching constant, and [Q] is the
typically cause either a less pronounced (positive or nega- quencher concentration. This concordance can be observed
tive) or no change in DNA solution viscosity.[26] The results by comparing the fluorescence quenching curve of IF0/IF to
obtained with our compounds reveal that while complex 4 the concentration of complex 4 (Figure 6, b). It may thus
(Figure 5) interacts with DNA in a classic intercalative be concluded that complex 4 binds to DNA mainly by me-
mode, the presence of complexes 1, 2, or 3 (data not shown) ans of intercalation. The value of Kapp is 1.72×106 –1,
has no effect on the relative viscosity of CT-DNA. This which corresponds to the copper(II) complex concentration
indicates that the interaction of these complexes follows the [Q] = 58.1 µ that causes a 50% quenching of the initial
general pattern of other complexes that bind in DNA EB fluorescence. This finding suggests that the interaction
grooves by either a partial or nonclassic intercalation.
of 4 with DNA is quite strong.
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Genotoxic Potential of N-(Benzothiazolyl)sulfonamide Copper(II) Complexes
FULL PAPER
pUC18 Cleavage
We then went on to study the activity of the complexes
as chemical nucleases using supercoiled (SC) pUC18 DNA
in cacodylate buffer (pH = 6.0) with ascorbate/H2O2 acti-
vation. The efficiency of the complexes has been compared
with that of both copper salt and the bis(o-phenanthroline)-
copper complex under the same conditions.
The results shown in Figure 7 indicate that the DNA
cleavage activity order is 4 Ͼ 2 Ͼ 1 Ͼ 3. The results also
reveal that the DNA scission mediated by the compounds
depends on complex concentration, with the cleavage being
more efficient at higher concentrations.
Figure 6. (a) Emission spectra of the DNA–EB system (100 µ CT-
DNA base pairs and 10 µ ethidium bromide) λex = 500.0 nm λem
= 530–650 nm, in the absence (continuous line) and in the presence
(discontinuous lines) of increasing amounts of complex 4 (0–
64 µ). (b) Emission quenching of DNA–EB system upon addition
of [Cu(L4)2(phen)] (4).
The presence of complexes 1, 2, or 3 (data not shown)
has no effect on the emission spectra of the DNA–EB sys-
tem under the same conditions.
Thermal Denaturation Results
The binding of complexes 1–4 to CT-DNA was then
studied from the thermal denaturation profile of DNA.
While complexes 1 and 2 produced no increase in the melt-
ing temperature (Tm) upon their addition to DNA (CT-
DNA Tm = 57 °C), complexes 3 and 4 increased the Tm to
75 and 71 °C, respectively. These results suggest a groove
binding for complexes 1 and 2 and intercalation for com-
plexes 3 and 4, thereby explaining the resulting increased
duplex stabilization.[28]
The behavior of complex 3 is noteworthy, especially since
Figure 7. Agarose gel electrophoresis of pUC18 plasmid DNA
the thermal denaturation experiments indicate that this treated with copper salt, the complexes, or bis(o-phenanthroline)-
copper(II). [Ascorbate/H2O2]:[tested compound] = 1:1. Incubation
compound can intercalate with DNA while fluorescence
time: 30 min (37 °C). (a) CuSO4 (b) [Cu(L1)2(phen)] (1) complex,
and viscosity results do not agree with this result. One pos-
(c) [Cu(L2)2(phen)MeOH] (2) complex, (d) [Cu(L3)2(phen)] (3)
complex, (e) [Cu(L4)2(phen)] (4) complex.
sible explanation for the duplex stabilization that is effected
by complex 3 is that specific hydrogen-bonding interactions
are present between the N-(benzothiazolyl)sulfonamide li-
gands and the bases or phosphate backbone of the DNA.
Another possibility is that deformations in the double-helix
structure that occur upon binding of the complexes result
in additional interstrand interactions.[29]
Lane 1: DNA/EcoRI+Hind III. Marker, 3; lane 2: supercoiled
DNA; lane 3: copper salt (a) or complexes (b–e) 1.5 µ; lane 4:
copper salt (a) or complexes (b–e) 3 µ; lane 5: copper salt (a) or
complexes (b–e) 4.5 µ; lane 6: copper salt (a) or complexes (b–e)
6 µ; lane 7: copper salt (a) or complexes (b–e) 7.5 µ; lane 8:
copper salt (a) or complexes (b–e) 9 µ; lane 9: [Cu(phen)2]2+ com-
plex 9 µ.
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Complexes 1 and 2 at 3 and 4.5 µ (see parts b and c in DNA cleavage in the presence of alternative H-atom do-
Figure 7, lanes 4 and 5) produced supercoiled DNA cleav- nors, which should scavenge radicals (such as OH·) in solu-
age, which was transformed into circular DNA (Form II). tion. To this end, we included standard scavengers of reac-
Linearized DNA was not observed under these conditions. tive oxygen intermediates in the electrophoretic process
This latter finding suggests that cleavage may occur ran- (Figure 8).
domly over the DNA since a significant portion of the plas-
mid already appears to be converted to Form II without
concurrent formation of Form III. For its part, Form III
resulted from multiple cleavage of Form II and appeared
only when full cleavage of Form I was practically achieved
(Figure 7, part b, lane 8 and part c, lane 6).
