Copper(II) complexes with sulfonamides derived from 2-picolylamine and their use as chemical nucleases

Article abstract of DOI:10.1016/j.ica.2005.11.032

Reaction between 2-picolylamine (2-aminomethylpyridine) with 2-mesitylenesulfonyl and 4-tert-butylbenzene sulfonyl chlorides leads to the formation of 2,4,6-trimethyl-N-[pyridin-2-ilmethyl]benzenesulfonamide (Hpmesa) and 4-ter-butyl-N-[pyridin-2-ilmethyl]benzenesulfonamide (Hptbsa). These compounds react with Cu(II) salts to yield coordination compounds with CuL ₂ stoichiometry. The immediate environment of the metal ion is a highly distorted tetrahedron, the sulfonamide ligands acting in a bidentate fashion. Compound Hpmesa crystallizes in monoclinic space group P2₁/c (number 14), with Z = 4; complex [Cu(pmesa)₂] crystallizes in monoclinic space group P1? (number 2), with Z = 4 and complex [Cu(ptbsa)₂] crystallizes in monoclinic space group Pca2₁ (number 29), with Z = 4. Both complexes have been characterized by spectroscopic methods. EPR measurements show rhombic spectra with parameters indicating that the unpaired electron is in d_x2-y2 orbital. The two complexes act as chemical nucleases in the presence of ascorbate/H₂O₂, the reactive oxygen species being the hydroxyl and the singlet oxygen-like species.

Full text of DOI:10.1016/j.ica.2005.11.032

Inorganica Chimica Acta 359 (2006) 1465–1472
www.elsevier.com/locate/ica
Copper(II) complexes with sulfonamides derived from
2-picolylamine and their use as chemical nucleases
a,
a
a
b
*
´
´
´
´
´
Benigno Macıas , Marıa V. Villa , Monica Salgado , Joaquın Borras ,
b
c
´
´
Marta Gonzalez-Alvarez , Francisca Sanz
a
Departamento de Quı´mica Inorga´nica, Facultad de Farmacia, Universidad de Salamanca, 37007 Salamanca, Spain
b
Departamento de Quı´mica Inorga´nica, Facultad de Farmacia, Universidad de Valencia, Valencia, Spain
c
Servicio de rayos X, Universidad de Salamanca, Salamanca, Spain
Received 6 September 2005; received in revised form 15 November 2005; accepted 23 November 2005
Available online 4 January 2006
Abstract
Reaction between 2-picolylamine (2-aminomethylpyridine) with 2-mesitylenesulfonyl and 4-tert-butylbenzene sulfonyl chlorides leads
to the formation of 2,4,6-trimethyl-N-[pyridin-2-ilmethyl]benzenesulfonamide (Hpmesa) and 4-ter-butyl-N-[pyridin-2-ilmethyl]benzene-
sulfonamide (Hptbsa). These compounds react with Cu(II) salts to yield coordination compounds with CuL2 stoichiometry. The imme-
diate environment of the metal ion is a highly distorted tetrahedron, the sulfonamide ligands acting in a bidentate fashion. Compound
Hpmesa crystallizes in monoclinic space group P21/c (number 14), with Z = 4; complex [Cu(pmesa)2] crystallizes in monoclinic space
ꢀ
group P1 (number 2), with Z = 4 and complex [Cu(ptbsa)2] crystallizes in monoclinic space group Pca21 (number 29), with Z = 4. Both
complexes have been characterized by spectroscopic methods. EPR measurements show rhombic spectra with parameters indicating that
the unpaired electron is in dx2ꢀy2 orbital. The two complexes act as chemical nucleases in the presence of ascorbate/H2O2, the reactive
oxygen species being the hydroxyl and the singlet oxygen-like species.
ꢀ 2005 Elsevier B.V. All rights reserved.
Keywords: Sulfonamides; Chemical nucleases; Cu(II) complexes; DNA cleavage
1. Introduction
substituted phen rings induce strands scission of DNA in
the presence of H2O2 and reductants [21–24].
Research on oxidative DNA cleavage by metal com-
plexes is an expanding area, which nowadays attracts lively
interest [1–4]. Owing to the fact that all these complexes
show their own selectivity for a cleavage mechanism, the
design of new DNA cleavage agents is of great interest.
A wide range of metal complexes involving nitrogen
ligands based on macrocycles have been used for DNA
cleavage [5–20]. It became well known that copper-1,10-
phenanthroline complex and the related complexes with
One of the most interesting aspects on which research is
developing is the influence of the ligands on the nuclease
activity of the complexes. We have previously reported
the synthesis and characterisation of several copper com-
plexes with sulfonamide ligands, which significantly inter-
act with plasmid pUC18 DNA [25–31]. Here, we report
the synthesis of sulfonamides from 2-picolylamine and 2-
mesitylenesulfonyl and 4-tert-butylbenzene sulfonyl chlo-
rides, which, upon coordination with copper cations, form
a stable five-member ring, an important requirement to
coordinate nucleic acids. Once these complexes have been
characterised by common physicochemical techniques for
coordination compounds, their nuclease activity with plas-
mid pUC18 has been checked.
