Functional superoxide dismutase mimics. Structural characterization and magnetic exchange interactions of copper(II)-N-substituted sulfonamide dimer complexes

Article abstract of DOI:10.1021/ic049718w

Dinuclear copper(II) complexes with N-substituted sulfonamide ligands as superoxide dismutase (SOD) mimics have been investigated. The new N-(thiazol-2-yl)toluenesulfonamide (Htz-tol) and N-(thiazol-2-yl)naphthalene- sulfonamide (Htz-naf) ligands have been prepared and structurally characterized. The complexes derived from these ligands, [Cu₂(tz-tol)₄] (1) and [Cu₂(tz-naf)₄] (2), have been synthesized, and their crystal structure, magnetic properties, and EPR spectra were studied in detail. In both compounds the metal centers are bridged by four nonlinear triatomic NCN groups. The coordination geometry of the coppers in the dinuclear entity of 1 and 2 is distorted square planar with two N-thiazole and two N-sulfonamido atoms. Magnetic susceptibility data show a strong antiferromagnetic coupling, with -2J = 121.3 cm^-1 for compound 1 and -2J = 104.3 cm^-1 for compound 2. The EPR spectra of the polycrystalline samples of compounds 1 and 2 have been measured at the X- and Q-band frequencies at different temperatures. Above 20 K the spectra are characteristic of S = 1 species with zero-field splitting parameter D = 0.230 cm^-1 for compound 1 and 0.229 cm^-1 for compound 2. The EPR parameters are discussed in terms of the known binuclear structures. The complexes exhibit high SOD activity, as shown by the low IC₅₀ values obtained with the xanthine/xanthine oxidase/NBT assay: 0.13 μM for compound 1; 0.17 μM for compound 2.

Full text of DOI:10.1021/ic049718w

Inorg. Chem. 2004, 43, 6805−6814
Functional Superoxide Dismutase Mimics. Structural Characterization
and Magnetic Exchange Interactions of Copper(II)
Sulfonamide Dimer Complexes†
−N-Substituted
R. Cejudo-Mar´ın, G. Alzuet, S. Ferrer, and J. Borra´s*
Departamento de Qu´ımica Inorga´nica, Facultad de Farmacia, UniVersidad de Valencia,
AVda. Vicent Andre´s Estelle´s s/n, 46100-Burjassot, Spain
A. Castin˜eiras
Departamento de Qu´ımica Inorga´nica, Facultad de Farmacia, UniVersidad de Santiago de
Compostela, 15703-Santiago de Compostela, Spain
E. Monzani and L. Casella
Dipartimento di Chimica Generale, UniVersita` di PaVia, Via Taramelli 12, 27100-PaVia, Italy
Received March 4, 2004
Dinuclear copper(II) complexes with N-substituted sulfonamide ligands as superoxide dismutase (SOD) mimics
have been investigated. The new N-(thiazol-2-yl)toluenesulfonamide (Htz-tol) and N-(thiazol-2-yl)naphthalene-
sulfonamide (Htz-naf) ligands have been prepared and structurally characterized. The complexes derived from
these ligands, [Cu2(tz-tol)4] (1) and [Cu2(tz-naf)4] (2), have been synthesized, and their crystal structure, magnetic
properties, and EPR spectra were studied in detail. In both compounds the metal centers are bridged by four
nonlinear triatomic NCN groups. The coordination geometry of the coppers in the dinuclear entity of 1 and 2 is
distorted square planar with two N-thiazole and two N-sulfonamido atoms. Magnetic susceptibility data show a
1
1
strong antiferromagnetic coupling, with
−2J
)
121.3 cm- for compound 1 and
−2J
)
104.3 cm- for compound
2. The EPR spectra of the polycrystalline samples of compounds 1 and 2 have been measured at the X- and
Q-band frequencies at different temperatures. Above 20 K the spectra are characteristic of S 1 species with
zero-field splitting parameter D
0.230 cm-1 for compound 1 and 0.229 cm-1 for compound 2. The EPR parameters
are discussed in terms of the known binuclear structures. The complexes exhibit high SOD activity, as shown by
)
)
the low IC50 values obtained with the xanthine/xanthine oxidase/NBT assay: 0.13
µM for compound 1; 0.17 µM
for compound 2.
Introduction
important role in the protection of cells against its toxicity
effects. However, in the case of an oxygen burst, these
enzymes are insufficient in vivo. The clinical efficacy of
SOD has been disappointing because SOD enzymes have
molecular weights too high to cross the cell membranes.
Studies on the reactivity of low molecular weight copper
complexes which have a SOD-like function have attracted
much attention for the development of SOD mimics and for
expanding the biomimetic chemistry of transition metal
complexes. For many years efforts have been made to obtain
compounds with high catalytic activity. Among the most
successful compounds possessing SOD activity are dinuclear
Superoxide anion radical is a metabolite of oxygen in vivo
and is considered to be a highly toxic species in many living
systems.1 It is involved in lipid peroxidation, DNA damage,
aging, cancer, and many other diseases. Superoxide dismu-
•-
tase (SOD) is an efficient scavenger of O2 and plays an
* Author to whom correspondence should be addressed. E-mail:
joaquin.borras@uv.es. Tel: ++34 963544530. Fax: ++34 963544960.
† Abbreviations: Htz-naf ) N-(thiazol-2-yl)naphthalenesulfonamide; Htz-
tol ) N-(thiazol-2-yl)toluenesulfonamide; MF+ ) blue formazane; NBT
) nitro blue tetrazolium; SOD ) superoxide dismutase.
(1) Sawyer, D. T.; Valentine, J. S. Acc. Chem. Res. 1981, 14, 393.
10.1021/ic049718w CCC: $27.50
Published on Web 09/15/2004
© 2004 American Chemical Society
Inorganic Chemistry, Vol. 43, No. 21, 2004 6805

Cejudo-Mar´ın et al.
copper complexes.2-4 Important requirements for SOD-like
activity appear to be a medium strength donor power and
the flexibility of the ligands. Interestingly, the presence of
coordination sites belonging to N heteroaromatic rings is
considered an important factor for gaining high SOD activity.
We have previously reported a variety of mononuclear Cu-
(II) compounds with sulfathiazole (4-amino-N-(thiazol-2-yl)-
benzenesulfonamide) and imidazole, imidazole derivatives
or pyridine that exhibit high SOD-like activity.5-7 Also, we
have prepared and fully characterized a dinuclear copper
compound of sulfathiazolato.8 In this compound, classified
as a type IV copper dimer,9 the two metal ions are bridged
by four nonlinear triatomic NCN groups. These triatomic
units are found in biological molecules such as adenine.10,11
More recently, we have described the crystal structure and
properties of a dicopper(II) complex with sulfamethazine
(4-amino-N-[4,6-dimethyl-2-pyrimidinyl]benzenesulfon-
amide) bridged by two carboxylato anions and by two NCN
fragments of the ligand.12 Both compounds display interesting
magnetic and EPR behavior. DFT studies have shown the
nature of the antiferromagnetic interaction between the two
copper(II) at a very short distance.
As an extension of our studies of sulfonamide derivatives
and their metal complexes, we have developed a new class
of N-substituted sulfonamide ligands based on thiazole units.
This type of ligands possess important features for obtaining
copper(II) complexes with high SOD-like activity: (i)
nonlinear NCN fragments and, thus, the possibility of
forming dinuclear compounds with relatively short Cu-Cu
distances; (ii) a coordination site with nitrogen heteroaromatic
donors; (iii) the ability of accommodating the copper ion in
different geometries.