At low concentrations of complex 3 (1.5, 3, and 4.5 µ,
lanes 3, 4, and 5 in Figure 7, d) the plasmid remained su-
percoiled. In contrast to the reactivity of 1 and 2, complex
3 was unable to mediate the conversion of supercoiled DNA
(Form I) at 3 and 4.5 µ (Figure 7, part d, lanes 4 and 5).
Upon increasing the concentration of 3 to 6 and 7.5 µ,
the supercoiled DNA was partially transformed into open
circular DNA (Figure 7, part d, lanes 6 and 7) whereas at
9 µ, DNA Form I was fully converted into Form II and
Form III (Figure 7, part d, lane 8).
Complex 4 was the most reactive of the complexes (Fig-
ure 7, e), proving to be a very efficient chemical nuclease.
At the very low concentration of 1.5 µ, the compound was
capable of mediating the conversion of supercoiled DNA to
its open circular form (lane 3 in Figure 7, e). In the concen-
tration range from 3 to 6 µ, complex 4 induced complete
degradation of the plasmid to yield the open circular and
linear forms (Figure 7, part e, lanes 4–6) while at 7.5 µ,
the supercoiled DNA was converted into Form II, Form III
and small linear fragments that appeared as a smear on the
gel (Figure 7, part e, lane 7). At a concentration of 9 µ,
Figure 8. Agarose gel electrophoresis of pUC18 plasmid treated
with 9 µ complexes and 9 µ ascorbate/H2O2 in the presence of
potential inhibitors. Incubation time 30 min (37 °C). (a) [Cu(L1)2-
(phen)] (1) complex, (b) [Cu(L2)2(phen)MeOH] (2) complex,
(c) [Cu(L3)2(phen)] (3) complex, (d) [Cu(L4)2(phen)] (4) complex.
Lane 1: DNA/EcoRI+Hind III. Marker, 3; lane 2: supercoiled
DNA; lane 3: complex without inhibitors; lane 4:
complex+DMSO (1 ); lane 5: complex+tert-butyl alcohol (1 );
lane 6: complex+NaN3(100 m); lane 7: complex+2,2,6,6 tet-
ramethyl-4-piperidone (100 m); lane 8: complex+distamycin
(8 µ); lane 9: complex+SOD (15 units).
only the linear Form III and small linear fragments were
observed (Figure 7, part e, lane 8). These products are in-
consistent with a mechanism of concerted cleavage of both
strains of the plasmid.
As no cleavage was found for CuSO4 under the same
conditions (lanes 3–8 of Figure 7, a), the DNA cleavage ac-
tivity of the compounds can be ascribed to the cooperative
effect between CuII and its ligands.
Since our compounds contain o-phenanthroline as a li-
gand, we have compared their nucleolytic activity with that
of bis(o-phenanthroline)copper complex, which is a well-
The presence of the hydroxyl radical scavengers dmso
known and very efficient chemical nuclease. A comparison and tert-butyl alcohol (lanes 4 and 5 of Figure 8a–d) signifi-
of lane 8 of Figure 7, part b (1, 9 µ), part c (2, 9 µ), part cantly inhibited the chemical nuclease activity of the com-
d (3, 9 µ), and part e (4, 9 µ) with lane 9 of Figure 7 (a) plexes. This suggests a cleavage pathway involving the for-
([Cu(phen)2]2+, 9 µ) clearly indicates that these complexes mation of the reactive hydroxyl radical.
present considerably higher nuclease activity than the bis(o-
Sodium azide (lane 6 of Figure 8, a–d) inhibited DNA
phenanthroline)copper complex. Under these conditions, cleavage by the compounds to some extent, a finding that
1
bis(o-phenanthroline)copper complex was only able to points to the participation of O2 in the reaction. However,
transform a small quantity of SC DNA into its open circu- except in the case of 4, 2,2,6,6-tetramethyl-4-piperidone, an-
lar form while the complexes mediated the full conversion other singlet oxygen scavenger, had no observable effect on
of the plasmid Form I to the Forms II and III, with com- the cleavage reaction (lanes 7), which clearly contradicts the
plex 4 producing small linear fragments. It is thus clear that initial result. This may be resolved by taking into account
the presence of an N-sulfonamide ligand increases nuclease that since the azide ion can act as a ligand of copper(II),
activity.
inhibition of the nuclease reaction probably occurs not by
The existence of diffusible radical species in the nuclease radical quenching, but rather by blocking the coordination
mechanism can be inferred by monitoring the quenching of site on copper that is involved in radical formation. We can
3828
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Eur. J. Inorg. Chem. 2006, 3823–3834

Genotoxic Potential of N-(Benzothiazolyl)sulfonamide Copper(II) Complexes
FULL PAPER
1
–
thus conclude that O2 is most likely not involved in the CuI(L) + O2 Ǟ CuII(L) + O2
reaction.[30]
–
O2 + H2O2 Ǟ O2 + OH– + OH·
Lane 9 of Figure 8 (a–d) shows a significant reduction of
the DNA cleavage of the complexes in the presence of the
SOD enzyme, which indicates that O2– is one of the reactive
species that actually breaks the DNA.