*
Corresponding author. Tel.: +34 923 294524; fax: +34 923 294515.
´
E-mail address: bmacias@usal.es (B. Macıas).
0020-1693/$ - see front matter ꢀ 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2005.11.032

1466
B. Macı´as et al. / Inorganica Chimica Acta 359 (2006) 1465–1472
2. Experimental
2.1. Chemicals and methods
[Cu(pmesa)2]: Anal. Calc. for CuC30H34N4O4S2: C,
56.09; H, 5.29; N, 8.72; Cu, 9.89. Found: C, 56.25; H,
5.42; N, 8.39; Cu, 9.71%. IR mmax: 1288 mas(SO2); 1139
ms(SO2); 549 d(SO2); 937 m(S–N); 666 m(C–S). UV–Vis
(nm): DMF solution, 276, 284, 410 and 652; solid state:
310, 413, 482, 691 and 1048.
[Cu(ptbsa)2]: Anal. Calc. for CuC32H38N4O4S2: C,
57.33; H, 5.67; N, 8.35; Cu, 9.48. Found: C, 57.39; H,
5.81; N, 8.28; Cu, 9.36%. IR mmax: 1282 mas(SO2); 1147
ms(SO2); 567 d(SO2); 969 m(S–N); 645 m(C–S). UV–Vis
(nm): DMF solution, 278, 306, 386 and 622; solid state:
259, 289, 404, 626 and 950.
2-Picolylamine and 2-mesitylenesulfonyl and 4-tert-
butylbenzene sulfonyl chlorides were provided by Aldrich
and copper salts by Fluka AG. All reagents used were of
analytical grade.
Chemical analyses for carbon, hydrogen and nitrogen
were performed on a 2400 elemental analyser from
Perkin–Elmer. Copper was determined on an inductively
coupled plasma (ICP) spectrometer (Perkin–Elmer model
2380 Plasma 2). FT-IR spectra were recorded using KBr
mulls and a Perkin–Elmer FT-IR 1730 instrument.
Room-temperature magnetic moment was measured by
the Evans method on a magnetic susceptibility balance
from Sherwood scientific LTD model MSB-Auto. Elec-
tron paramagnetic resonance spectra were recorded at
Q-band frequencies with a Bruker ER 200D. Electronic
spectra in the solution of DMF were recorded on a
Hewlett–Packard 8452A diode spectrophotometer (200–
800 nm) and in the solid state on a Perkin–Elmer
Lambda 35 spectrophotometer (200–1100 nm). Molecular
masses were measured by Servicio de Masas (Universidad
de Salamanca) by the FAB method with samples held on
a nitrobenzyl alcohol (NBA) matrix in a VG TS-250
apparatus.
2.3. X-ray determination structure
X-ray diffraction data were collected at room tempera-
ture on a four circle Seifert XRD 3003 SC diffractometer
˚
(Cu Ka, k = 1.54180 A), with a graphite monochromator
and a x-2h scan was done. The unit cell parameters were
determined by least squares refinement on the 2h values
of 25 strong well centered reflections in the range
16ꢁ < 2h < 40ꢁ. Scattering factors for neutral atoms and
anomalous dispersion correction for Cu, C, N, O and S
were taken from ‘‘International Tables for X Ray Crystal-
lography’’ [32]. Structures of the compounds were resolved
ꢀ
by direct methods and refined in the space groups P21/c, P1
y Pca21, respectively. Full-matrix least-squares refinement
with anisotropic thermal parameters for non-H atoms
2
2.2. Synthesis of the compounds
was carried out by minimizing xðF 2o ꢀ F c2Þ . Refinement
on F2 for all reflections, weighted R factors (Rx), and all
goodness of fit S are based on F2, while conventional R fac-
tors (R) are based on F; R factors based on F2 are statisti-
cally about twice as large as those based on F, and R
factors based on all data will be even larger.
All calculations were performed using CRYSOM [33]
software for data collection, XRAY8O [34] for data reduc-
tion, SHELXTLe [35] to resolve and refine the structure and
to prepare material for publication. Crystal data and some
details of the structural determination are given in Table 1.
Selected bond distances and angles are collected in Tables 2
and 3.
Sulfonamides, both Hpmesa and Hptbsa, were prepared
by reaction of 2-picolylamine with 2-mesitylenesulfonyl or
4-tert-butylbenzenesulfonyl chlorides, following the
method described elsewhere [27,29]. Elemental chemical
analysis data are in agreement with the formulae
C15H18N2O2S and C16H20N2O2S, respectively. Yield: 69%
and 73%.
Hpmesa: Anal. Calc. for C15H18N2O2S: C, 62.06; H,
6.21; N, 9.65. Found: C, 61.98; H, 6.11; N, 9.60%. IR mmax
3203 m(N–H); 1325 mas(SO2); 1160 ms(SO2); 536 d(SO2); 1002
m(S–N); 656 m(C–S). FAB+/MS spectra, m/z = 291.
