Copper complexes with sulfonamides derivatives of natural amino acids as chemical nucleases

Article abstract of DOI:10.1002/zaac.200700318

Three new coordination compounds have been prepared by reacting copper salts with sulfonamides derived from amino acids. Their molecular structures have been determined with the aid of single crystal X-ray diffraction. The copper cations are fourcoordinated in all cases and the local environment is almost planar. Compound [Cu(ts-gly)(NH₃)₂] ·H ₂O (1) crystallizes in monoclinic space group P2₁/c (n° 14) with Z = 4 and with unit cell parameters a = 6.0490(12) A, b = 24.719(5) A, c = 9.159(2) A, α = γ = 90°, and β = 94.69(3)°. Compound [Cu(ts-isoleu)(NH₃)₂] (2) crystallizes in monoclinic space group C2/c (n° 8) with Z = 15 and with unit cell parameters a = 30.800(6) A, b = 6.5510(13) A, c = 15.830(3) A, α = β = 90°, and β= 100.19(3)°. Compound Na[Cu(ts-gly)(gly)] · 2H₂O (3) crystallizes in triclinic space group P-I (n° 2) with Z = 2 and with unit cell parameters a = 5.6220(11)A, b = 7.775(2) A, c = 18.704(4) A, α = 94.61(3)°, β= 98.32(3)°, and γ = 98.29(3)°. The study of nuclease activity with Plasmid pUC18 shows that all three compounds act as chemical nucleases, although complex Na[Cu(ts-gly)(gly)] ·2H₂O is more efficient in splitting the DNA chains than the other two compounds.

Full text of DOI:10.1002/zaac.200700318

DOI: 10.1002/zaac.200700318
Copper Complexes with Sulfonamides Derivatives of Natural Amino Acids as
Chemical Nucleases
a,
a
a
b
c
´
´
´
´
´
Benigno Macıas *, Marıa V. Villa , Laura Sanchez-Mesonero , Francisca Sanz , Joaquın Borras , and Marta
c
´
´
Gonzalez-Alvarez
a
Salamanca/Spain, Departamento de Quımica Inorganica. Facultad de Farmacia and b Servicio de Rayos X, Universidad de Salamanca
´
´
c
´
´
Valencia/Spain, Departamento de Quımica Inorganica. Facultad de Farmacia. Universidad de Valencia
Received April 26th, 2007; revised June 20th, 2007.
th
˜
Dedicated to Professor Alfonso Castineiras on the Occasion of his 65 Birthday
˚
Abstract. Three new coordination compounds have been prepared
by reacting copper salts with sulfonamides derived from amino ac-
ids. Their molecular structures have been determined with the aid
of single crystal X-ray diffraction. The copper cations are four-
coordinated in all cases and the local environment is almost planar.
Compound [Cu(ts-gly)(NH3)2]·H2O (1) crystallizes in monoclinic
space group P21/c (no 14) with Z ϭ 4 and with unit cell parameters
15.830(3) A, α ϭ γ ϭ 90°, and β ϭ 100.19(3)°. Compound
Na[Cu(ts-gly)(gly)]·2H2O (3) crystallizes in triclinic space group
P-1 (no 2) with Z ϭ 2 and with unit cell parameters a ϭ
˚
˚
˚
5.6220(11) A, b ϭ 7.775(2) A, c ϭ 18.704(4) A, α ϭ 94.61(3)°, β ϭ
98.32(3)°, and γ ϭ 98.29(3)°. The study of nuclease activity with
plasmid pUC18 shows that all three compounds act as chemical
nucleases, although complex Na[Cu(ts-gly)(gly)]·2H2O is more
efficient in splitting the DNA chains than the other two com-
pounds.
˚
˚
˚
a ϭ 6.0490(12) A, b ϭ 24.719(5) A, c ϭ 9.159(2) A, α ϭ γ ϭ 90°,
and β ϭ 94.69(3)°. Compound [Cu(ts-isoleu)(NH3)2] (2) crystallizes
in monoclinic space group C2/c (no 8) with Z ϭ 15 and with unit
˚
˚
cell parameters
a
ϭ
30.800(6) A,
b
ϭ
6.5510(13) A,
c
ϭ
Keywords: Copper; Sulfonamides; Nuclease activity
Introduction
Our group has published numerous papers on the nucle-
ase activity of copper(II) complexes in the presence of
reducing agents [19Ϫ28]. The present paper focuses on cop-
per(II) complexes with sulfonamides derived from natural
amino acids. It should be noted that sulfonamides and their
various derivatives are used extensively in medicine because
of their pharmacological properties, which include antibac-
terial activity [29]. The compounds reported on herein were
studied with the aid of spectroscopic, magnetic, and struc-
tural measurements. Once the complexes had been charac-
terized, we assayed their nuclease activity with plasmid
pUC18 and found that these particular compounds were
similar to other copper complexes that have been described
previously in the literature [30Ϫ34].