The proposed mechanism for our compounds is similar
to that for the related Cu–N-quinolinsulfonamide-o-phen-
anthroline complex.[11]
In summary, the four complexes are very efficient chemi-
cal nucleases at low concentrations and in the presence of
a low concentration of H2O2/ascorbate. In particular,
[Cu(L4)2(phen)] (4) is a promising compound with much
higher activity than that exhibited by the paradigmatic
bis(o-phenanthroline)copper(II) complex. The high effi-
ciency of 4 is probably due to the fact that its intercalation
binding to DNA permits ROS formation near the deoxy-
ribose moiety. Studies with inhibiting reagents suggest that
the cleavage mechanism for these compounds is different
from that proposed by Sigman for the bis(o-phenan-
throline)copper(II) complex.
In the presence of the minor groove binder distamycin
(lanes 8), the complexes displayed no apparent inhibition of
DNA damage. This suggests that there are no minor bind-
ing preferences for the complexes.[31]
The mechanism of the DNA strand scission of bis(o-
phenanthroline)copper(II) in the presence of reducing
agents indicates that upon its reduction, the copper(II)
complex binds to DNA, thereby liberating a phenan-
throline.[32] Hydrogen peroxide then forms a copper–oxene
reactive species that attacks the C1–H of the -ribose. How-
–
ever, for complexes 1, 2, 3, and 4, OH· and O2 are the
active oxygen species involved in the reaction. The involve-
ment of these ROS excludes the formation of a copper–
oxene such as that proposed by Sigman for bis(o-phenan-
throline)copper(II).[32]
Genotoxicity Assay in Yeast Cells
Because of the presence of hydroxyl and superoxide radi-
cals and taking into account that the complexes are not able
per se to induce any effect on the double stranded DNA,
but that the cleavage process can be induced with the ad-
dition of activating agents at very low concentrations, we
propose that the DNA damage can occur either through a
Fenton-type reaction:
Sensitivity of the RAD54-yEGFP and RNR2-yEGFP
Reporter System to EMS at Different Incubation Times
Previous studies have shown that the brightness of the
fluorescence signal obtained from yeast strains carrying the
RAD54-yEGFP promoter-reporter construct on the plas-
mid correlates with exposure to increasing concentrations
of MMS (methyl methane sulfonate).[15] EMS (ethyl meth-
ane sulfonate) and MMS are alkylating agents that can act
on DNA and lead to damage and mutagenesis. EMS is reg-
ularly used as the reference substance for assessing the mu-
tagenic and DNA-damaging potential of chemicals.[14] In
order to test the sensitivity of the various transformed
CuII(L) + e– Ǟ CuI(L)
CuI(L) + H2O2 Ǟ CuII(L) + OH– + OH·
or a Haber–Weiss reaction:
CuII(L) + e– Ǟ CuI(L)
Figure 9. Comparison of EMS-dependent fluorescence in different Saccharomyces cerevisiae strains carrying the RAD54-yEGFP or the
RNR2-yEGFP constructs. Intensity of the fluorescence signal is dependent on incubation time (0, 5, 10, 15, 20, 25, or 30 min; 0 min is
represented as the background). The fluorescence signal displayed has been normalized for cell density.
Eur. J. Inorg. Chem. 2006, 3823–3834
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M. González-Álvarez, G. Alzuet, L. del Castillo, J. Borrás, M. Liu-González
FULL PAPER
strains to this paradigmatic mutagenic substance, we have duce a fluorescence signal in the selected CML128-RNR2-
performed fluorescence measurements in response to EMS yEGFP system. The response obtained in the presence of
treatment with yeast cell cultures carrying the RAD54- CuCl2 or [Cu(phen)2]2+ was studied for comparative pur-
yEGFP or RNR2-yEGFP constructs. These assays have poses while EMS was used as a positive control. Figure 10
also been carried out with the aim of selecting the most shows the intensity of the fluorescence signals induced by
sensitive transformed stain for subsequent evaluation of the EMS, compounds 1–4, and CuCl2 and bis(o-phenan-
genotoxicity of copper(II) complexes.
throline)copper(II) over the system after 20 hours of incu-
The results, shown in Figure 9, demonstrate that after bation.
treatment with EMS, only a weak increase in fluorescence
is observed with the various RAD54-yEGFP constructions.