Hptbsa: Anal. Calc. for C16H20N2O2S: C, 63.15; H,
:
6.58; N, 9.21. Found: C, 63.02; H, 6.49; N, 9.19%. IR mmax
:
2.4. Cleavage of pUC18 by copper complexes
3127 m(N–H); 1329 mas(SO2); 1165 ms(SO2); 550 d(SO2);
1006 m(S–N); 632 m(C–S). FAB+/MS spectra, m/z = 305.
The mesitylene derivative was obtained in its nitrate
form, C15H19N2O2S Æ NO3, as crystals suitable for molecu-
lar structure determination by single-crystal X-ray.
The copper complexes were obtained through direct
reaction of sulfonamides with copper salts. A portion
(5 ml) of an aqueous solution containing 1 mmol copper
acetate was dropwise added to 2 mmol of the sulfonamide
dissolved in 20 ml methanol. Slow evaporation of the
brown solution yields the corresponding green crystalline
complexes. The yield was 80%. However, in order to obtain
crystals suitable for X-ray diffraction structural determina-
tion, they were recrystallised in DMF.
Reactions were carried out by mixing 6 lL of complexes
or copper salt solutions (20, 40, 60 or 80 lM) in DMF:H2O
[1:1], 1 lL of pUC18 (750 lM in nucleotides), 7 lL of H2O,
3 lL of ascorbate and 3 lL of H2O2 100-fold excess relative
to the complex concentration. After allowing the samples
to incubate at 37 ꢁC for 60 min, 3 lL of a quenching buffer
solution (0.25% bromophenol blue, 0.25% xylene cyanole
and 30% glycerol) was added. Agarose gel electrophoresis
was performed using 0.8% gel in 0.5 · TBE buffer
(0.045 M Tris, 0.045 M boric acid and 1 mM EDTA) con-
taining 2 lL/100 mL of a solution of ethidium bromide
(EB) (10 mg/mL) for 2 h at 80 V. The gel was photo-
graphed on a capturing system Gel Printer plus TDI.

B. Macı´as et al. / Inorganica Chimica Acta 359 (2006) 1465–1472
1467
Table 1
Crystal data and structure refinement for ligands and copper complexes
Hpmesa
[Cu(ptbsa)2]
[Cu(pmesa)2]
Empirical formula
Formula weight
Temperature (K)
C
353.39
293(2)
1.54180
15H19N3O5S
CuC32H38N4O4S2
670.32
293(2)
CuC30H34N4O4S2
642.27
293(2)
˚
Wavelength (A)
1.54180
1.54180
ꢀ
Crystal system, space group
Unit cell dimensions
monoclinic, P21/c
triclinic, P1
orthorhombic, Pca21
˚
a (A)
22.727(5)
8.128(2)
9.066(2)
90
95.92(3)
90
1665.8(6)
4, 1.405
11.632(2)
16.935(3)
19.365(3)
115.23(3)
95.77(3)
98.81(3)
3350.9(12)
4, 1.329
8.400(2)
16.458(3)
21.992(4)
90
90
90
3040.3(11)
4, 1.403
2.636
˚
b (A)
˚
c (A)
a (ꢁ)
b (ꢁ)
c (ꢁ)
3
˚
Volume (A )
Z, calculated density (Mg/m3)
Absorption coefficient (mmꢀ1
F(0 0 0)
)
2.010
744
2.414
1404
1340
Crystal size
h Range for data collection (ꢁ)
Limiting indices
0.25 · 0.20 · 0.10 mm
1.95–60.00
0.20 · 0.15 · 0.08 mm
2.57–60.00
0 < h < 13,
ꢀ17 < k < 17,
ꢀ19 < l < 19
9692/3801
full-matrix least-squares on F2
9689/0/788
0.20 · 0.15 · 0.10 mm
2.68–60.00
0 < h < 9,
0 < k < 18,
0 < l < 24
9958/2526
ꢀ23 < h < 23,
ꢀ9 < k < 9,
0 < l < 10
Reflections collected/observed
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2r (I)]
R indices (all data)
2509/1865
full-matrix least-squares on F2
2446/0/221
full-matrix least-squares on F2
2340/1/377
0.967
0.831
0.808
R1 = 0.0513, wR2 = 0.1171
R1 = 0.0719, wR2 = 0.1265
0.544 and ꢀ0.337
R1 = 0.0620, wR2 = 0.1246
R1 = 0.1597, wR2 = 0.1814
0.589 and ꢀ1.179
R1 = 0.0286, wR2 = 0.0571
R1 = 0.0503, wR2 = 0.0644
0.189 and ꢀ0.182
ꢀ3
˚
Largest difference in peak and hole (e A
)
Table 2
Selected bond lengths (A) and angles (ꢁ) for ligand and complexes
˚
Hpmesa
[Cu(ptbsa)2]
[Cu(pmesa)2]
Bond lengths
N(1)–S(1)
N(1)–C(10)
N(2)–C(11)
N(2)–C(15)
Bond lengths
N(1)–S(1)
N(1)–C(21)
N(2)–C(22)
N(2)–C(26)
Bond lengths
N(1)–S(1)
N(1)–C(19)
N(2)–C(20)