Chart 1
purification. Elemental Analyses (C, H, N, S) were carried out at
the microanalytical laboratory of the Instituto Tecnolo´gico de
Qu´ımica (Universidad Polite´cnica de Valencia, Valencia, Spain).
The infrared spectra (ν ) 400-4000 cm-1) were obtained on KBr
pellets using a Mattson Satellite FTIR spectrophotometer. Electronic
spectra were recorded on samples dispersed in Nujol mulls (solid)
and on acetonitrile and acetone solutions using a Shimadzu 2101
PC spectrophotometer. The kinetic measurements were performed
on a HP 8452A spectrophotometer. Electrochemical measurements
were made using a Princeton Applied Research model 273A
potentiostat/galvanostat. Cyclic voltammograms were obtained for
acetone solutions of the complexes containing tetrabutylammonium
perchlorate as the supporting electrolyte under argon atmosphere.
The electrochemical cell employed was of a standard three-electrode
configuration: platinum working electrode; platinum wire counter
electrode; Ag-AgCl reference electrode. All the potentials were
transformed to NHE scale. EPR measurements on ground crystals
were carried out at variable temperature on a Bruker ELEXSYS
spectrometer, operating at X-band and Q-band frequencies. Frozen-
solution EPR spectra of the complexes were recorded in acetonitrile
and acetone at 100 K. The variable-temperature magnetic suscep-
tibility measurements on microcrystalline samples were taken with
a Quantum Design MPMS2 SQUID susceptometer equipped with
a 55 kG magnet, operating at 10 kG in the range of 1.8-400 K.
The susceptometer was calibrated with (NH4)2Mn(SO4)2‚12H2O.
Corrections for the diamagnetism were estimated from the Pascal
constants.
Synthesis of the Ligands: N-(Thiazol-2-yl)toluenesulfonamide
(Htz-tol) and N-(Thiazol-2-yl)naphthalenesulfonamide (Htz-naf).
A solution containing 2.0 g of 2-aminothiazole and 4.55 g of the
corresponding sulfonyl chloride (p-toluenesulfonyl chloride or
naphthalenesulfonyl chloride) in 12 mL of pyridine was heated to
60 °C for 1 h. The mixture was poured into 20 mL cold water and
then stirred for several min. The resulting product was recrystallized
from ethanol. Crystals suitable for X-ray diffraction were formed
after 6 months. Yield: 72%, based on C10H10N2S2O2 (Htz-tol) (Mr
) 254.34). Anal. Calcd: C, 47.24; H, 3.94; N, 11.02; S, 25.20.
In this paper, we report the synthesis, crystal structures,
magnetic properties, and SOD-like activity of two new
dinuclear compounds possessing the above characteristics,
[Cu2[tz-tol)4] (Htz-tol ) N-(thiazol-2-yl)toluenesulfonamide)
and [Cu2[tz-naf)4] (Htz-naf ) N-(thiazol-2-yl)naftalene-
sulfonamide) (see Chart 1).
Experimental Section
Found: C, 47.81; H, 3.95; N, 10.81; S, 24.78. IR (KBr) (νmax
/
Materials and Physical Methods. All reagents and chemicals
were purchased from commercial sources and used without further
cm-1): 1541 (thiazole ring); 1329 and 1147 (SO2); 934 (S-N).
Yield: 65%, based on C13H10N2S2O2 (Htz-naf) (Mr ) 290.35). Anal.
Calcd: C, 53.42; H, 3.42; N, 9.60; S, 21.92. Found: C, 53.84; H,
3.38; N, 9.41; S, 21.81. IR (KBr)(νmax/cm-1): 1540 (thiazole ring);
1306, 1279, 1141, and 1118 (SO2); 928 (S-N).
(2) Tabbi, G.; Driessen, V. L.; Reedikj, J.; Bonomo, R. P.; Veldman, N.;
Spek, A. L. Inorg. Chem. 1997, 36, 1168.
(3) Zhang, J.-J.; Luo, Q.-H.; Long, De-L.; Chen, J.-T.; Li, F.-M.; Liu,
A.-D. J. Chem. Soc., Dalton Trans. 2000, 1893.
(4) Feng, C.-J.; Luo, K.-H.; Wang, Z.-L.; Shen, M.-C.; Wang, H.-W.;
Zhao M.-H. J. Inorg. Biochem. 1999, 75, 1.
(5) Casanova, J.; Alzuet, G.; Borra´s, J.; Latorre, J.; Sanau´, M.; Garc´ıa-
Granda, S. J. Inorg. Biochem. 1995, 60, 219.
(6) Casanova, J.; Alzuet, G.; Borra´s, J.; Carugo, O. J. Chem. Soc., Dalton
Trans. 1996, 2239.
(7) Casanova, J.; Alzuet, G.; Ferrer, S.; Latorre, J.; Ramirez, J. A.; Borra´s,
J. Inorg. Chim. Acta 2000, 304, 170.
(8) Casanova, J.; Alzuet, G.; Latorre, J.; Borra´s, J. Inorg. Chem. 1997,
36, 2052.
(9) Hathaway, B. J. Copper. In ComprehensiVe Coordination Chemistry;
Wilkinson G., Gillard R. D., Cleverty, J. A., Eds.; Pergamon Press:
New York, 1987; Vol. 5.
Synthesis of the Complexes. (a) [Cu2[tz-tol)4] (1). A 100 mL
volume of an ethanolic solution containing 1 mmol of Htz-tol (0.254
g) was added to 50 mL of an ethanolic solution containing 0.5 mmol
of Cu(CF3COO)2 (0.15 g). The resulting yellow mixture was left
to stand at room temperature. After 7-8 days well-shaped dark
red crystals suitable for X-ray diffraction were formed. Yield: 51%,
based on Cu2C40H36N8S8O8 (Mr ) 1140.33). Anal. Calcd: C, 42.13;
H, 3.18; N, 9.83; S, 22.49. Found: C, 41.89; H, 2.93; N, 9.73; S,
22.82. IR (KBr) (νmax/cm-1): 1471 (thiazole ring); 1322, 1140 (SO2);
948 (S-N). Solid-state electronic spectra (λmax, nm): 510 (LF).
UV-vis: in CH3CN, λmax ) 490 nm (ꢀ ) 1070 mol-1 dm3 cm-1);
in acetone, λmax ) 495 nm (ꢀ ) 992 mol-1 dm3 cm-1).
(10) de Meester, P.; Goodgame, D. M. L.; Price, K. A.; Skapski, A. C.
Nature 1971, 229, 191.
(11) de Meester, P.; Skapsk, A. C. J. Chem. Soc A 1971, 2167.
(12) Gutierrez, L.; Alzuet, G.; Borra´s, J.; Castin˜eiras, A.; Rodriguez-Fortea,
A.; Ruiz, E. Inorg. Chem. 2001, 40, 3089.