The number of reports published in the literature on the
practical use of transition metal complexes as chemical nu-
cleases is considerable [1Ϫ8]. One of the metals most widely
used for this purpose is copper, as it can undergo redox
reactions to produce free radicals that are able to split
nucleic acid chains. The mechanism governing this process
involves the formation of reactive oxygen species (ROS)
such as the hydroxyl radical, the superoxide ion, and singlet
oxygen [9Ϫ18]. In their attempts to elucidate the manner in
which these copper complexes react with DNA, researchers
have found that the reaction can take place not only
through intercalation between the base pairs, but also
through interactions with the minor or major groove of the
nucleic acids.
Experimental Section
Because of the importance of copper(II) complexes in
this type of biological processes, their synthesis and activity
have been studied from various perspectives, with their po-
tential applications inspiring researchers to investigate both
new ligands and more active complexes.
Materials and Methods
4-Toluene sulfonyl chloride was provided by Aldrich; copper salts
and amino acids were provided by Fluka AG. All reagents used
were of analytical grade.
Plasmid pUC18 (0.25 µg/µL) in TE (tris 10 mM and EDTA 1 mM,
pHϭ 8.0) was purchased from Roche Diagnostics, Germany. Solu-
tions of the metal complexes and other reagents for strand scission
experiments were prepared fresh daily.
´
* Dr. Benigno Macıs
´
´
Departamento de Quımica Inorganica. Facultad de Farmacia
Universidad de Salamanca
Chemical analyses for carbon, hydrogen, and nitrogen were per-
formed on a Perkin-Elmer 2400 elemental analyzer. Copper was
determined on an inductively coupled plasma (ICP) spectrometer
Salamanca/Spain
Tel.: ϩ34-923-294524; fax: ϩ34-923-294515.
E-mail address: bmacias@usal.es
Z. Anorg. Allg. Chem. 2007, 633, 1937Ϫ1944
 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1937

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B. Macıas, M. V. Villa, L. Sanchez-Mesonero, F. Sanz, J. Borras, M. Gonzalez-Alvarez
(Perkin-Elmer model 2380 Plasma 2). FT-IR spectra were recorded
(3) were resolved with the aid of direct methods and refined in the
monoclinic space groups P-1 and P21/c, respectively. The Patterson
method was used to resolve the structure of CuC13H23N3O4S (2),
which was then refined in the monoclinic space group C2/c. Full
matrix least-squares refinement with anisotropic thermal param-
eters for non-H atoms was carried out by minimizing ω(Fo -Fc ) .
Refinement on F2 for all reflections, weighted R factors (Rω), and
all goodness of fit S are based on F2 while conventional R factors
(R) are based on F. Interestingly, R factors based on F2 are statisti-
cally almost twice as large those based on F whereas R factors
based on all data are even larger.
with KBr mulls on a Perkin-Elmer FT-IR 1730 instrument. The
Evans method was used to measure magnetic moments at room-
temperature on a magnetic susceptibility balance from Sherwood
Scientific (LTD model MSB-Auto). Electron paramagnetic reson-
ance spectra were recorded at Q-band frequencies with a Bruker
ER 200D spectrometer. In DMF (dimethyl formamide) and meth-
anol solution, electronic spectra were recorded on a Hewlett Pack-
ard 8452A diode spectrophotometer (200-800 nm); in solid state
they were recorded on a Perkin Elmer Lambda 35 spectrophoto-
meter (200-1100 nm).
2
2 2
Calculations were performed with CRYSOM [37] software for data
collection, XRAY8O [38] for data reduction, and SHELXTLTM
[39] for resolving and refining the structures as well as for preparing
material for publication. Crystal data and several details of the
structural determination are given in Table 1. All crystallographic
data have been deposited at the Cambridge Crystallographic
Data Centre, 912 Union Road, Cambridge CB2 1EZ, UK
(fax: ϩ44-1223-336033; e-mail: deposit@ccdc.cam.ac.uk or
www: http://www.ccdc.cam.ac.uk) [CCDC: 644679-644681].
Synthesis of complexes
The N-(p-Toluenesulfonyl) amino acid derivatives, (p-ts-gly) and
(p-ts-isoleu), were obtained by means of a direct reaction between
p-toluenesulfonyl chloride and the amino acid in accordance with
a previously described method [35].