As an alternative sensor system, the RAD54 promoter
was replaced by the promoter of the RNR2 gene. RNR2, a
small subunit of ribonucleoside diphosphate reductase es-
sential for the synthesis of nucleotide precursors, was shown
to be induced by MMS treatment.[15] EMS was also able to
induce measurable levels of yEGFP fluorescence driven by
RNR2, where the fluorescence signal obtained with the
RNR2 promoter was stronger than that observed with the
RAD54 promoter. This result is consistent with data pre-
sented by Jelinsky and Samson[33] and Afanassiev et al.[14]
The best results were obtained with the CML128-RNR2
strain, which was consequently chosen to test the ability of
the copper compounds to cause DNA damage.
As can be seen in Figure 10, complexes 3 and 4 induced
fluorescence. In particular, complex 4, which had been
found to be the most efficient chemical nuclease, showed a
significant induction of the fluorescence reporter system,
which indicates that this compound is able to produce DNA
cleavage inside the cell. The signal was much stronger than
that of complex 3. Other substances that have been de-
scribed in the literature as having induced fluorescence sig-
nals over similar systems include cisplatin (a strong DNA
cleaving agent), camptothecin (an inhibitor of topoisomer-
ase I), aphidicolin (an inhibitor of DNA polymerases α and
δ), and actinomycin D (an intercalator that inhibits mainly
transcription, but also replication to some extent).[14]
Complexes 1, 2, and bis(o-phenanthroline)copper(II),
which possesses strong DNA cleaving activity, did not acti-
vate the RNR2 promoter-driven yEGFP expression Also,
the results show that CuCl2 was unable to induce a fluores-
cence signal. These negative results observed with com-
plexes 1, 2, and bis(o-phenanthroline)copper(II), all of
which are able to produce pUC18 DNA cleavage, could be
because of enzymatic inactivation, lack of cell penetration,
detoxification, or other specific properties of the yeast.
Some substances known to have mutagenic or DNA-dam-
aging potential, including psoralen, benzpyrene, tetracy-
cline, colchicine, bleomycin, and mitomycin C, are unable
to induce activity in similar systems.[14]
Sensitivity of the CML128-RNR2-yEGFP Reporter System
to Copper Compounds at Different Concentrations
The induction of a fluorescence signal driven by pro-
moters that respond to DNA damage in the yeast S. cerevis-
iae provides a system to monitor the activity of substances
that possess mutagenic potential at low concentrations. This
method not only allows for the recognition of mutagenic
agents that are able to produce DNA damage to living sys-
tems, but it can also be used to determine the biologically
active concentrations of these substances, since the yeast
system provides all the typical features of eucaryotic cell
architecture and metabolism.
Because of the high genotoxic potential of 4, our next
task will be to evaluate the therapeutic use of this com-
We thus tested the ability of compounds 1, 2, 3, and 4, pound. With this aim, the potential antitumor activity of 4
previously shown to be strong DNA-cleaving agents, to in- will be tested on human tumor cell lines.
Figure 10. Fluorescence response profiles after treatment of CML128-RNR2-yEGFP with increasing concentrations of EMS, complexes
1, 2, 3, or 4, [Cu(phen)2]2+, or CuCl2. Diagrams show the dose response of fluorescence after 20 h of incubation (0  is represented as
the background).
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Genotoxic Potential of N-(Benzothiazolyl)sulfonamide Copper(II) Complexes
FULL PAPER
21.07; found C 55.11; H 3.97; N 9.26; S 21.14. IR (KBr): ν = 1560
˜
Conclusions
(thiazole); 1314, 1146 ν(SO2); 960 ν(S–N) cm–1. FAB: m/z = 305
[M+]. UV: λmax = 302 (πǞπ*) nm.
Ternary copper(II) complexes with N-substituted sulfon-
amide ligands and o-phenanthroline have been prepared
and structurally characterized with the aid of X-ray crystal-
lography. The DNA cleavage activities of these complexes
have been studied and compared with that of bis(o-phenan-
throline)copper(II). All the complexes act as efficient chem-
ical nucleases and their activities were found to be higher
than that of the paradigmatic bis(o-phenanthroline)cop-
per(II) complex. The most active cleaving agent was found
to be [Cu(L4)2(phen)](4), which was able to mediate DNA
scission at very low concentrations (1.5 µ) in the presence
of low concentrations of activating agents. The significant
activity of this complex can be ascribed to its efficient bind-
ing to DNA, as deduced from viscosity, fluorescence, and
thermal denaturation studies. The sensitivity of the
CML128-RNR2-yEGFP reporter system to copper com-
pounds was tested to evaluate the genotoxic potential (i.e.,
the potential to inflict DNA damage) of the compounds at
the cellular level. The significant induction of fluorescence
by complex 4 indicated a high genotoxic potential for this
compound. This fact makes 4 a very promising DNA dam-
aging agent for further studies on human tumor cell lines.