(b) [Cu2(tz-naf)4] (2). The [Cu2(tz-naf)4] compound was obtained
by following the procedure described above, but in this case the
6806 Inorganic Chemistry, Vol. 43, No. 21, 2004

Functional Superoxide Dismutase Mimics
Htz-naf ligand was dissolved in 100 mL of acetone. Yield: 25%,
based on Cu2C52H36N8S8O8 (Mr ) 1284.45). Anal. Calcd: C, 48.68;
H, 2.82; N, 8.72; S, 19.97. Found: C, 48.93; H, 2.73; N, 8.63; S,
19.82. IR (KBr) (νmax/cm-1): 1467 (thiazole ring); 1322, 1298, 1144,
1123 (SO2); 954 (S-N). Solid-state electronic spectra (λmax, nm):
500 (LF). UV-vis: in CH3CN, λmax ) 500 nm (ꢀ ) 882 mol-1
dm3 cm-1); in acetone, λmax ) 498 nm (ꢀ ) 1272 mol-1 dm3 cm-1).
X-ray Structure Determination. (a) N-(Thiazol-2-yl)toluene-
sulfonamide (Htz-tol). Diffraction data were collected on an area
detector equipped Kappa CCD single-crystal diffractometer, using
a combination of æ and ω scans.13 Data were integrated, scaled,
and averaged by the HKL2000 package.14 Crystal structures were
solved by Patterson methods, using the program DIRDIF.15 An
empirical absorption correction was applied using XABS2.16
Anisotropic least-squares refinement was carried out with SHELXL-
97.17 Atomic scattering factors were taken from ref 18. Plots were
made with the ORTEP package.19 All calculations were made at
the Scientific Computer Center of the University of Oviedo and
on the X-ray group computers.
(b) N-(Thiazol-2-yl)naphthalenesulfonamide (Htz-naf). An
orange prismatic crystal of C13H10N2O2S2 was mounted on a glass
fiber and used for data collection. Cell constants and an orientation
matrix for data collection were obtained by least-squares refinement
of the diffraction data from 25 reflections in the range of 12.66 <
θ < 37.42° on an Enraf Nonius MACH3 automatic diffractometer.20
Data were collected at 293 K using Cu KR radiation (λ ) 1.541 84
Å) and the ω/2θ-scan technique and corrected for Lorentz and
polarization effects.21 A semiempirical absorption correction (ω-
scans) was made.22 The structure was solved by direct methods,23
which revealed the position of all non-hydrogen atoms, and refined
on F2 by a full-matrix least-squares procedure using anisotropic
displacement parameters.17 All hydrogen atoms were located from
difference Fourier maps and included as fixed contributions riding
on attached C atoms with isotropic thermal parameters 1.3 times
those of the respective C atoms. Atomic scattering factors were
taken from ref 18. Molecular graphics were made with PLATON24
and SCHAKAL.25
absorption using SADABS (transmission factors: 0.925-0.797).27
The structure was solved by direct methods and refined by full-
matrix least-squares techniques against F2 using the program
SHELXS-97.17 Positional and anisotropic atomic displacement
parameters were refined for all non-hydrogen atoms. Hydrogen
atoms were placed geometrically, and positional parameters were
refined using a riding model. Isotropic atomic displacement
parameters for hydrogen atoms were constrained to be 1.2 (1.5 for
methyl groups). Atomic scattering factors were taken from ref 18.
Molecular graphics were made with PLATON99.24
(d) [Cu2(tz-naf)4]. A red-brown block crystal of [Cu(C26H18-
N4O4S4)2]2 was mounted on a glass fiber and used for data
collection. Cell constants and an orientation matrix for data
collection were obtained by least-squares refinement of the dif-
fraction data from 25 reflections in the range of 11.210° < θ <
30.406° on an Enraf-Nonius CAD4 automatic diffractometer.20 Data
were collected using the ω-scan technique and corrected for Lorentz
and polarization effects.21 A semiempirical absorption correction
(ω-scan) was made.22 The structure was solved by Patterson and
Fourier methods,23 which revealed the position of all non-hydrogen
atoms, and refined on F2 by a full-matrix least-squares procedure
using anisotropic displacement parameters.17 Hydrogen atoms were
introduced in the calculation in idealized positions [d(C-H) ) 0.92
Å], and their atomic coordinates were recalculated after each cycle.
They were given isotropic thermal parameters 20% higher than
those of the carbon to which they are attached. Criteria of a
satisfactory complete analysis were the value of the ratios of rms
shift to standard deviation less than 0.001 and no significant features
in final difference maps. Atomic scattering factors were taken from
ref 18. Molecular graphics were from PLATON24 and SCHAKAL.25
A summary of the crystal data, experimental details, and
refinement results for Htz-tol, Htz-naf, complex 1, and complex 2
are listed in Table 1.
SOD Assay. The SOD activity of the copper compounds was
studied according to a modification of the xanthine/xanthine oxidase
assay,28 with NBT as a superoxide anion detecting agent.29 The
assay was carried out in 50 mM phosphate buffer, pH 7.8, in
thermostated optical cells at 25.0 ( 0.1 °C. The kinetics of reduction
of nitro blue tetrazolium (NBT) to blue formazane (MF+) was
monitored through the absorbance changes with time at 560 nm.
In a typical experiment, solutions of NBT (0.23 mM) and xanthine
(0.28 mM) were mixed in the cuvette and the reaction was started
by addition of a concentrated xanthine oxidase solution (final
volume 1600 µL). The xanthine oxidase concentration must be
optimized to find the appropriate amount that causes a linear
absorbance variation with time during the reading period (usually
60 s). Under these conditions the turnover concentration of
superoxide remains constant during the assay. We found that with
the optimized xanthine oxidase concentration a straight line is
observed when ∆A560/min is lower than 0.03. With larger initial
rates the absorbance variation with time follows a parabolic
behavior. The effect of the Cu complexes on the NBT reduction
rates was evaluated by adding small amounts of a concentrated
solution of each complex to the assay solution so that its final
concentration in the cuvette varied from 0.05 to 10 µM. The
(c) [Cu2(tz-tol)4]. A red plate crystal of [Cu(C10H9N2O2S2)2]2
was mounted on a glass fiber and used for data collection. Crystal
data were collected using a Bruker SMART CCD 1000 diffractom-
eter. The data were processed with SAINT26 and corrected for
(13) Nonius, BV, Collect; Enraf-Nonius: Delft, The Netherlands, 1997-
2000.
(14) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307.
(15) Beurskens, P. T.; Beurskens, G.; de Gelder, R.; Garc´ıa-Granda, S.;
Gould, R. O.; Israel, R.; Smits, J. M. M. The DIRDIF-99 program
system; Technical Report of the Crystallography Laboratory; University
of Nijmegen: Nijmegen, The Netherlands, 1999.
(16) Parkin, S.; Moezzi, B.; Hope, H. J. Appl. Crystallogr. 1995, 28, 53.
(17) Sheldrick, G. M. SHELXL-97. Program for the Refinement of Crystal
Structures; University of Go¨ttingen: Go¨ttingen, Germany, 1997.
(18) International Tables for Crystallography; Kynoch Press: Birmingham,
U.K., 1992 (present distributor Kluwer Academic Publishers, Dor-
drecht, The Netherlands); Vol. C, Tables 4.2.6.8 and 6.1.1.4.
(19) Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.
(20) Nonius, BV CAD4-Express Software, ver. 5.1/1.2; Enraf-Nonius: Delft,
The Netherlands, 1994.
(21) Kretschmar, M. GENHKL Program for the Reducction of CAD4
Diffractometer Data; University of Tuebingen: Tuebingen, Germany,
1997.
(22) North, A. C. T.; Phillips, D. C.; Mathews, F. S. Acta Crystallogr.
1968, A24, 351.