Complexes [Cu(ts-gly)(NH3)2]·H2O (1) and [Cu(ts-isoleu)(NH3)2]
(2) were prepared through reaction of Cu(NO3)2 ·3H2O (1 mmol in
10 mL MeOH) with a solution consisting of (p-ts-gly) or (p-ts-iso-
leu) (2 mmol in 10 mL MeOH) and 2 mL of concentrated aqueous
NH3. Slow evaporation of the solvent from the resulting solution
led to dark blue crystals, which were adequate for molecular struc-
ture determination by means of X-ray diffraction (yield 60 %).
Cleavage of pUC18 plasmid by the complexes
Reactions were performed by mixing 7 µL of cacodylate buffer,
6 µL of complex solution in final concentrations ranging from 6 to
24 µM, 1 µL of pUC18 DNA solution (0.25 µg/µL), and 6 µL of
ascorbate solution in either a 25-fold excess relative to the concen-
tration complex (for complexes 1 and 2) or in a 2.5-fold excess (for
complex 3). The mixtures were incubated for 60 min (complexes 1
and 2) and 30 min (complex 3) at 37 °C. The mixtures were then
quenched with 3 µL of a buffer solution consisting of 0.25 %
bromophenole blue, 0.25 % xylene cyanole, and 30 % glycerol. The
solution was then subjected to electrophoresis on a 0.8 % agarose
gel in 0.5 ϫ TBE buffer (0.045 M tris, 0.045 M boric acid, and
1 mM EDTA) containing 2 µL/100 mL of an ethidium bromide
solution (10 mg/mL) at 80V for approx. 2 h. The gel was photo-
graphed on a capturing gel printer plus TDI.
Anal. for CuC9H17N3O5S (molecular mass ϭ 342.79) C 31.12 (calc.
31.50); H 5.02 (4.96); N 12.09 (12.26); Cu 18.68 (18.54) %.
IR (KBr cmϪ1): 3365 (νN-H); 1587 (νasCOO); 1398 (νsCOO); 1309 (νasSO2); 1134
(νsSO2); 577 (δSO2); 815 (νS-N); 668 (νC-S). UV-Vis: (nm) 650.
Anal. for CuC13H23N3O8S (molecular massϭ380.94) C 41.18 (calc.
40.95); H 6.44 (6.04); N 11.67(11.03); Cu 16.91 (16.66) %.
IR (KBr cmϪ1): 3389 (νN-H); 1590 and 1554 (νasCOO); 1430 (νsCOO); 1337
(νasSO2); 1128 (νsSO2); 577 (δSO2); 809 (νS-N); 672 (νC-S). UV-Vis: (nm) 650.
Complex Na[Cu(ts-gly)(gly)]·2H2O (3) was prepared by adding a
1 M NaOH solution to a solution of p-ts-gly (2 mmol in 10 mL
MeOH) and glycine (2 mmol in 10 mL MeOH) until pHϭ8 is re-
ached. While this initial solution was being stirred, another solu-
tion containing 2 mmol Cu(NO3)2 ·3H2O in 10 mL MeOH was ad-
ded dropwise. Slow evaporation of the solvent at room temperature
from the resulting solution gave rise to intense blue crystals ad-
equate for molecular structure determination by means of X ray
diffraction (yield 65 %). Anal. for CuNaC11H15N2O8S (molecular
mass ϭ 421.8) C 31.18 (calc. 31.29); H 3.53(3.56); N 6.71(6.64);
Cu 15.18(15.04) %.
To test for the presence of reactive oxygen species (ROS) generated
during strand scission, reactive oxygen intermediate scavengers
were added to the reaction mixtures. The scavengers used were
DMSO (0.4 M), tert-butyl alcohol (0.4 M), sodium formate
(0.4 M), 2,2,6,6-tetramethyl-4-piperidone (0.4 M), strichnine
(0.4 M), SOD (15 units), Tiron (10 mM), and catalase (10 µg/mL).
Assays in the presence of either the minor groove binder distamycin
(8 µM), the major groove binder methyl green (2.5 µL of a 0.01 mg/
ml solution), or the stabilizer of copper (I) forms neocuproine
(160 µM) were also performed. Samples were treated as described
above.
IR (KBr cmϪ1): 3350 (νN-H); 1620 and 1590 (νasCOO); 1391 (νsCOO); 1374
(νasSO2); 1141 (νsSO2); 587 (δSO2); 815 (νS-N); 667 (νC-S). UV-Vis: (nm) 610.