N(2)–C(24)
1.611(3)
1.459(4)
1.338(4)
1.340(5)
1.596(6)
1.599(10)
1.481(14)
1.372(15)
1.29(2)
1.444(10)
1.343(10)
1.351(10)
N(10)–S(10)
1.588(6)
N(10)–C(210)
N(20)–C(220)
N(20)–C(260)
1.472(10)
1.344(10)
1.343(10)
Bond angles
C(10)–N(1)–S(1)
C(11)–N(2)–C(15)
Bond angles
C(21)–N(1)–S(1)
C(26)–N(2)–C(22)
Bond angles
C(19)–N(1)–S(1)
C(24)–N(2)–C(20)
120.9(2)
123.0(3)
116.6(5)
116.1(8)
118.2(8)
121.8(10)
C(210)–N(10)–S(10)
C(260)–N(20)–C(220)
115.4(5)
117.0(7)
Mechanistic aspects of DNA cleavage reaction were
investigated using inhibiting reagents such as hydroxyl
radical scavengers (DMSO, tert-butyl alcohol, sodium for-
mate and potassium iodide); singlet oxygen scavengers
(sodium azide, 1,4-diaza-benzene[2.2.2]octane (DABCO)
and 2,2,6,6,-tetramethyl-4-piperidone); superoxide radical
scavenger (SOD); the Cu(I) chelating agent neocuproine;
the minor groove binder distamycin and the major groove
binder methyl green. Samples were treated as described
above.
Replicates. Unless otherwise indicated, each result
described in this paper is representative of at least three
separated experiments.
3. Results and discussion
Reaction of 2-picolynamine with sulfonyl chloride yields
sulfonamides whose formula is shown in Fig. 1, their
molecular masses have been measured by mass spectrome-
try. The band at 3200 cmꢀ1 recorded in the FT-IR spectra

1468
B. Macı´as et al. / Inorganica Chimica Acta 359 (2006) 1465–1472
of d–d transitions of d9 species in a geometry between tet-
rahedral and square-planar [37].
Table 3
˚
Selected bond lengths (A) and angles (ꢁ) for the complexes
[Cu(ptbsa)2]
[Cu(pmesa)2]
3.1. Description of the crystal structures
Bond lengths
Cu(1)–N(1)
Cu(1)–N(2)
Cu(1)–N(3)
Cu(1)–N(4)
Bond lengths
Cu(1)–N(1)
Cu(1)–N(2)
Cu(1)–N(3)
Cu(1)–N(4)
1.901(6)
1.981(7)
1.900(6)
1.987(6)
1.906(11)
2.035(9)
1.885(11)
2.016(9)
The structure of the free Hpmesa ligand is shown in
Fig. 2; from the data in Table 2, we can observe that the
bond distances and bond angles are only slightly modified
upon coordination to the copper cations.
The structures of the copper complexes, Figs. 3 and 4,
are very similar to each other, as expected bearing in mind
that the differences between both ligands are in sections of
their formula quite far from the coordinating sites. The
copper cations are coordinated by four nitrogen atoms,
adopting a highly distorted tetrahedral geometry. The sul-
fonamide ligands are bidentate in both complexes, leading
to stable five-member rings.
Cu(10)–N(10)
Cu(10)–N(20)
Cu(10)–N(30)
Cu(10)–N(40)
1.934(6)
1.994(6)
1.919(7)
1.989(7)
Bond angles
Bond angles
N(3)–Cu(1)–N(1)
N(3)–Cu(1)–N(4)
N(1)–Cu(1)–N(4)
N(3)–Cu(1)–N(2)
N(1)–Cu(1)–N(2)
N(4)–Cu(1)–N(2)
159.2(3)
N(3)–Cu(1)–N(1)
N(3)–Cu(1)–N(4)
N(1)–Cu(1)–N(4)
N(3)–Cu(1)–N(2)
N(1)–Cu(1)–N(2)
N(4)–Cu(1)–N(2)
152.3(2)
82.4(3)
104.7(3)
105.2(3)
82.2(3)
81.8(5)
110.2(4)
108.4(5)
82.4(4)
139.7(3)
132.41(15)
The distances between the copper cation and the nitro-
gen atoms adjacent to the sulfonyl group are slightly
N(30)–Cu(10)–N(10)
N(30)–Cu(10)–N(40)
N(10)–Cu(10)–N(40)
N(30)–Cu(10)–N(20)
N(10)–Cu(10)–N(20)
N(40)–Cu(10)–N(20)
159.5(3)
81.7(3)
103.9(3)
105.4(3)
83.3(3)
˚
˚
˚
shorter (1.885 A, 1.906 A for the mesitylene derivative, y
˚
1.900 A, 1.901 A for the terbutylbenzene) than the corre-
˚
˚
sponding distances for picolyamine (2.016 A, 2.035 A for
˚
˚
139.9(3)
the mesitylene derivative, and 1.981 A, 1.987 A for the
terbutylbenzene) but in any case, within the expected range
for copper complexes with this sort of ligands [13,38,39].