(26) Bruker SMART and SAINT, Area Detector Control and Integration
Software; Bruker Analytical X-ray Instruments Inc.: Madison, WI,
1997.
(23) Sheldrick, G. M. Acta Crystallogr. 1990, A46, 467.
(24) Spek, A. L. PLATON. A Multipurpose Crystallographic Tool; Utrecht
University: Utrecht, The Netherlands, 2000.
(25) Keller, E. SCHAKAL-97. A computer program for the graphic
representation of molecular and crystallographyc models; University
of Freiburg i. Br.: Freiburg, Germany, 1997.
(27) Sheldrick, G. M. SADABS, Program for Empirical Absorption
Correction of Area Detector Data; University of Goettingen: Goet-
tingen, Germany, 1997.
(28) Beauchamp, C.; Fridovich I. Anal. Biochem. 1971, 44, 276.
(29) Bielski, B. H. J.; Shiue, G. G.; Bajuk, S. J. Phys. Chem. 1980, 84,
830.
Inorganic Chemistry, Vol. 43, No. 21, 2004 6807

Cejudo-Mar´ın et al.
Table 1. Crystal and Structure Refinement Data for Htz-tol, Htz-naf, [Cu(tz-tol)2]2 (1), and [Cu(tz-naf)2]2 (2)
param
Htz-tol
10H10N2O2S2
254.34
293(2)
Htz-naf
13H10N2O2S2
290.35
293(2)
[Cu(tz-tol)2]2 (1)
40H36Cu2N8O8S8
1140.33
291(2)
[Cu(tz-naf)2]2 (2)
empirical formula
fw
C
C
C
C
52H36Cu2N8O8S8
1284.45
temp, K
213(2)
wavelength, Å
cryst system
space group
1.5418
monoclinic
P21/a
1.541 84
monoclinic
P21/c (No. 14)
0.710 73
monoclinic
C2/c (No. 15)
a ) 27.174(3)
1.541 84
triclinic
P1h (No. 2)
a ) 12.8308(8), R ) 66.679(15)
unit cell dimens, Å and deg
a ) 13.21320(7)
a ) 12.8179(15)
b ) 6.5384(3), â ) 110.135(2) b ) 9.1246(11), â ) 90.993(7) b ) 10.1931(10), â ) 122.025(2) b ) 14.964(2), â ) 82.525(11)
c ) 13.68250(5)
1109.83(5)
4; 1.522
4.253
c ) 11.0332(8)
1290.2(2)
4; 1.495
3.742
c ) 20.025(2)
4702.6(8)
4; 1.611
1.320
c ) 15.048(3), γ ) 83.314(9)
2624.0(7)
2; 1.626
4.513
V, Å3
Z; calcd density, Mg/m3
abs coeff, mm-1
F(000)
528
600
2328
1308
cryst size, mm
θ range for data collcn, deg
limiting indices
0.35 × 0.32 × 0.30
6.77-69.69
16 e h e 15, -7 e k e 6,
-16 e l e 16
0.32 × 0.20 × 0.20
3.45-72.89
-15 e h e 15, 0 e k e 11,
0 e l e 13
0.18 × 0.16 × 0.06
0.15 × 0.10 × 0.05
1.77-28.06
5.34-65.03
-35 e h e 35, -13 e k e 12,
-13 e l e 26
-1 e h e 15, -17 e k e 17,
-17 e l e 17
10245/8918 [R(int) ) 0.0613]
65.03; 99.8
reflecn collcd/unique
completeness to θ deg; %
abs corr
3307/2058 [R(int) ) 0.0292] 5452/2582 [R(int) ) 0.0050] 15546/5654 [R(int) ) 0.1197]
69.69; 98.8
empirical
0.3907 and 0.2881
72.89; 100.0
Ψ-scans
0.5216 and 0.3806
28.06; 98.9
SADABS
0.9250 and 0.7971
Ψ-scan
max and min transmn
refinement method
data/restraints/params
goodness-of-fit on F2
final R indices [I > 2σ(I)]
R indices (all data)
0.8058 and 0.5509
full-matrix least squares on F2
8918/0/703
full-matrix least squares on F2 full-matrix least squares on F2 full-matrix least squares on F2
2058/0/169
2.096
2582/0/173
1.086
5654/0/298
0.711
1.020
R1 ) 0.0463, wR2 ) 0.1598 R1 ) 0.0348, wR2 ) 0.0971 R1 ) 0.0505, wR2 ) 0.0662
R1 ) 0.0518, wR2 ) 0.1982 R1 ) 0.0435, wR2 ) 0.1039 R1 ) 0.1960, wR2 ) 0.0884
R1 ) 0.0487, wR2 ) 0.1042
R1 ) 0.1028, wR2 ) 0.1224
0.527 and -0.525
largest diff peak and hole, e‚Å-3 0.371 and -0.625
0.271 and -0.300
0.338 and -0.418
Scheme 1
distorted tetrahedral configuration with bond angles in the
range from 104 to 119°. The C-C bond distances of the
toluene ring in Htz-tol and those of the naphthalene ring in
Htz-naf can be regarding as normal.
In the Htz-naf ligand the thiazole and naphthalene rings
are nearly perpendicular to each other (angle between planes,
82.69°). The structure is stabilized by a strong hydrogen bond
formed by the Nthiazole and Nsulfonamido (N-H‚‚‚N distance,
2.849(2) Å).
absorbance change at 560 nm with time upon addition of the xantine
oxidase solution (as optimized in the previous step) was followed.
Blank experiments were carried without xanthine oxidase to correct
for the absorbance readings.
Description of the Crystal Structures of [Cu2(tz-tol)4]
(1) and [Cu2(tz-naf)4] (2). The molecular drawings of the
[Cu2(tz-tol)4] (1) and [Cu2(tz-naf)4] (2) complexes with the
atomic numbering scheme are shown in Figures 1 and 2,
respectively. Selected structural parameters for the two
compounds are listed in Table 2. The structures of 1 and 2
consist of dimer units. In both complexes each copper atom
of the dinuclear entity is four-coordinated with a slightly
distorted square planar environment. The copper(II) ions are
linked to two thiazole N atoms of two sulfonamidate ligands
(tz-tol- in 1 and tz-naf- in 2) and two sulfonamido N atoms
from other two sulfonamidate anions. In the coordination
polyhedra the Nthiazole atoms are in trans position. In the
dinuclear species the metal ions are bridged by four triatomic
NCN groups. For the two compounds the metal-ligand
distances Cu-Nthiazole (ca. 1.94 Å in 1 and ca. 1.96 Å in 2)
are shorter than the Cu-Nsulfonamido ones (ca. 2.07 Å in 1
and ca. 2.03 Å in 2). These bond lengths compare well with
those for previously reported N-substituted sulfonamide
copper complexes.5,6,8,12 Bond angles of the CuN4 chro-
mophores are close to 90° and range from 87.98(15) to 92.30-
(15)° in 1 and from 86.98(15) to 90.89(15)° in 2. In
compound 1 the Cu(II) ion is displaced 0.2182(21) Å above
the mean plane formed by N(11), N(21a), N(12a), and N(22)
atoms. In complex 2 the Cu1 atom is placed 0.1847(20) Å
above the plane formed by N(12), N(21), N(32), and N(41)
Results and Discussion
Crystal Structure of the Ligands. ORTEP drawings of
the Htz-tol and Htz-naf ligands, bond distances (Å), and
angles (deg) are deposited as Supporting Information. In
general, angles and bond distances of the two ligands do
not show significant features compared with related sulfon-
amides.30,31 It is known that N-substituted sulfonamides exist
as two tautomeric forms, amido and imido (see Scheme 1).