N-(6-Chlorobenzothiazol-2-yl)benzenesulfonamide (HL1) and N-(6-
Chlorobenzothiazol-2-yl)toluenesulfonamide (HL2): These com-
pounds were obtained and characterized as previously re-
ported.[23,34]
Synthesis of the Complexes
[Cu(L1)2(phen)] (1), [Cu(L2)2(phen)MeOH] (2), [Cu(L3)2(phen)] (3),
and [Cu(L4)2(phen)](4): Aqueous NaOH (0.5 , 2 mL) was added
to a solution of the ligand (1 mmol) in methanol (40 mL). A sus-
pension of CuSO4 (0.5 mmol) and 1,10-phenanthroline monohy-
drate (0.5 mmol) in methanol was then added, and the resulting
mixture was stirred for ca. 30 min at 40 °C. After cooling, the mix-
ture was filtered to remove the precipitate that formed. Well-shaped
crystals of 1, 3, and 4 were obtained from the filtrate by slow evapo-
ration at room temperature for several months. Crystals of complex
2 were also formed, but they were not of sufficiently high quality
for structural determination. Reaction in methanol by means of
slow diffusion afforded good quality crystals of 2 after several days.
Crystals of all the complexes were isolated by means of filtration,
washed with MeOH, and dried under vacuum.
Complex 1: Yield: 0.142 g, 32%. C38H26Cl2CuS4N6O4: calcd. C
51.09, H 2.93, N 9.41, S 14.36; found C 51.15, H 2.96, N 9.39, S
14.28. IR (KBr): ν = 1465 (thiazole); 1279, 1129 ν(SO ); 961
˜
2
ν(S–N) cm–1. Solid UV/Vis: λmax = 355 (πǞπ*), 652 sh (d–d) nm.
Complex 2: Yield: 0.124 g, 26%. C41H32Cl2CuS4N6O5: calcd. C
51.76, H 3.39, N 8.83 S 13.48; found C 51.65, H 3.42, N 8.91 S
Experimental Section
13.28. IR(KBr): ν = 1462 (thiazole); 1295, 1139 ν(SO ); 968
˜
2
Materials: Reagents and solvents were commercially available and
were used without further purification. pUC18 was purchased from
Roche Diagnostics. Calf thymus DNA (CT-DNA) was supplied by
Sigma–Aldrich.
ν(S–N) cm–1. Solid UV/Vis: λmax = 338 (πǞπ*), 543, 702 (d–d) nm.
Complex 3: Yield: 0.152 g, 37%. C38H26CuS4N6O4: calcd. C 55.49,
H 3.19 N 10.22 S 15.60; found C 55.32, H 3.15 N 10.10 S 15.67.
IR (KBr): ν = 1432 (thiazole); 1276, 1144 ν(SO ); 968 ν(S–N) cm–1.
˜
2
Physical Methods: Elemental analyses (C,H,N,S) were performed
with a Carlo–Erba AAS instrument. IR spectra (KBr disks) were
obtained with a Mattson satellite FT-IR in the range 4000–
400 cm–1. Fast atomic bombardment (FAB) mass spectra were ob-
tained with a VG Autospec spectrometer with 3-nitrobenzyl alcohol
as the matrix. Diffuse reflectance spectra (nujol mulls) of the com-
plexes were carried out with a Shimadzu UV-2101 PC spectropho-
tometer. Electronic Paramagnetic Resonance (EPR) spectra were
taken at the X-band frequency at room temperature with a Bruker
ELEXSYS Spectrometer.
Solid UV/Vis: λmax = 371 (πǞπ*), 671 sh (d–d) nm.
Complex 4: Yield: 0.115 g, 27%. C40H30CuS4N6O4: calcd. C 56.49
H 3.56 N 9.88 S 15.08. found C 56.53 H 3.42 N 9.88 S 15.08.
IR(KBr): ν = 1489–1441 (thiazole); 1276, 1134 ν(SO ); 967
˜
2
ν(S–N) cm–1. Solid UV/Vis: λmax = 337 (πǞπ*), 665 sh (d–d) nm.
X-ray
0.04×0.04×0.08 mm
0.2×0.08×0.24 mm
Crystallography:
A
green
blue
brown
crystal
crystal
crystal
measuring
measuring
measuring
(1),
(2),
a
a
0.04×0.1×0.2 mm (3), and
a
dark blue crystal measuring
¯
Synthesis of the Ligands
0.14×0.12×0.08 mm (4), and either a triclinic space group P1 (1)
or a monoclinic space group P21/c (2, 3, and 4) (as determined
from systematic absences) were used. Data collection was per-
formed at 293 K with a Nonius Kappa-2000 single crystal dif-
fractometer, with Cu-Kα radiation (λ = 1.54184 Å) for complexes
1, 3, and 4 or with a Nonius Kappa CCD single crystal dif-
fractometer with Mo-Kα radiation (λ = 0.71073 Å) for complex 2.