The bond angles formed by copper and the nitrogen
atoms belonging to the same ligand (close to 82ꢁ) are mark-
edly smaller (probably because strains induced by the sul-
fonamide molecule itself) than the angles formed by
copper with nitrogen atoms from different ligands, espe-
cially with nitrogen atoms adjacent to the sulfonyl group
(close to 150ꢁ), probably because of repulsion between
these groups.
Fig. 1. Structure of the Hpmesa (top) and Hptbsa (bottom).
of ligands Hpmesa and Hptbsa disappears in the spectra of
the corresponding complexes, due to nitrogen deprotona-
tion upon coordination with copper cations. Such a reac-
tion is also responsible for the shift towards lower
wavenumbers of the bands due to stretching modes of
SO2 moiety and the S–N bond, because of electron transfer
to the copper cations via the nitrogen atoms of the sulfon-
amide ligands.
The magnetic moments at room temperature of both
complexes are 1.83 BM, very close to the spin-only mag-
netic moment [36]. The electronic spectra in solution shows
a broad band at 625–650 nm (e = 105 Mꢀ1 cmꢀ1) due to a
d–d transition, together with
a
more intense
(e = 5500 Mꢀ1 cmꢀ1) band at 400 nm, originated by a
ligand metal charge transfer process (LMCT).
Two other intense bands, ca. 275 and 290 nm, are due to
transitions between energy levels of the ligands. The diffuse
reflectance spectrum shows a broad band where two max-
ima at ca. 650 and 1000 nm can be identified, characteristic
Fig. 2. ORTEP diagram for the Hpmesa ligand.

B. Macı´as et al. / Inorganica Chimica Acta 359 (2006) 1465–1472
1469
Fig. 3. ORTEP diagram for the [Cu(pmesa)2] complex.
Fig. 4. ORTEP diagram for the [Cu(ptbsa)2] complex.
From the equations corresponding to the planes
3.2. Electronic paramagnetic resonance spectroscopy
defined by the nitrogen atoms and the copper cation, we
can conclude that for the mesitylene derivative the copper
The electronic paramagnetic resonance spectra in the Q-
band (measured on ground crystals samples) at room tem-
perature for the two complexes show that both complexes
are rhombic. The spectrum of the [Cu(pmesa)2] complex is
shown in Fig. 5. The EPR parameters calculated from sim-
ulation [40] are gx = 2.033, gy = 2.1165 and gz = 2.2472 for
the [Cu(pmesa)2] and gx = 2.043, gy = 2.089 and gz = 2.25
for the [Cu(ptbsa)2] complex. The value of R = (gy – gx)/
(gz ꢀ gy) is 0.64 for the [Cu(pmesa)2] complex and 0.28 for
the [Cu(ptbsa)2] complex, suggesting that the unpaired
electron is in the dx2ꢀy2 orbital. The rhombic nature of the
˚
atom is 0.45 A above the line joining atoms N1 and N3,
˚
and 0.82 A below atoms N2 and N4; from these values
and the bond angles, it is clear that the tetrahedron is
strongly distorted. A similar geometry is found for the
tertbutylbenzene derivative, where the copper cations are
˚
˚
0.35 A above the line joining atoms N1 and N3, and
0.68 A below the line joining atoms N2 and N4. Summa-
rizing, the local environment around the copper cation
can be considered mid-way between tetrahedral and
square-planar.

1470
B. Macı´as et al. / Inorganica Chimica Acta 359 (2006) 1465–1472
increased, the intensity of the circular supercoiled DNA
(Form I) band decreases, while that for the nicked (Form
I) and linear DNA (Form III) apparently increases. For
the [Cu(ptbsa)2] complex, the circular supercoiled DNA
(form I) was found for complex concentrations up to
18 lM (lane 10), and when it was larger than 24 lM (lane
11) the circular supercoiled DNA (Form I) band was com-
pletely disappeared. With a concentration of 18 lM the
three DNA forms coexist, while at 24 lM only Forms II
and fIII are observed. For [Cu(pmesa)2] complex, only
when its concentration is 24 lM the supercoiled circular
DNA (Form I) disappears. At this concentration, a mix-
ture of the Forms II and I are observed. In order to com-
pare the nuclease activity of uncoordinated copper with
those of the complexes, lanes 4–7 correspond to Cu(II)
solutions at the same concentration as for the complexes.
We can observe that the complexes are more active as nuc-
leases than free Cu(II). On the other hand, if lanes 11 and
15 are compared, the [Cu(ptbsa)2] complex results slightly
more active than [Cu(pmesa)2] complex. Finally, the cop-
per–phenanthroline complex is more active than both com-
plexes (see lanes 11, 15 and 16). The nuclease activity
shown by these complexes is larger than that shown by sim-
ilar Cu complexes with sulfonamides derived from 8-amin-
oquinoleine, but lower than that shown by complexes by
mixed sulfonamide and o-phenanthroline or bipyridyl
ligands [27,29].