In the Htz-tol ligand the C(1)-N(1) bond distance [1.320-
(4) Å] is slightly shorter than the C(1)-N(2) one [1.342(4)
Å], which seems to indicate that Htz-tol adopts mainly the
imido form. However, for the Htz-naf ligand these bond
lengths are found to be equal [C1-N1, 1.338(2) Å; C1-
N2, 1.330(2) Å] suggesting an equilibrium between both
amido and imido forms. In the two ligands, the dimensions
of all the C-N distances do not support the existence of
distinct CdN (average 1.316 Å) and C-N (average 1.416
Å) bonds due to an extensive π electron pair delocalization
through the C(1)-N(1), C(1)-N(2), and C(2)-N(2) bonds.
Atoms around the sulfonamide S atom are arranged in a
(30) Casanova, J.; Alzuet, G.; Ferrer, S.; Borra´s, J.; Garc´ıa-Granda, S.;
Perez-Carren˜o, E. J. Inorg. Biochem. 1993, 51, 689.
(31) Pedregosa, J. C.; Casanova, J.; Alzuet, G.; Borra´s, J.; Garc´ıa-Granda,
S.; Gutie´rrez Rodr´ıguez. A. Inorg. Chim. Acta 1995, 232, 117.
6808 Inorganic Chemistry, Vol. 43, No. 21, 2004

Functional Superoxide Dismutase Mimics
Figure 1. Molecular structure of [Cu2(tz-tol)4].
Figure 2. Molecular structure of [Cu2(tz-naf)4].
atoms, which are arranged in a regular square plane, and
the Cu2 atom is located 0.1998(21) Å above the mean plane
formed by N(11), N(22), N(31), and N(42) atoms. The
Cu‚‚‚Cu distances, 2.7224(13) Å in 1 and 2.6257(10) Å in
2, are in the range found for dinuclear copper(II) com-
pounds with four NCN bridges.9-11 It is noteworthy that the
metal-metal distance in 2 is very short, comparable with
the Cu‚‚‚Cu distance in the structurally related [Cu2-
(sulfathiazolato)4] complex.9 In [Cu2(tz-tol)4] (1) and [Cu2-
(tz-naf)4] (2) the two CuN4 planes of each compound show
a parallel arrangement. For 1 the dihedral angle between the
two CuN4 planes is 0° since this complex has a binary axis
perpendicular to the center of Cu‚‚‚Cu distance, and for 2
the dihedral angle between the two CuN4 mean planes is
1.49(21)°.
the angle subtended by two planes, each encompassing the
copper and two donor atoms.32 For strictly square planar
complexes with D4h symmetry, the value is 0°, while, for
regular tetrahedral complexes, the angle is 90°. The tetra-
hedrality value of 24.29(22)° for the [Cu2(tz-tol)4] compound
indicates a higher distortion from the square planar geometry
than the tetrahedrality values of 16.28(24)° [Cu1] and 17.40-
(26)° [Cu2] for the [Cu2(tz-naf)4] complex.
The tz-tol- anions in 1 and the tz-naf- anions in 2 act as
bridging bidentate ligands, linking one Cu(II) by the thiazole
N atom and the other Cu(II) through the sulfonamidate N
atom. In general, the internal geometry of the tz-tol- and
tz-naf- compares well with that of the corresponding
protonated ligands (Htz-tol, Htz-naf); only a slight lengthen-
ing of the Cthiazole-Nsulfonamido bond distances and a slight
The square planar geometry of the copper(II) ions in both
compounds presents tetrahedral distortion. Tetrahedrality for
any tetracoordinate copper complex can be characterized by
(32) Battaglia, L. P.; Bonamartini-Corradi, A.; Marcotrigiano, G.; Menabue,
L.; Pellacani, G. C. Inorg. Chem. 1979, 18, 148.
Inorganic Chemistry, Vol. 43, No. 21, 2004 6809

Cejudo-Mar´ın et al.
Table 2. Selected Bond Distances (Å) and Angles (deg) for
[Cu(tz-tol)2]2 (1) and [Cu(tz-naf)2]2 (2)a
[Cu(tz-tol)2]2
Cu(1)-N(21)#1
Cu(1)-N(11)
Cu(1)-N(12)#1
Cu(1)-N(22)
Cu(1)-Cu(1)#1
[Cu(tz-naf)2]2
1.942(4)
1.947(4)
2.067(4)
2.088(4)
Cu(1)-N(21)
1.965(4)
1.969(4)
2.024(4)
2.047(4)
2.6257(10)
1.963(4)
1.988(4)
2.018(4)
2.055(4)
Cu(1)-N(41)
Cu(1)-N(32)
Cu(1)-N(12)
2.7224(13) Cu(1)-Cu(2)
Cu(2)-N(11)
Cu(2)-N(31)
Cu(2)-N(22)
Cu(2)-N(42)
N(21)#1-Cu(1)-N(11) 178.04(17) N(21)-Cu(1)-N(41) 173.89(17)
N(21)#1-Cu(1)-N(12)#1 90.11(16) N(21)-Cu(1)-N(32) 86.98(15)
N(11)-Cu(1)-N(12)#1 87.98(15) N(41)-Cu(1)-N(32) 90.89(15)
N(21)#1-Cu(1)-N(22) 89.63(15) N(21)-Cu(1)-N(12) 90.19(15)
Figure 3. Temperature dependence of øM (b) and øMT (O) for [Cu2(tz-
tol)4]. The solid lines represent the fitted function.
N(11)-Cu(1)-N(22)
92.30(15) N(41)-Cu(1)-N(12) 90.38(15)
N(12)#1-Cu(1)-N(22) 155.71(15) N(32)-Cu(1)-N(12) 164.78(15)
N(21)#1-Cu(1)-Cu(1)#1 91.08(14) N(11)-Cu(2)-N(31) 173.13(16)
N(11)-Cu(1)-Cu(1)#1
88.99(13) N(11)-Cu(2)-N(22) 90.47(16)
process CuIICuI/CuI2. In the reverse scan, an anodic peak at
-41 mV due to the two copper(I) oxidation is observed. The
irreversibility of the electrochemical processes was thought
to be caused by changes in the coordination sphere around
the metal center upon reduction.
N(12)#1-Cu(1)-Cu(1)#1 78.50(11) N(31)-Cu(2)-N(22) 87.75(16)
N(11)-Cu(2)-N(42) 89.14(16)
N(31)-Cu(2)-N(42) 90.76(16)
N(22)-Cu(2)-N(42) 164.07(15)
a Symmetry transformation used to generate equivalent atoms: (#1) -x,
y, -z + 1/2.
Magnetic Properties. Magnetic susceptibility measure-
ments of 1 and 2 were performed on crystals in the
temperature range 2-350 K. The temperature dependence
of the magnetic susceptibility/Cu of the [Cu2(tz-tol)4] com-
pound is shown in Figure 3. The [Cu2(tz-naf)4] complex
exhibits the same magnetic behavior (data not shown). The
dominant features of the plots are a maximum around 100
K and a rapid decrease to zero at lower temperatures. The
solid curve in Figure 3 is the best fit of the data to the
Bleaney-Bowers equation34 for the exchange-coupled cop-
per(II) dimers,
shortening of the Cthiazole-Nthiazol bond lengths due to
deprotonation and coordination can be appreciated.