The crystal-detector distance was fixed at 35 mm (1, 3, and 4) or
32.1 mm (2) and a total of 106 (1), 228 (2), 430 (3), and 91 (4)
images were collected by means of the oscillation method, with a
scan angle per frame of 2° (1, 3, and 4) or 1.7° (2) oscillation and
20 s (1), 15 s (2), 26 s (3), and 2 s (4) exposure time per image. The
data collection was carried out and calculated with the program
Collect.[35] Data reduction and cell refinements were performed
with the programs HKL Denzo and Scalepack.[36]
N-(Benzothiazol-2-yl)benzenesulfonamide (HL3) and N-(Benzothi-
azol-2-yl)toluenesulfonamide (HL4): A solution containing 2-amino-
benzothiazole (1 g) and benzenesulfonyl chloride (2.5 g) or tolu-
enesulfonyl chloride (2.5 g) in pyridine (6 mL) was heated at reflux
for 1 h. The mixture was added to cold water (10 mL) and stirred
for several minutes. The resulting solid was recrystallized from eth-
anol. After several weeks, slow evaporation of the filtrate gave rise
to single crystals suitable for X-ray diffraction.
N-(Benzothiazol-2-yl)benzenesulfonamide (HL3): Yield: 1.89 g,
98.9%. C13H10N2S2O2 (290.35): calcd. C 53.77; H 3.47; N 9.65; S
20.08; found C 53.88; H 3.26; N 9.69; S 20.05. IR (KBr): ν = 1561
˜
(thiazole); 1317–1304, 1146 ν(SO2); 959 ν(S–N) cm–1. FAB: m/z =
291 [M+]. UV: λmax = 309 (πǞπ*) nm.
N-(Benzothiazol-2-yl)toluenesulfonamide (HL4): Yield: 1.88 g,
Unit cell dimensions were determined from 295 (1), 10423 (2), 3916
93.0%. C14H12N2S2O2 (304.38): calcd. C 55.24; H 3.97; N 9.20; S (3), and 597 (4) reflections between θ = 0.999° and 39.973° (1),
Eur. J. Inorg. Chem. 2006, 3823–3834
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3831

M. González-Álvarez, G. Alzuet, L. del Castillo, J. Borrás, M. Liu-González
FULL PAPER
0.998° and 28.7° (2), 0.999° and 63.691° (3), and 0.99° and 36.05°
(4). Multiple observations were averaged: for complex 1 (Rmerge
Metal Complexes–DNA Interactions
=
Viscosity Measurements: Viscosity experiments were carried out
with a Cannon 25 L97 viscosimeter immersed in a Julabo thermo-
statted water bath maintained at 25.0 0.1 °C. Solutions of the
complexes (1–10 µ) in cacodylate buffer (pH = 6.0) were added
to a calf thymus DNA (50 µ base pairs) solution in cacodylate
buffer. The flow times were measured with a digital stopwatch. The
flow measurements were performed in triplicate. Data were pre-
sented as (η/η0)1/3 vs. the ratio of the concentration of complex and
DNA, where η is the viscosity of DNA in the presence of the com-
plex and η0 is the viscosity of DNA alone. Viscosity values were
calculated from the observed flow time of a DNA-containing solu-
tion corrected from the flow time of buffer alone (t0), η = t – t0.
0.0000) this resulted in 1712 unique reflections of which 1189 were
observed with IϾ2σ(I), for complex 2 (Rmerge = 0.030) this resulted
in 10601 unique reflections of which 6891 were observed with
IϾ2σ(I), for complex 3 (Rmerge = 0.0000) this resulted in 4086
unique reflections of which 2439 were observed with IϾ2σ(I), and
for complex 4 (Rmerge = 0.0000) this resulted in 879 unique reflec-
tions of which 732 were observed with IϾ2σ(I). Final mosaicity
was 0.35(1), 0.43 (2), 0.50 (3), and 0.509 (4). All data completeness
was 74.2% (1), 98.2% (2), 84.0% (3), and 95.5% (4).
Crystal structures were solved by direct methods, either with the
program SIR-97 (1, 2, and 3) or SHELXS-86 (4).[37] Anisotropic
least-squares refinements were carried out with SHELXL-97.[38] All
non-hydrogen atoms were anisotropically refined while hydrogen
atoms were located geometrically. The final cycle of full-matrix le-
ast-squares refinement based on 1712 (1), 10601 (2), 4086 (3), and
879 (4) reflections and 494 (1), 536 (2), 561 (3), and 284 (4) param-
eters converged to a final value of R1 [F2 Ͼ2σ(F2)] = 0.052, wR2
[F2 Ͼ2σ(F2)] = 0.130, R1 (F2) = 0.082, wR2 (F2) = 0.150 for complex
1; R1 [F2 Ͼ2σ(F2)] = 0.046, wR2 [F2 Ͼ2σ(F2)] = 0.127, R1 (F2) =
0.090, wR2 (F2) = 0.170 for complex 2; R1 [F2 Ͼ2σ(F2)] = 0.044,
wR2 [F2 Ͼ2σ(F2)] = 0.094, R1 (F2) = 0.092, wR2 (F2) = 0.112 for
complex 3; and R1 [F2 Ͼ2σ(F2)] = 0.036, wR2 [F2 Ͼ2σ(F2)] = 0.097,
R1 (F2) = 0.045, wR2 (F2) = 0.105 for complex 4. Final difference
Fourier maps showed no peaks higher than 0.261 e/Å3 nor deeper
than –0.397 e/Å3 for complex 1, no peaks higher than 0.678 e/Å3
to 1.06 Å to H14d nor deeper than 1.5 e/Å3 to 0.85 Å to Cu for
complex 2, no peaks higher than 0.197 e/Å3 to 1.14 Å to N1c nor
deeper than –0.405 e/Å3 to 0.98 to Cu1 for complex 3, and no peaks
higher than 0.411 e/Å3 nor deeper than –0.167 e/Å3 for complex 4.