Results and Discussion
Crystal Structures Determination
Spectroscopic properties
X-ray diffraction data were collected on a four circle Seifert XRD
3003 SC diffractometer (CuKα, λ ϭ 1.54180 A) with a graphite
˚
The IR spectra clearly show the two most relevant groups
present in the complexes. The band that had been recorded
for the free ligand at 1718 cmϪ1 (p-ts-gly) and 1702 cmϪ1
(p-ts-isoleu), which is due to the stretching vibration of the
CϭO double bond, is absent from the spectra of the com-
plexes, with the carboxylate group giving rise to new bands
at 1590 cmϪ1 (antisymmetric vibration) and 1400 cmϪ1
monochromator at room temperature and with a ω-2θ scan. The
unit cell parameters were determined through least-squares refine-
ment on the 2θ values of 25 strong, well-centered reflections in the
range 16° < 2θ < 40°. Scattering factors for neutral atoms and
anomalous dispersion correction for Cu, Na, C, N, O, and S were
taken from the “International Tables for X Ray Crystallography”
[36]. The structures of CuC9H17N3O5S (1) and CuNaC11H15N2O8S
1938
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 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2007, 1937Ϫ1944

Copper Complexes with Sulfonamides Derivatives
Table 1 Summary of crystal parameters, data collection and refinement for the two crystal structures.
[Cu(ts-gly)(NH3)2]·H2O
[Cu(ts-isoleu)(NH3)2]
Na[Cu(ts-gly)(gly)]·2H2O
Empirical formula
Formula weight
Temperature /K
CuC9H17N3O5S
342.86
293(2)
CuC13H23N3O4S
380.94
293(2)
CuNaC11H15N2O8S
421.84
293(2)
˚
Wavelength /A
1.54180
1.54180
1.54180
Crystal system
Space group
monoclinic
P21/c
monoclinic
C2/c
triclinic
P1
¯
Unit cell dimensions
˚
a /A
6.0490(12)
24.719(5)
9.159(2)
30.800(6)
6.5510(13)
15.830(3)
5.6220(11)
7.775(2)
18.704(4)
˚
b /A
˚
c /A
α /°
β /°
γ /°
94.61(3)
98.32(3)
98.29(3)
796.2(3)
94.69(3)
100.19(3)
3
˚
Volume /A
1364.9(5)
3143.7(11)
Z
4
8
2
Calculated density/g cmϪ3
Absorption coefficient /mmϪ1
F(000)
1.668
3.897
708
1.610
3.400
1592
1.760
3.857
430
Crystal size /mm
θ range for data collection/°
Limiting indices
Reflections collected / observed
Refinement method
Data / restraints / parameters
Goodness-of-fit on F2
Final R indices [I > 2σ(I)]
R indices (all data)
Absolute structure parameter
0.10 ϫ 0.15 ϫ 0.20
3.58Ϫ59.97
0.10 ϫ 0.12 ϫ 0.18
2.92Ϫ64.93
0.08 ϫ 0.12 ϫ 0.20
2.40Ϫ59.98
0ՅhՅ6, 0ՅkՅ27, Ϫ10ՅlՅ0
1246 / 931
Ϫ33ՅhՅ34, Ϫ7ՅkՅ7, Ϫ16ՅlՅ0
4937 / 1999
Ϫ6ՅhՅ6, Ϫ8ՅkՅ8, Ϫ20ՅlՅ0
2367 / 2184
full-matrix least-squares on F2
1244 / 2 / 182
full-matrix least-squares on F2
2622 / 0 / 198
full-matrix least-squares on F2
2359/ 0 / 226
0.999
1.059
1.191
R1 ϭ 0.0549, wR2 ϭ 0.1411
R1 ϭ 0.0734, wR2 ϭ 0.1532
0.0045(8)
R1 ϭ 0.0680, wR2 ϭ 0.2013
R1 ϭ 0.0844, wR2 ϭ 0.2182
0.0010(2)
R1 ϭ 0.0586, wR2 ϭ 0.1571
R1 ϭ 0.0642, wR2 ϭ 0.1680
0.0058(15)
Ϫ3
˚
Largest diff. peak and hole /e A
0.389 and Ϫ0.495
1.293 and Ϫ1.663
1.462 and Ϫ0.942
(symmetric vibration). The bands resulting from the sym-
metric and antisymmetric vibration modes of the SO2 group
are recorded in positions very close to those for the ligands.
The electronic spectra recorded in the solid state with the
DR technique show a broad band at about 650 nm in the
visible range for the complexes with two coordinating am-
monia molecules, but closer to 610 nm for the complex with
coordinating glycine. These bands are present in essentially
the same positions in the spectra recorded in solution, re-
gardless of whether methanol or DMF is used as solvent.
This finding indicates that the immediate environment of
the copper cations is the same in both solution and solid
state. This difference in the position of the absorption maxi-
mum correlates nicely with the differences found for the ef-
fective magnetic moments: 1.75 BM for the ammonia com-
plexes, which is very close to the expected “spin only” value
of 1.73 BM, and 1.94 BM for the glycine complex. These
differences probably arise from the different environment of
the copper cation in each complex; while it is N3O in the
first series of complexes, it is N2O2 in the second complex
[40, 41].