Spectroscopic and Electrochemical Properties. The IR
spectra of the Cu(II) complexes show similar pattern of
bands. Compared with the IR spectra of the free ligands,
the most remarkable difference occurs in the band corre-
sponding to the stretching vibration of the thiazole ring,
which is shifted from 1540 cm-1 in the free ligands to ca.
1470 cm-1 in the complexes. The ν(SO2)as and ν(SO2)s bands
do not show significant shifts with respect to those of the
ligands. The characteristic band corresponding to the ν(S-
N) appears to be shifted by ca. 14 cm-1 (compound 1) and
26 cm-1 (compound 2) to higher frequencies.
øM ) (Ng2â2/kT)[3 + exp(-2J/kT)]-1(1 - F) +
FNg2â2/4kT + øTIP (1)
The diffuse reflectance spectra of the two complexes show
a very broad and intense band around 500 nm, characteristic
of a CuN4 chromophore with the copper ion in a square
planar environment.33 The electronic spectra of the complexes
in CH3CN and in acetone display a maximum at ca. 490 nm
for 1 and at ca. 500 nm for 2. As no significant changes
occur in the position of d-d bands upon dissolution of the
complexes, this indicates the absence of significant modifica-
tions in the coordination sphere of the metal ions.
which results from a consideration of the eigenvalues of H
) -2JS1S2 and where the symbols have the usual meanings.
An excellent fit was obtained when -2J ) 121.3 cm-1, g )
2.19, øTIP ) 120 × 10-6, F ) 0.022, and R ) 3.5 × 10-5
for compound 1 and -2J ) 104.3 cm-1, g ) 2.13, øTIP
)
120 × 10-6, F ) 0.006, and R ) 1.2 × 10-4 for compound
2.
The antiferromagnetic coupling constant of both com-
pounds is larger than that of the related [Cu2(sulfathiazolato)4]
complex (-2J ) 61.5 cm-1). No direct correlation is found
between the magnitude of the coupling constant and the
Cu‚‚‚Cu distance (1, 2.7224 Å; 2, 2.6257 Å; [Cu2(sulfa-
thiazolato)4], 2.671 Å). Moreover, it should be noted that
the antiferromagnetic exchange coupling constants of 1 and
2 are smaller than that of copper(II) dimers with adenine
and its derivatives.10,11 In this sense, the magnetic behavior
of [Cu2(tz-tol)4] and [Cu2(tz-naf)4] is in good agreement with
the theoretical results previously reported by us12 for di-
nuclear copper(II) compounds with NCN bridges belonging
to ligands with two condensed ring (adenine) or with one
single ring (sulfathiazole or sulfamethazine).
The electrochemical behavior of both complexes was
investigated by cyclic voltammetry in acetonitrile solution.
When we scan toward the negative potentials, the cyclic
voltammogram curve of complex 1 is characterized by one
irreversible electrochemical signal at Epc ) -394 mV that
corresponds to the reduction CuII /CuI2. Accordingly, in the
2
reverse scan, a sharp anodic peak is found at -24 mV which
can be attributed to oxidation of two copper(I) ions. For
complex 2, the cathodic part of the cyclic voltammogram
reveals two successive irreversible electrochemical signals.
The first weak signal at -198 mV is assigned to the reduction
CuII /CuIICuI and the second at -377 mV to the reduction
2
(33) Ravichandran, V.; Chacko, K. K.; Aoki, A.; Yamazaki, H.; Ruiz-
Sa´nchez, J.; Suarez-Varela, J.; Lo´pez-Gonzalez, J. D.; Salas-Peregr´ın,
J. M.; Colacio-Rodriguez, E. Inorg. Chim. Acta 1990, 173, 107.
(34) Bleaney, B.; Bowers, K. D. Proc. R. Soc. London, Ser. A 1952, 214,
451.
6810 Inorganic Chemistry, Vol. 43, No. 21, 2004

Functional Superoxide Dismutase Mimics
samples, two transitions allowed by the selection rule (∆ms
) (1) will result in each principal direction and six
resonance fields can be determined. Furthermore, a transition
with (∆ms ) (2) is also obtained. The six resonance fields
1
2
1
2
1
2
are Hx , Hx , Hy , Hy , Hz , and Hz , whose equations are
proposed by Wasserman et al.36 for a rhombic symmetry (E
* 0). For an axial symmetry (E ) 0), when D < hν, as is
usual in the case of copper dimers, four ∆ms ) (1 transitions
along with a half-field (∆ms ) (2) transition are allowed.
1
1
2
2
These four resonance fields are Hz , Hxy , Hxy , and Hz , which
are given by the following equations:
Hz1 ) (ge/gz)(H0 - D′)
(3)
(4)
Hxy1 ) (g /g )2H (H - D′)
e
z
0
0
Hxy2 ) (g /g )2H (H + D′)
(5)
(6)
e
z
0
0
Hz2 ) (ge/gz)(H0 + D′)
The X-band EPR spectrum of [Cu2(tz-tol)4] (1) at room
temperature (Figure 4a) shows transitions at 932, 1874, 4324,
and at 4822 G, the last one occurring as a shoulder. At the
Q-band, the spectrum of 1 displays peaks at 8980, 10 800,
and 12 960 G (Figure 4b). The X-band EPR spectrum of
[Cu2(tz-naf)4] (2) at room temperature (Figure 4a) exhibits
peaks at 996, 2009, and 4261 G and a shoulder at 4800 G.
At the Q-band the spectrum of 2 shows transitions at 9000,
10 700, 12 970, and 13 850 G (Figure 4b). These signals,
1
1
2
2
assigned to the resonance fields Hz , Hxy , Hxy , and Hz ,
respectively, are indicative of copper centers with an axial
symmetry, in agreement with the crystal structures. In
addition, the formally forbidden transition (∆ms ) (2), the
so-called half-field spin transition, is observed at the Q-band
at 5607 G for [Cu2(tz-tol)4] and at 5580 G for [Cu2(tz-naf)4].
Figure 4. (a) X-band powder EPR spectra of [Cu2(tz-tol)4] and [Cu2(tz-
naf)4] at 290 K. (b) Q-band powder EPR spectra of [Cu2(tz-tol)4] and [Cu2-
(tz-naf)4] at 290 K.
EPR. The solid-state EPR spectra of the complexes
obtained at room temperature at the X- and Q-band (Figure
4) show signals typical for coupled binuclear complexes,35,36
together with the characteristic signal for a monomeric
species at ∼3300 G (Figure 4a) and at ∼11 300 G (Figure
4b). The formation of a binuclear complex is connected with
antiferromagnetic coupling of two Cu(II) ions, leading to a
singlet ground state and an excited spin triplet state. The
energy difference, 2J, between these states depends on the
strength of the interaction. If the triplet state is thermally
accessible (2J ≈ kT ≈ 200-400 cm-1) paramagnetism is
observed and the EPR spectra could be satisfactorily
described using the interactive spin Hamiltonian for isolated
Cu(II) dimers (S ) 1):
1
1
2
2
From the observed transitions Hz , Hxy , Hxy , and Hz and
the half-field spin transitions37 the calculated parameters are
the following: D ) 0.230 cm-1, g| ) 2.35, g ) 2.08
(compound 1); D ) 0.229 cm-1, g| ) 2.25, g ) 2.07
(compound 2). The D value is of the same order of magnitude
as that of the structurally related [Cu2(stz)4] complex.9
The X-band EPR spectra of both complexes were recorded
at different temperatures. The spectra of the [Cu2(tz-naf)4]
(2) complex at 290, 70, and 20 K are shown in Figure 5.