Semi-empirical absorption correction was applied using the pro-
gram XABS2[39] for complex 1. Geometrical calculations were
made with PARST.[40] The crystallographic plots were made with
ORTEP.[41] A summary of crystallographic data of the complexes
is given in Table 2.
Fluorescence Quenching of Ethidium Bromide Intercalated to DNA
by Interaction of the Copper Complexes with DNA: The fluorescence
spectra were recorded with a JASCO FP-6200 spectrofluorimeter
at room temperature. The experiments were conducted by adding
copper(II) complex solutions at various concentrations (0–64 µ)
to samples containing 100 µ calf thymus DNA base pairs and
10 µ ethidium bromide in cacodylate buffer (pH = 6.0). All the
samples were excited at 500 nm and emission was recorded at 530–
650 nm.
Thermal Denaturation Experiments: DNA-melting experiments
were carried out by monitoring the absorbance (260 nm) of CT-
DNA (100 µ base pairs) at different temperatures in the absence
and presence of the complexes in a 4:1 DNA/complex ratio. Mea-
surements were performed with an Agilent 8453 UV/Vis spectro-
photometer equipped with a Peltier temperature controlled sample
cell and driver (Agilent 89090A). The solution containing the ter-
nary complex and CT-DNA in phosphate buffer (1 m phosphate,
2 m NaCl, pH = 7.2) was continuously stirred and heated with a
rate of temperature increase of 1 °C/min. The temperature interval
studied ranged from 25 to 90 °C.
pUC18 DNA Cleavage: A typical reaction for complexes was under-
taken by mixing a cacodylate buffer (0.1 , 7 µL, pH 6.0), pUC18
(1 µL, 0.25 µg/µL), a solution of the tested complex (6 µL) at in-
The supplementary crystallographic data for this paper is contained
in CCDC-299259, -299260, -299261, and -299262. These data can creasing concentrations between 1.5 µ and 9 µ, ascorbate (3 µL),
be obtained free of charge from The Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
and a H2O2 (3 µL) twofold molar excess relative to the concentra-
tion of the complex in cacodylate buffer. The mixtures were allowed
Table 2. Crystal data and structure refinement for [Cu(L1)2(phen)] (1), [Cu(L2)2(phen)MeOH] (2), [Cu(L3)2(phen)] (3), and [Cu(L4)2-
(phen)] (4).
Compound
1
2
3
4
Empirical formula
Formula mass [g/mol]
C38H26Cl2CuN6O4S4
893.33
C41H32Cl2CuN6O5S4
958.41
C38H26CuN6O4S4
822.43
C20H15Cu0.5N3O2S2
425.24
Crystal system
Space group
a [Å]
b [Å]
c [Å]
α [°]
β [°]
triclinic
P1
monoclinic
P21/c
17.4880(2)
11.7090(2)
20.5440(2)
90
95.7960(7)
90
4185.23(10)
4
monoclinic
P21/c
11.6840(3)
18.0120(5)
17.2570(3)
90
101.2430(11)
90
3562.08(15)
4
monoclinic
P21/c
12.0450(7)
11.3450(6)
17.8480(10)
90
126.072(3)
90
1971.34(19)
4
¯
10.8510(9)
11.4360(7)
15.6000(12)
86.714(3)
81.697(3)
89.291(3)
1912.4(2)
2
γ [°]
V [Å3]
Z
λ [Å]
1.54184
4.551
1.551
0.71073
0.903
1.521
1.54184
3.485
1.534
1.54184
3.167
1.433
µ [mm–1]
ρcalcd. [mg/m3]
T [K]
293(2)
293(2)
293(2)
293(2)
R1
wR2
0.0523
0.1304
0.0458
0.1271
0.0437
0.0941
0.0361
0.0971
3832
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2006, 3823–3834

Genotoxic Potential of N-(Benzothiazolyl)sulfonamide Copper(II) Complexes
FULL PAPER
to stand for 30 min at 37 °C. After that, a quench buffer solution
(3 µL) consisting of bromophenole blue (0.25%), xylene cyanole
(0.25%), and glycerol (30%) were added. The solution was then
subjected to electrophoresis on a 0.8% agarose gel in 0.5×TBE
buffer (0.045  Tris, 0.045  boric acid, and 1 m EDTA) contain-
ing 2 µL/100 mL of a solution of ethidium bromide (10 mg/mL) at
80 V for about 2 h. The gel was photographed on a capturing gel
printer plus TDI.
without salts, 5 g of ammonium sulfate, and 20 g of glucose per
liter) with the suitable requirements were inoculated into a fresh
sample of the yeast strain to give approximately 1×106 cells/mL.