Na[Cu(ts-gly)(gly)]·2H2O is rhombic with gx (2.045) < gy
(2.080) < gz (2.250) and R ϭ 0.20, which suggests that the
2
2
unpaired electron is in the dx Ϫy orbital.
Crystal Structures
The molecular structures of the complexes, along with their
unit cells, are shown in Figures 1 to 6 and selected bond
distances and angles are shown in Table 2. The copper cat-
The Q-band spectra of the polycrystalline samples of the
complexes were recorded at room temperature. Powder
samples of [Cu(ts-gly)(NH3)2]·H2O (1), [Cu(ts-iso-
leu)(NH3)2] (2), and Na[Cu(ts-gly)(gly)]·2H2O (3) were pre-
pared from single crystals. The Q-band EPR spectra of
[Cu(ts-gly)(NH3)2]·H2O and [Cu(ts-isoleu)(NH3)2] are axial
spectra, with gʈ (2.160; 2.223, respectively) > g֊ (2.055;
2.053, respectively). This indicates a mainly copper(II)
Figure 1 Molecular structure of the [Cu(ts-gly)(NH3)2]·H2O
2
2
dx Ϫy orbital ground state. The Q-band EPR spectrum of
complex.
Z. Anorg. Allg. Chem. 2007, 1937Ϫ1944
 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.zaac.wiley-vch.de
1939

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B. Macıas, M. V. Villa, L. Sanchez-Mesonero, F. Sanz, J. Borras, M. Gonzalez-Alvarez
˚
Table 2 Selected bond length /A and angles /° for copper complexes.
[Cu(ts-gly)(NH3)2]·H2O
[Cu(ts-isoleu)(NH3)2]
Na[Cu(ts-gly)(gly)]·2H2O
Bond lengths
Bond lengths
Bond lengths
Cu(1)-N(1)
Cu(1)-O(4)
Cu(1)-N(2)
Cu(1)-N(3)
1.983(9)
1.954(7)
1.981(9)
1.989(8)
Cu(1)-N(1)
Cu(1)-O(4)
Cu(1)-N(2)
Cu(1)-N(3)
1.975(5)
1.937(5)
1.971(6)
2.21(2)
Cu(1)-N(1)
Cu(1)-O(4)
Cu(1)-N(2)
Cu(1)-O(5)
1.945(4)
1.945(3)
1.982(4)
1.972(3)
Bond angles
Bond angles
Bond angles
O(4)-Cu(1)-N(1)
O(4)-Cu(1)-N(3)
N(1)-Cu(1)-N(3)
O(4)-Cu(1)-N(2)
N(1)-Cu(1)-N(2)
N(3)-Cu(1)-N(2)
83.1(3)
88.2(4)
170.8(3)
172.2(3)
99.5(3)
89.6(4)
O(4)-Cu(1)-N(1)
O(4)-Cu(1)-N(3)
N(1)-Cu(1)-N(3)
O(4)-Cu(1)-N(2)
N(1)-Cu(1)-N(2)
N(3)-Cu(1)-N(2)
82.3(2)
92.8(5)
171.0(5)
171.2(2)
97.6(2)
86.2(5)
O(4)-Cu(1)-N(1)
O(4)-Cu(1)-O(5)
N(1)-Cu(1)-O(5)
O(4)-Cu(1)-N(2)
N(1)-Cu(1)-N(2)
O(5)-Cu(1)-N(2)
84.17(15)
89.33(14)
169.47(15)
168.9(2)
102.1(2)
83.0(2)
Figure 2 Stereoview of the unit cell for [Cu(ts-gly)(NH3)2]·H2O
complex.
Figure 4 Stereoview of the unit cell for [Cu(ts-isoleu)(NH3)2]
complex.
Figure
3 Molecular structure of the [Cu(ts-isoleu)(NH3)2]
complex.
ions are four-coordinated in all cases, with a CuN3O group
in the complexes with coordinating ammonia in addition to
the sulfonamide ligand and a CuN2O2 group for the com-
plex with coordinating glycine. The local environment is al-
most planar, as the angles formed by the copper atom with
Figure 5 Molecular structure of the Na[Cu(ts-gly)(gly)]·2H2O
complex.