In order to identify the oxygen radical species that
cleaves the DNA strands, a new electrophoretic study
was carried out with radical scavengers. Fig. 7 shows that
the hydroxyl scavengers (dmso, tert-butyl alcohol, sodium
formate and potassium iodide) (lanes 5–8) inhibit the
DNA cleavage, suggesting that the diffusible hydroxyl rad-
ical is one of the ROS (reactive oxygen species) responsible
for DNA breakage. For singlet oxygen scavengers (sodium
azide, DABCO and 2,2,6,6 tetramethyl piperidone) (lanes
9–11) an inhibition of DNA cleavage is observed for
sodium azide and 2,2,6,6 tetramethyl piperidone; however,
DABCO has nof inhibition effect, probably due to the slow
kinetic of the singlet oxygen and DABCO [42,43]. The
superoxide dismutase enzyme (lane 12) increases the
DNA breakage with the [Cu(ptbsa)2] complex, while it
experimental
simulated
1.05 104
1.1 104
1.15 104
G gauss
1.2 104
Fig. 5. Q-band EPR spectra (experimental and simulated) of the
[Cu(pmsa)2] complex.
spectra is in agreement with the tetrahedral distortion of the
coordination polyhedron in both complexes [27,29].
3.3. Cleavage of plasmid pUC18 DNA
DNA recognition by transition metal complexes has
been studied by the DNA cleavage chemistry, as it is asso-
ciated with redox-active or photoactivated metal complexes
[41]. DNA cleavage is controlled by relaxation of super-
coiled circular form of pUC18 into nicked circular form
and linear forms. When circular plasmid DNA is submitted
to electrophoresis, the fastest migration is observed for the
supercoiled form (Form I). If one strand is cleaved, the
supercoils will relax to produce a slower-moving open cir-
cular form (Form II). If both strands are cleaved, a linear
form (Form III) will be generated that migrates at an inter-
mediate speed.
DNA cleavage was analyzed by monitoring the conver-
sion of supercoiled DNA (form I) to nicked DNA (Form
II) and linear DNA (Form III) in aerobic conditions. As
shown in Fig. 6, when the complex concentration is
Fig. 6. Agarose gel electrophoresis of pUC18 plasmid DNA treated with [Cu(ptbsa)2] and [Cu(pmesa)2] (in dmf/H2O [1:1]) in the presence of 50-fold
excess of ascorbate/H2O2 tandem relative to complex concentration. Incubation time: 1 h (37 ꢁC). Lane 1: kDNA/EcoR1 + HindIII Marker; lane 2:
supercoiled DNA (control); lane 3: pUC18 DNA + reductors; lane 4: CuCl2 20 lM (6 lM final); lane 5: CuCl2 40 lM (12 lM final); lane 6: CuCl2 60 lM
(18 lM final); lane 7: CuCl2 80 lM (24 lM final); lane 8: complex [Cu(ptbsa)2] 20 lM (6 lM final); lane 9: [Cu(ptbsa)2] 40 lM (12 lM final); lane 10:
complex [Cu(ptbsa)2] 60 lM (18 lM final); lane 11: complex [Cu(ptbsa)2] 80 lM (24 lM final); lane 12: complex [Cu(pmesa)2] 20 lM (6 lM final); lane 13:
complex [Cu(pmesa)2] 40 lM (12 lM final); lane 14: complex [Cu(pmesa)2] 60lM (18 lM final); lane 15: complex [Cu(pmesa)2] 80 lM (24 lM final); lane
16: complex [Cu(phen)2] 80 lM (24 lM final).

B. Macı´as et al. / Inorganica Chimica Acta 359 (2006) 1465–1472
1471
Fig. 7. Agarose gel electrophoresis of pUC18 plasmid DNA treated with 80 lM (24 lM final) [Cu(ptbsa)2] (top) and [Cu(pmesa)2] (bottom) in the
presence of 50-fold excess of ascorbate/H2O2 tandem and potential inhibitor agents. Incubation time 1 h (37 ꢁC). Lane 1: kDNA/EcoR1 + HindIII
Marker; lane 2: supercoiled DNA (control); lane 3: pUC18 DNA + reductors; lane 4: complex; lane 5: complex + dmso (0.4 M); lane 6: complex + tert-
butyl alcohol (0.4 M); lane 7: complex + sodium formate (0.4 M); lane 8: complex + potassium iodide (0.4 M); lane 9: complex + sodium azide (0.4 M);
lane 10: complex + DABCO (0.4 M); lane 11: complex + 2,2,6,6-tetramethyl-4-piperidone (0.4 M); lane 12: complex + SOD (15 units); lane 13:
complex + neocuproine (160 lM); lane 14: complex + neocuproine (1 mM); lane 15: complex + distamycin 8 (lM); lane 16: complex + methyl green
(0.00125 mg/mL, 1/10 of [pUC18].