The cultures were incubated overnight at 28 °C with vigorous shak-
ing. The cultures were then washed twice in 50 m potassium phos-
phate buffer (pH = 7.0) and resuspended in this buffer to a final
concentration of 5×107 cells/mL. EMS (30 µL) was added to 1 mL
of cells and incubated for 30 min at 30 °C. 100 µL samples were
obtained at different times (0, 5, 10, 15, 20, 25, and 30 min) during
the incubation. The EMS mutagenesis was stopped in each cell
suspension by adding an equal volume of a freshly made 10% (w/
v) filter-sterilized solution of sodium thiosulfate. The suspensions
were then mixed well and centrifuged, after which the cells were
collected and washed twice with sterile water. The cells were then
inoculated in liquid medium and incubated at 28 °C for 20 h on an
orbital shaker. For cell fluorescence measurements, the cell cultures
were diluted (1:10 in sterile distilled water) and transferred directly
to cuvettes. Samples with cell cultures with no mutagenic agents
were taken as blanks. Fluorescence measurements were performed
with a Jasco FP-6200 spectrofluorometer. The excitation and emis-
sion wavelengths were set to 488 and 511 nm, respectively, with a
slit width of 10 nm. Fluorescence values were normalized for the
absorption values of each cell. The normalized fluorescence values
were then plotted against the substance concentration and incu-
bation time. All calculations and plots were made with MS Excel.
To test for the presence of reactive oxygen species (ROS) generated
during strand scission, various reactive oxygen intermediate scaven-
gers were added to the reaction mixtures. The scavengers used were
superoxide dismutase (15 units), DMSO (1 ), tert-butyl alcohol
(1 ), sodium azide (100 m), and 2,2,6,6-tetramethyl-4-piperidone
(100 m). An assay in the presence of the minor groove binder
distamycin (8 µ) was also performed. Samples were treated as de-
scribed above.
Genotoxicity Assays
Construction of a Plasmid Containing yEGFP Under the Transcrip-
tional Control of the RNR2 or RAD54 Promoter: PCR was used to
amplify the RNR2 and RAD54 promoters from genomic Saccharo-
myces cerevisiae DNA with the primers shown in Table 3. pUG35
plasmid was digested with Sal I and Sac I to remove the original
promoter of yEGFP. The PCR products were digested with the
same restriction enzymes and were ligated into the pUG35 to pro-
duce pUG35-RNR2 or pUG35-RAD54. The yeast strains used in
this study, listed in Table 4, were grown on a YNB medium (1.68 g
of YNB without aa and without salts, 5 g of ammonium sulfate,
and 20 g of glucose per liter) with the required amino acids.
Evaluation of Genotoxicity Produced by the Complexes: Single colo-
nies of the selected transformed yeast strains grown on selective
medium were used to inoculate 5 mL of medium and then grown
to mid log phase (0.4 AU at 600 nm). The cells were resuspended
in fresh medium to a final concentration of 0.1 AU. Solutions of
EMS, the copper salt, or the test compounds at increasing concen-
trations were distributed in parallel. The tubes were incubated at
28 °C for 20 h on an orbital shaker at 120 rpm. Fluorescence mea-
surements were performed as described above except that, in this
case, the first measurement was taken at 0 h and repeated at 4, 16,
and 20 h of incubation.
Table 3. Primers used in the construction of the RAD54-yRGFD
or RNR2-yEGFD promoter-reporter plasmid.
RAD54-3SAL GGC CGT TCA CCG TCG ACT ATT CC
RAD54-5SAC AAA ATA TTG AGC TCG AAG ATC TGT CC
RNR2-3SAL GGA CAA TGC GTC GAC AGC AG
RNR2-5SAC TAG CCA GAG CTC TGC ATT ACG C
Acknowledgments
Table 4. Yeast strain used.
J. B. and G. A. acknowledge financial support from the Spanish
CICYT (CTQ2004-03735). M. G.-A. wishes to thank the city gov-
ernment of Valencia (Spain) for a Carmen and Severo Ochoa post-
doctoral fellowship.
Strain
Relevant genotype
BY4741
MATa his3-δ1 leu2-δ0 met15-δ0 ura3-δ0
MATa his3-δ200 ura3-52 leu3-δ1
MATa ura3-1 ade2-1 leu2-3,112 trp1-1 his3-11,15
MATa leu2-3,112 ura3-52 trp1-1 his4 can1r
MATa/MATα ura3-52/ura3-52 trp1-289/trp1-289
leu2-3,112/leu2-3,112 his3δ1/his3δ1
CML235
W303-1A
CML128
CEN.PK2
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Received: April 5, 2006
Published Online: July 25, 2006
3834
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2006, 3823–3834