1940
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 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2007, 1937Ϫ1944

Copper Complexes with Sulfonamides Derivatives
Figure 7 Agarose gel electrophoresis of pUC18 plasmid DNA
treated with copper salt, the complexes [Cu(ts-gly)(NH3)2]·H2O (1)
and [Cu(ts-isoleu)(NH3)2] (2) and the bis(o-phenantroline)cop-
per(II) and 25 fold-excess of ascorbate. Incubation time 60 min
(37 °C)
1. λDNA/EcoRIϩHindIII Marker; 2. control; 3. Control ϩ ascor-
bate; 4. CuSO4 6 µM; 5. CuSO4 12 µM; 6. CuSO4 18 µM; 7. CuSO4
24 µM; 8. Complex 1 6 µM; 9. Complex 1 12 µM; 10. Complex 1
18 µM; 11. Complex 1 24 µM; 12. Complex 2 6 µM; 13. Complex 2
12 µM; 14. Complex
2 18 µM; 15. Complex 2 24 µM; 16.
[Cu(phen)2]2ϩ 24 µM
pUC18 in 0.1 M cacodylate buffer (pHϭ 6.0) and with as-
corbate activation. The DNA cleavage activity of the com-
plexes was assayed from the conversion of supercoiled
DNA (SC, Form I) to nicked circular (NC, Form II) or
linearized DNA (LC, Form III).
Figure
6 Stereoview of the unit cell for Na[Cu(ts-
gly)(gly)]·2H2O complex.
trans donor atoms are close to or greater than 170°. The
angle formed by glycine bonded to the toluensulfonyl group
in complex Na[Cu(ts-gly)(gly)]·2H2O (84.17°) is similar to
that measured for the complex in which only glycine coordi-
nates the copper cation (83°). The other two angles are
larger due to the lack of restrictions from bidentate ligands.
For the other two complexes, in which there are two coor-
dinating ammonia molecules, the angle formed by glycine
and copper is also close to 83°. Both the Cu-O and the
Cu-N distances are very similar in all complexes, and are
all within the range expected for this type of complexes
[31, 34].
The unit cell drawings allow us to locate the positions of
both the water molecules and the sodium cations in those
complexes containing these species. The complex derived
from isoleucine crystallizes without water molecules. This is
probably because of the larger size of the organic chain, but
may also be due to its greater hydrophobic effect. As
for the copper cations, they are basically aligned with
each other. From the unit cell for complex Na[Cu(ts-
gly)(gly)]·2H2O, it is clear that the molecules are oriented
parallel to the a axis and that they are twisted 180° to one
another along the b axis, with octahedrally-coordinated Na
cations between the layers. The various structural units in
complex [Cu(ts-gly)(NH3)2]·H2O have a similar distri-
bution, with the H2O molecules between them.
Figure 7 shows the results of the addition of [Cu(ts-
gly)(NH3)2]·H2O (1) or [Cu(ts-isoleu)(NH3)2] (2) to pUC18
DNA in the presence of a 25-fold excess of ascorbate for
60 min (pH ϭ 6.0). For comparative purposes, reactions
were also carried out with CuII sulfate, which is known to
cause extensive DNA cleavage in the presence of ascorbate/
H2O2 [42]. The extent of the cleavage reaction with increas-
ing concentrations of the complex (6, 12, 18, and 24 µM) is
given in lanes 8-11 for complexes [Cu(ts-gly)(NH3)2]·H2O
and in lanes 12-15 for complex [Cu(ts-isoleu)(NH3)2]. Both
complexes show strong nuclease activity, as is obvious from
the data in lanes 11 and 15, which indicate that the super-
coiled DNA has disappeared and only forms II and III are
present. In contrast, the copper(II) sulfate solutions (lanes
4-7) cause DNA damage only at the highest concentration,
indicating that the [Cu(ts-gly)(NH3)2]·H2O and [Cu(ts-iso-
leu)(NH3)2] complexes are more potent chemical nucleases.
Nevertheless, both of these complexes exhibit less nuclease
activity than the bisphenantroline copper(II) complex (lane
16). Figure 8 shows the electrophoretic results of the ad-
dition of the Na[Cu(ts-gly)(gly)]·2H2O (3) complex to
pUC18 DNA in the presence of a 2.5-fold excess of ascorb-
ate for 30 min (pH ϭ 6). This complex shows potent nucle-
ase ability, as can be inferred from the fact that at reducing
agent concentrations lower than those used in the experi-
ment with the other two complexes, only forms II and III
of the DNA appear (see lanes 8-11). Obviously, the nuclease
activity of the copper sulfate is lower than that of the cop-
per complex. Moreover, the Na[Cu(ts-gly)(gly)]·2H2O com-
plex shows cleavage activity similar to that of the bisphen-
antroline copper(II) complex (lane 12), the known para-
digm of artificial chemical nucleases.