Cu+
Cu+
+ Ox
1.- Cu2+
2.- Cu+
+
+
Red
ing stable five-member rings, giving rise to a tetrahedral-
square planar coordination around the metal site. The
two complexes here studied constitute a further evidence
of the ability of some Cu(II) complexes to interact with
and to split DNA chains. Such an interaction is somewhat
dependent on the nature of the hydrocarbon side-chain
(mesitylene or 4-tert-butylbenzene). This means that fur-
ther studies should be carried out in order to insight into
the structure–activity relationships.
DNA
DNA
3.- Cu+
DNA + H2O2
Copper-Oxo species
Cu+
OOH
Cu2+ OH
DNA
DNA
5. Supplementary material
Crystallographic data (excluding structure factors) for
the structures reported in this paper have been deposited
with the Cambridge Crystallographic Data Center as sup-
plementary material (CCDC-278680-278682). Copies of
the data can be obtained free of charge on application to
CCDC, 12 Union Road, Cambridge CB21EZ, UK
(e-mail: deposit@ccdc.cam.uk).
DNA cleavage
Scheme 1. Possible reaction pathway for DNA cleavage.
inhibits the nucleolytic process with the [Cu(pmesa)2] com-
plex. In both cases, the participation of the superoxide
anion cannot be ruled out, since the reaction of SOD with
ꢀ
O2 produces hydrogen peroxide, which seems to be neces-
Acknowledgements
sary for the reaction. Neocuproine, that forms a very stable
complex with copper(I) ion, inhibits the DNA breakage at
both concentrations studied, indicating that copper(I) is
necessary to obtain the ROS species. Finally, the binders
of minor and major grooves, distamycin and methyl green,
also diminishes the DNA cleavage, suggesting that the
complexes can interact through both grooves.
´
The authors thank ‘Ministerio de Ciencia y Tecnologıa’
and ‘FEDER funds’, grant BQU2002-03965, for financial
support. M.G-A and J.B. thank ‘Ministerio de Ciencia y
Tecnologıa’ and ‘FEDER funds’, Project CTQ2004-
03735. M.G-A thanks Ayuntamiento de Valencia for the
‘‘Carmen and Severo Ochoa’’ grant.
´
From these last experiments, the following mechanism
based on that reported by Sigman [44] can be proposed
(see Scheme 1).
References
[1] A.M. Pyle, J.K. Barton, Prog. Inorg. Chem. 38 (1990) 413.
[2] A. Sigel, H. Sigel, (Eds.), Probing of nucleic acids by metal on
complexes of small molecules, in: Metal Ions in Biological Systems,
1996, p. 33.
4. Conclusions
Sulfonamides derived from 2-picolylamine are able to
act as bidentate ligands coordinating Cu(II) cations, form-
[3] A. Sigel, H. Sigel, (Eds.), Metal ions and their complexes in
medication, in: Metal Ions in Biological Systems, 2004, p. 41.

1472
B. Macı´as et al. / Inorganica Chimica Acta 359 (2006) 1465–1472
´
´
´
´
´
[4] C. Liu, M. Wang, T. Zhang, H. Sun, Coord. Chem. Rev. 248 (2004)
147.
[5] C.P. Raptopoulou, S. Paschalidou, A.A. Pantazaki, A. Terzis, S.P.
Perlepes, Th. Lialiaris, E.G. Bakalbassis, J. Mrozinski, D.A. Kyriaki-
dis, J. Inorg. Biochem. 71 (1998) 15.
[25] B. Macıas, I. Garcıa, M.V. Villa, J. Borras, M. Gonzalez-Alvarez, A.
Castineiras, J. Inorg. Biochem. 96 (2003) 367.
˜
´
´
´
´
´
[26] B. Macıas, I. Garcıa, M.V. Villa, J. Borras, M. Gonzalez-Alvarez, A.
Castineiras, Inorg. Chim. Acta 353 (2003) 139.
˜
´
´
[27] B. Macıas, M.V. Villa, E. Fiz, I. Garcıa, A. Castineiras, M.
˜
´
´ ´
Gonzalez-Alvarez, J. Borras, J. Inorg. Biochem. 88 (2002) 101.
[6] C. Liu, J. Zhou, Q. Li, L. Wang, Z. Liao, H. Xu, J. Inorg. Biochem.
75 (1999) 233.
´
´
´
´
[28] M. Gonzalez-Alvarez, G. Alzuet, J. Borras, B. Macıas, M. Olmo, M.
[7] K.H. Reddy, P.S. Reddy, P.R. Babu, Transit. Met. Chem. 25 (2000)
505.
Liu-Gonza´lez, F. Sanz, J. Inorg. Biochem. 89 (2002) 29.
´
´
´
[29] B. Macıas, M.V. Villa, I. Garcıa, A. Castineiras, J. Borras, R.
˜
´
Cejudo-Marın, Inorg. Chim. Acta 342 (2003) 241.