The spectra of [Cu2(tz-tol)4] (1) in the same conditions are
similar (data not shown). At 10 K the EPR of the two
complexes are silent. For both compounds, the peaks in the
EPR spectrum at room temperature are clearly much broader
than those of the spectra at 70 K, a temperature close to that
of the maximum in the magnetic measurements. At high
temperatures, the triplet states are relatively more populated
and the dimer signals are broader due to short electron-spin
relaxation times (T1e) and to intermolecular interactions.
When the temperature decreased to 20 K, the spectrum
became narrower and seven hyperfine features with a relative
intensity of 1:2:3:4:3:2:1, due to two interacting Cu(II) ions
H ) gâBS + DSz2 + E(Sx2 - Sy2) - 2D/3
(2)
Here, D and E are the zero field splitting parameters, â is
the Bohr magneton, and x, y, and z are the principal axes of
the coordinating system, fixed with respect to the Cu‚‚‚Cu
bond. Wasson et al.35 showed that, in the case of powder
(35) Wasson, J. R.; Chin-I, S.; Trapp, C. Inorg. Chem. 1968, 7, 469.
(36) Wasserman, E.; Snyder, L. C.; Yager, W. A. J. Chem. Phys. 1964,
41, 1763.
(37) Eaton, S. A.; More, K. M.; Sawant, B. M.; Eaton, G. R. J. Am. Chem.
Soc. 1983, 105, 6550.
Inorganic Chemistry, Vol. 43, No. 21, 2004 6811

Cejudo-Mar´ın et al.
Figure 7. SOD activity study of the complex [Cu2(tz-tol)4]: Dependence
absorbance changes with time at 560 nm as a function of [Cu2(tz-tol)4]
concentration.
Figure 5. X-band powder EPR spectra of [Cu2(tz-naf)4] at 290, 70, and
20 K.
Figure 8. SOD activity study of the complex [Cu2(tz-naf)4]: Dependence
absorbance changes with time at 560 nm as a function of [Cu2(tz-naf)4]
concentration.
1
Figure 6. Temperature dependence of the intensity of the Hz EPR signal
for the [Cu2(tz-tol)4] complex.
characteristic of mononuclear species with a square planar
CuN4 chromophore. In the parallel region a hyperfine
structure with A| value of 151 × 10-4 cm-1 is observed. Also,
in the perpendicular region, nine signals attributed to the
superhyperfine coupling of the four nitrogen with AN ) 14
× 10-4 cm-1 are clearly resolved.39 In frozen acetone solution
the X-band EPR spectrum of complex 1 exhibits weak signals
at 1966 G and at 4281 G. These signals are almost coincident
with the corresponding ones in the EPR spectrum of the
polycrystalline sample which indicates that the dimeric
species remains in solution.
Superoxide Dismutase Activity. Kinetic studies of the
SOD reaction by [Cu2(tz-tol)4] and [Cu2(tz-naf)4] complexes
were carried out using the xanthine/xanthine oxidase/NBT
assay system. NBT acts as superoxide detecting agent
through its reduction to MF+. Figures 7 and 8 show that the
rate of the absorbance development at 560 nm with time is
reduced in the presence of [Cu2(tz-tol)4] and [Cu2(tz-naf)4],
respectively, in a concentration-dependent behavior. This
indicates that in the presence of both dinuclear complexes
the steady-state concentration of superoxide is reduced; thus,
(2nI + 1; n ) 2 and I ) 3/2) are observed in the low-field
components. The separation between these lines is ap-
proximately 67 G and is just a half of the hyperfine splitting
evident on the line corresponding to the free copper ion. This
is just what is to be expected from a line, the origin of which
is due to an exchange interaction.38
For evaluation of the exchange interaction parameter, 2J,
the variation of the intensity of the EPR signals with the
temperature was studied for the two complexes. Figure 6
1
shows the integral intensity of the Hz peak at the tempera-
tures from 293 to 20 K for compound 1. The relative intensity
of the absorption is given by35
R
1/T(e-2J/kT)/(1 + 3e-2J/kT
)
(7)
From the plots, the values of -2J ) 123.76 cm-1 for [Cu2-
(tz-tol)4] and -2J ) 94.87 cm-1 for [Cu2(tz-naf)4] were
obtained; these values are very close to those obtained from
magnetic measurements.
In frozen acetonitrile solution the X-band EPR spectrum
of complex 1 shows a very weak signal whose pattern is
(38) Slichter, C. P. Phys. ReV. 1955, 99, 479.
(39) Wieserma, A. K.; Windle, J. J. J. Phys. Chem. 1964, 68, 2316.
6812 Inorganic Chemistry, Vol. 43, No. 21, 2004

Functional Superoxide Dismutase Mimics
Table 3. SOD Activity of Copper Complexes
[Cu2(tz-tol)4] and [Cu2(tz-naf)4] exhibit significant catalytic
activity toward the dismutation of superoxide.
compd
[Cu2(tz-tol)4]
IC50 (µM)
ref
In the presence of the copper complex the mechanism of
the reduction of NBT to MF+ comprises several reactions.
The system xanthine/xanthine oxidase produces superoxide
ion that undergoes self-degradation via a bimolecular reaction
but also reacts with NBT to produce MF+. At the same time
0.13 ( 0.02
0.17 ( 0.04
0.5
present work
present work
40, 41
42
42
42
43-46
[Cu2(tz-naf)4]
[Cu(im)Cu(pip)2]3+
[Cu2(bdpi)(CH3CN)2]3+
[Cu2(Me4bdpi)(H2O)2]3+
[CuZn(bdpi)(CH3CN)2]3+
native CuZnSOD
0.32
1.1
0.24
•-
0.0012-0.0081
the copper complex interacts with O2 catalyzing its
dismutation to molecular oxygen and hydrogen peroxide; this
reaction reduces the superoxide concentration in solution and,
thus, the MF+ production rates (i.e. slowing down absorbance
variation). The development of hydrogen peroxide in the
presence of the copper complexes could give rise to the
formation of reactive species which may be responsible for
a new path for the formation of MF+. In fact, Figures 7 and
8 show that even in the presence of a large concentration of
the copper complexes, the MF+ formation rate does not fall
completely to zero but reaches a small, constant value. This
residual absorbance variation can be accounted for consider-
ing that in the presence of high concentration of copper
complex all superoxide is rapidly dismutated in solution (to
a very little steady-state concentration) to O2 and H2O2. The
peroxide generated, through the formation of copper active
species, is responsible for the residual color development
observed.
Here the constant depends on the xanthine, xanthine oxidase,
and NBT concentrations and on the MF+ extinction coef-
ficient; K is the affinity constant of superoxide for the copper
complexes, [Cu]TOT is the total concentration of copper
complex (free complex plus superoxide bound complex), and
f indicates the relative efficiency of reaction 11, with respect
to reaction 9, in the formation of MF+. Equation 12 fits well
the experimental data. The parameters obtained are the
following: for [Cu2(tz-tol)4], K ) 7.5 ( 1.0 µM-1, f ) 0.05
( 0.02; for [Cu2(tz-naf)4], K ) 6.0 ( 1.0 µM-1, f ) 0.04 (
0.03.