Nuclease Activity of complexes and mechanistic
investigations
Oxidative DNA cleavage mediated by the compounds
The ability of the complexes to cleave DNA has been inves-
tigated by means of gel electrophoresis with supercoiled
Z. Anorg. Allg. Chem. 2007, 1937Ϫ1944
 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.zaac.wiley-vch.de
1941

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´
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B. Macıas, M. V. Villa, L. Sanchez-Mesonero, F. Sanz, J. Borras, M. Gonzalez-Alvarez
shown in Figure 9(a,b,c). The involvement of reactive oxy-
gen species was investigated with standard hydroxyl radical
scavengers (DMSO, tert-butyl alcohol, and sodium formi-
ate), singlet oxygen scavengers (2,2,6,6 tetramethyl-4-piperi-
done and strichnine), and superoxide radical scavengers (Ti-
ron and superoxide dismutase enzyme). The groove binding
preferences were tested in the presence of the minor groove
binder distamycin and the major groove binder methyl
green. The reduction of CuII to CuI during the cleavage
process was evaluated with neocuproine, a ligand that che-
lates strongly to copper(I).
Figure 8 Agarose gel electrophoresis of pUC18 plasmid DNA
treated with copper salt, the complex Na[Cu(ts-gly)(gly)]·2H2O (3)
and the bis(o-phenantroline)copper(II) and 2.5 fold-excess of ascor-
bate. Incubation time 30 min (37 °C)
Upon addition of DMSO, tert-butyl alcohol, and sodium
formate, a strong inhibition of the DNA cleavage mediated
by the three complexes is observed, thus indicating that hy-
droxyl radical is involved in the cleavage process (lanes 4-6,
Figure 9). The addition of tetramethylpiperidone or strichn-
ine (lanes 7 and 8, Figure 9) also decreases the cleavage
1. λDNA/EcoRIϩHindIII Marker; 2. control; 3. Control ϩ ascor-
bate; 4. CuSO4 6 µM; 5. CuSO4 12 µM; 6. CuSO4 18 µM; 7. CuSO4
24 µM; 8. Complex 3 6 µM; 9. Complex 3 12 µM; 10. Complex 3
18 µM; 11. Complex 3 24 µM; 12. [Cu(phen)2]2ϩ 12 µM
1
efficiency, which indicates that either O2 or a singlet oxy-
Mechanistic studies
gen-like entity is one of the active oxygen intermediates re-
sponsible for the cleavage. SOD (lane 9 of the Figures 9)
produces various behaviors, depending on the complex.
While complexes [Cu(ts-gly)(NH3)2]·H2O and Na[Cu(ts-
gly)(gly)]·2H2O cause increased DNA damage in the pres-
ence of SOD, no inhibition is observed with complex
[Cu(ts-isoleu)(NH3)2]. Adding Tiron to the reaction mixture
slightly decreases the DNA damage, which suggests that the
superoxide anion is involved in the DNA scission (lane 10,
Figure 9). It may be that superoxide decomposes, giving rise
to hydrogen peroxide that can develop into a hydroxyl rad-
ical and, as a consequence, increase the DNA cleavage. Cat-
alase also inhibited the nucleolytic process, a finding which
confirms that hydrogen peroxide is also necessary in the
DNA strand scission (lane 14, Figure 9).
The mechanism by which the compounds cleave pUC18
DNA was studied with the aid of inhibiting reagents. Re-
sults for complexes [Cu(ts-gly)(NH3)2]·H2O (1), [Cu(ts-iso-
leu)(NH3)2] (2), and Na[Cu(ts-gly)(gly)]·2H2O (3) are
Treatment of pUC18 DNA with distamycin prior to the
addition of the three complexes inhibits the cleavage reac-
tion (lane 12, Figure 9). In contrast, prior treatment with
methyl green has little effect on DNA strand scission (lane
13, Figure 9). From these results we can infer that all three
complexes have a preference for minor groove binding.
Finally, neocuproine inhibits the DNA damage (lane 11,
Figure 9), probably as a consequence of the stability of the
copper(I) neocuproine complex.
In general, two mechanisms can be proposed for CuII
complexes that mediate oxidative DNA cleavage, one in-
volving hydroxyl radicals and another involving metal-oxo
species.
Bocarsly et al. [43Ϫ45] suggest that hydroxyl radicals are
generated by a variety of chemical and physical processes.
Transition metal-mediated hydroxyl radical production is
known to occur via a number of routes, including the
Fenton mechanism and the Haber-Weiss reaction.
Figure 9 Agarose gel electrophoresis of pUC18 plasmid treated
with (a) complex 1 24 µM, ascorbate 25x, (b) complex 2 24 µM,
ascorbate 25x, (c) complex 3 24 µM ascorbate 2.5x, and ascorbate
in the presence of potential inhibitors. Incubation time 60 min
(37 °C) for complexes 1 and 2 or 30 min (37 °C) for complex 3.