[8] J. Liu, H. Zhang, C. Chen, H. Deng, T. Lu, L. Ji, Dalton Trans.
(2003) 114.
´
´
´
´
[30] M. Gonzalez-Alvarez, G. Alzuet, J. Borras, B. Macıas, A. Castineiras,
˜
[9] K.J. Humphreys, K.D. Karlin, S.E. Rokita, J. Am. Chem. Soc. 124
(2002) 6009.
Inorg. Chem. 42 (2003) 2992.
´
´
´
´
[31] B. Macıas, M.V. Villa, F. Sanz, J. Borras, M. Gonzalez-Alvarez, G.
[10] C.A. Detmer III, F.V. Pamatong, J.R. Bocarsly, Inorg. Chem. 36
(1997) 3676.
[11] C. Hemmert, M. Pitie, M. Renz, H. Gornitzka, S. Soulet, B.
Meunier, J. Biol. Inorg. Chem. 6 (2001) 14.
[12] K.J. Humphreys, A.E. Johnson, K.D. Karlin, S.E. Rokita, J. Biol.
Inorg. Chem. 7 (2002) 835.
[13] S.T. Frey, H.H. Sun, N.N. Murthy, K.D. Karlin, Inorg. Chim. Acta
242 (1996) 329.
[14] V.G. Vaidyanathan, B.U. Nair, J. Inorg. Biochem. 93 (2003) 271.
[15] A.M. Thomas, M. Nethaji, S. Mahadevan, A.R. Chakravarty, J.
Inorg. Biochem. 94 (2003) 171.
Alzuet, J. Inorg. Chem. 99 (2005) 1441.
[32] International Tables for Crystallography, Vol. C, Kluwer Academic
Publishers, Dordrecht, 1995.
´
[33] M. Martınez-Ripoll, F.H. Cano, An Interactive Program for Oper-
ating Rich. Sefert Single-crystal Four-circle Diffractometers, Institute
of Physical Chemistry Rocasolano, CSIC, Serrano 119, Madrid,
Spain, 1996.
[34] J.M. Stewart, F.A. Kundell, J.C. Baldwin, The X-RAY80 System.
Computer Science Center, University of Maryland, College Pard,
MD, USA, 1990.
[35] Siemens SHELXTLe Version 5.0, Siemens Analytical X-ray Instruments
Inc., Madison, WI 53719, 1995.
´
[16] A. Garcıa-Raso, J.J. Fiol, B. Adrover, V. Moreno, I. Mata, E.
Espinosa, E. Molins, J. Inorg. Biochem. 95 (2003) 77.
[17] X. Wang, H. Chao, H. Li, X. Hong, L. Ji, X. Li, J. Inorg. Biochem.
98 (2004) 423.
[18] A.M. Thomas, M. Nethaji, A.R. Chakravarty, J. Inorg. Biochem. 98
(2004) 1087.
[36] B.N. Figgis, M.A. Hitchman, in: Ligand Field Theory and its
Applications, Wiley–VCH, New York, 2000.
[37] A.B.P. Lever, Inorganic Electronic Spectroscopy, second ed., Elsevier,
New York, 1984.
[38] J. Casanova, G. Alzuet, S. Ferrer, J. Latorre, J.A. Ramirez, J.
´
Borras, Inorg. Chim. Acta 304 (2000) 170.
[19] T. Hirohama, Y. Kuranuki, E. Ebina, T. Sugizaki, H. Arii, M. Chikira,
P.T. Selvi, M. Palaniandavar, J. Inorg. Biochem. 99 (2005) 1205.
[20] H. Zhang, C.S. Liu, X.H. Bu, M. Yang, J. Inorg. Biochem. 99 (2005)
1119.
[21] D.S. Sigman, M.D. Kuwabara, C.B. Chen, T.W. Bruice, Meth.
Enzymol. 208 (1991) 414.
[22] O. Zelenko, J. Gallagher, Y. Xu, D.S. Sigman, Inorg. Chem. 37
(1998) 2198.
[23] S.N. Morehouse, H. Suliman, J. Haff, D. Nguyen, Inorg. Chim. Acta
297 (2000) 411.
´
[39] J. Casanova, G. Alzuet, J. Latorre, J. Borras, Inorg. Chem. 36 (1997)
2052.
[40] WINEPR-Simfonia 1.25 Bruker Analytic Gmbh, Kalrsruhe, FRG,
1994–96.
[41] A.S. Sitlani, E.C. Long, A.M. Pyle, J.K. Barton, J. Am. Chem. Soc.
114 (1992) 2303.
[42] H. Doi, H. Iwasaki, Y. Masubuchi, R. Nishigaki, T. Horie,
Chemico-Biol. Interact. 140 (2002) 109.
[43] C. Ouannes, T.J. Wilson, J. Am. Chem. Soc. 90 (1968) 6527.
[44] L.E. Marsall, D.R. Graham, K.A. Reich, Biochemistry (1981) 244.
[24] R.G. Bhirud, T.S. Srivastava, Inorg. Chim. Acta 179 (1991) 125.