Lane 1. λDNA/EcoR1ϩHindIII Marker; 2. control; 3. complex; 4.
complex ϩ dmso (0.4 M); 5. complex ϩ tert-butyl alcohol (0.4 M);
6. complex ϩ sodium formate (0.4 M); 7. complex ϩ 2,2,6,6 tetra-
methylpiperidone (0.4 M); 8. complex ϩ strichnine (0.4 M); 9. com-
plex ϩ SOD (15 units); 10. complex ϩ tiron (0.4 M); 11. complex
ϩ neocuproine (160 µM); 12. complex ϩ distamycin (8 µM);
13. complex ϩ methyl green (2.5 µL of a 0.01 mg/ml solution);
14. complex ϩ catalase (2.6 units)
In the Fenton mechanism, the metal acts as a catalyst for
hydroxyl radical generation from hydrogen peroxide:
CuIIL ϩ ascH2 Ǟ CuIL ϩ ascHϪ
CuIL ϩ H2O2 Ǟ CuIIL ϩ OHϪ ϩ OHț
1942
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 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2007, 1937Ϫ1944

Copper Complexes with Sulfonamides Derivatives
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The Haber-Weiss mechanism involves the reaction between
the superoxide anion with hydrogen peroxide:
CuIL ϩ O2 Ǟ CuIIL ϩ O2
Ϫ
O2 ϩ H2O2 Ǟ O2 ϩ OHϪ ϩ OHț
Ϫ
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Inorg. Chim. Acta 2004, 357, 2315.
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Sigman, however, asserts that bis(o-phenantroline)cop-
per(II), the classic nuclease system, follows a different
mechanistic pathway [46] in which reduction of the copper
to the cuprous state leads to the generation of the superox-
Ϫ
ide anion. The O2 then dismutates to H2O2 and dioxygen.
The hydrogen peroxide goes on to react with another equiv-
alent of cuprous ion to produce a hydroxyl radical-like spec-
ies, which may be metal-bound. It is this species, which
could be considered analogous to metal-oxo species, that is
responsible for initiating the DNA strand scission [47].
Bocarsly et al. [43Ϫ45] have compared their proposed
mechanism with that of Cu-phenanthroline. They conclude
that the differences between the two mechanisms are most
likely related to the electronic differences between the
ligands. Their mechanism is related to complexes with
ligands that present an σ character while the mechanism
proposed by Sigman is related to complexes with ligands
that have a π character (phen).
[18] Y. Li, Y. Wu, J. Zhao, P. Yang, J. Inorg. Biochem. 2007, 101,
283.
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´
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´
[19] M. Gonzalez-Alvarez, G. Alzuet, J. Borras, B. Macıas, M.
´
Olmo, M. Liu-Gonzalez, F. Sanz, J. Inorg. Biochem. 2002,
89, 29.
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´
[20] B. Macıas, M. V. Villa, E. Fiz, I. Garcıa, A. Castin˜eiras, M.
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Gonzalez-Alvarez, J. Borras, J. Inorg. Biochem. 2002, 88, 101.
´
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[21] B. Macıas, M. V. Villa, I. Garcıa, A. Castin˜eiras, J. Borras, R.
´
Cejudo-Marın, Inorg. Chim. Acta 2003, 342, 241.
´
In the title complexes, both mechanisms can be proposed.
This explains why not only the hydroxyl radical scavengers,
but also the scavengers of singlet oxygen or singlet oxygen-
like species inhibit the DNA damage and strand breakage.
´
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[22] B. Macıas, I. Garcıa, M. V. Villa, J. Borras, M. Gonzalez-Alva-
˜
rez, A. Castineiras, J. Inorg. Biochem. 2003, 96, 367.
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[23] M. Gonzalez-Alvarez, G. Alzuet, J. Borras, B. Macıas, A. Cas-
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tineiras, Inorg. Chem. 2003, 42, 2992.
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[24] B. Macıas, M. V. Villa, F. Sanz, J. Borras, M. Gozalez-Alvarez,
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Acknowledgments. Authors acknowledge financial support from the
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[25] M. Gonzalez-Alvarez, G. Alzuet, J. L. Garcıa-Gimenez, B.
´
Junta de Castilla y Leon (SA056A05). J. B. and M. G.-A. acknowl-
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Macıas, J. Borras, Z. Anorg. Allg. Chem. 2005, 631, 2181.
edge financial support from the Spanish CICYT (CTQ2004-
03735).
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[26] B. Macıas, M. V. Villa, M. Salgado, J. Borras, M. Gonzalez-
Alvarez, F. Sanz, Inorg. Chim. Acta 2006, 359, 1465.
[27] J. Borras, G. Alzuet, M. Gonzalez-Alvarez, J. L. Garcıa-
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 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2007, 1937Ϫ1944