The most important result is that both complexes ef-
ficiently catalyze superoxide dismutation below the micro-
molar concentration range. Moreover, despite the relatively
large standard deviation, the f values show that the mecha-
nism via hydrogen peroxide is 20 times less efficient in
promoting MF+ formation with respect to that of reaction
10. To better emphasize this result, it is convenient to express
the SOD activity of the complexes as IC50 values (the
concentration of the complex required to attain 50% inhibi-
tion of the reduction). The IC50 value can be obtained from
the reciprocal of the K values. Table 3 compares the data
obtained for [Cu2(tz-tol)4] and for [Cu2(tz-naf)4] with the IC50
values of the best copper SOD mimics so far reported40-42
and the value for native SOD43-46 (note that the smaller the
IC50 value, the higher the SOD activity). The IC50 value
exhibited by the present complexes is smaller than those
reported for any copper SOD mimics so far and approaches
the activity of the enzyme. The remarkable SOD activity
observed with [Cu2(tz-tol)4] and [Cu2(tz-naf)4] is probably
related to the presence of two coupled copper(II) ions in close
proximity. One Cu(II) may resemble the role of Zn(II) in
the native SOD which, through the imidazolate bridge,
controls the electron density at the redox active Cu(II).
The complete kinetic study of the SOD activity of [Cu2-
(tz-tol)4] and [Cu2(tz-naf)4] is complicated by the large
number of processes involved. But our experimental condi-
tions allow some simplifications. The xanthine oxidase and
NBT concentrations have been optimized to give a linear
absorbance increase during the assay; i.e., the superoxide
concentration in solution remains stationary during the assay.
If we assume that the binding of superoxide by the copper
complexes is fast, the fraction of free and copper-bound
superoxide is ruled by the copper complex concentration and
•-
by the affinity constant (K) for the copper complex/O2
adduct formation. The free superoxide reacts with NBT
through the classical mechanism while the copper complex/
•-
O2 adduct produces MF+ in a less efficient reaction via
hydrogen peroxide generation:
•-
xanthine + xanthine oxidase f O2
O2•- + NBT f MF+
(8)
(9)
Appendix
The effect of the copper complexes on the MF+ formation
in the xanthine oxidase/NBT assay can be accounted for
considering the following mechanism:
O2•- + Cu a CuO2
(10)
(11)
CuO2 f f H2O2 f f MF+
(40) Bienvenue, E.; Choua, S.; Lobo-Racio, M.-A.; Marzin, C.; Pacheco,
P.; Seta, P.; Tarrago, G. J. Inorg. Biochem. 1995, 57, 157.
(41) Weser, U.; Schubotz, L. M.; Lengfelder, E. J. Mol. Catal. 1981, 13,
249.
(42) Ohtsu, H.; Shimazaki, Y.; Odani, A.; Yamauchi, O.; Mori, W.; Itoh,
S.; Fukuzumi, S. J. Am. Chem. Soc. 2000, 122, 5733.
(43) McCord, J. M.; Fridovich, I. J. Biol. Chem. 1969, 244, 6049.
(44) Butler, J.; Koppenol, W. H.; Margoliasch, E. J. Biol. Chem. 1982,
257, 10747.
Here Cu is either [Cu2(tz-tol)4] or [Cu2(tz-naf)4] and CuO2
represents the adduct between superoxide and [Cu2(tz-tol)4]
or [Cu2(tz-naf)4]. The kinetic parameters for the SOD activity
of the complexes were obtained by fitting the absorbance
changes with time with eq 12 (see Appendix for its
derivation):
(45) Muller, J.; Schubl, D.; Maischle-Mosser, C.; Strahle, J.; Wesser, U.
J. Inorg. Biochem. 1999, 75, 63.
(46) Kovala-Demertzi, D.; Galani, A.; Demertzis, M. A.; Skoulika, S.;
Kotoglou, C. J. Inorg. Biochem. 2004, 98, 358.
1+ fK[Cu]TOT
dAbs
dt
) constant ‚
(12)
1 + K[Cu]TOT
Inorganic Chemistry, Vol. 43, No. 21, 2004 6813

Cejudo-Mar´ın et al.
•-
•-
xanthine + xanthine oxidase f O2
O2•- + Cu a CuO2
(S1)
(S2)
(S3)
(S4)
[O2 ]
TOT
[O2•-] )
(S7)
(S8)
1 + K[Cu]TOT
•-
[O2
]TOTK[Cu]TOT
O2•- + NBT f MF+
[CuO2] )
1 + K[Cu]TOT
CuO2 f f H2O2 f f MF+
•-
where [O2
]
is the total superoxide concentration (free
TOT
plus copper bound superoxide) as supplied by the xanthine
oxidase/xanthine system.
Here Cu is the copper complex and CuO2 represents its
adduct with superoxide. Reaction S1 provides, under the
conditions employed, a constant flux of superoxide. The MF+
formation occurs through reactions S3 and S4. The rate of
reaction S3 depends on the NBT concentration (which
remains approximately constant during the assay) and on the
fraction of free superoxide present in solution and is given
by
The global formation rate of MF+ is given by the sum of
rateS3 and rateS4; thus, combining equations from S5 to S8,
we obtain
1 + fK[Cu]TOT
•-
rate ) RS3[O2
]
(S9)
TOT
1 + K[Cu]TOT
The spectral variation due to the formation of MF+ with
time can be obtained from eq S9 through the Lambert-Beer
equation. If the steady-state concentration of superoxide is
•-
rateS3 ) RS3[O2
]
(S5)
•-
maintained constant during the experiment, [O2
]TOT can be
combined with RS3, the extinction coefficient of the product,
where RS3 depends on [NBT]. The rate of reaction S4
depends on the fraction of superoxide bound to the copper
complex. If we indicate with f the relative efficiency of
reaction S4 with respect to reaction S3, the rate of the former
process is
and the cuvette path length to give a single constant:
1 + fK[Cu]TOT
1 + K[Cu]TOT
dAbs
dt
constant ‚
(S10)
Acknowledgment. J.B., G.A., and S.F. acknowledge
financial support from the Spanish CICYT (Grant BQU2001-
3173-C02-01), and R.C.-M. acknowledges support by the
“MEBIOS” Marie Curie training site. The authors also thank
COSTD21 for support, S. Garc´ıa-Granda and H. Rodriguez-
Prieto (University of Oviedo) for crystallographic determi-
nation of the Htz-tol ligand, and J. A. Real (University of
Valencia) for magnetic measurements.
rateS4 ) fRS3[CuO2]
(S6)
In the proposed scheme, reaction S2 is considered a fast
equilibrium step (faster than reactions S3 and S4) so the
concentrations of O2•-, CuO2, and Cu are ruled by the affinity
constant K and the mass balance on superoxide and the
copper complex. Since during the assay the steady-state
concentration of superoxide is maintained very low, the
fraction of the complex bound to the anion can be considered
much lower than the total copper complex, [Cu]TOT ([Cu]TOT
) [Cu] + [CuO2] ≈ [Cu]TOT). Therefore, we can write
Supporting Information Available: Details of the X-ray
structure determinations of Htz-tol, Htz-naf, and the complexes as
CIF files. The material is available free of charge via the Internet
at http://pubs.acs.org.
IC049718W
6814 Inorganic Chemistry, Vol. 43, No. 21, 2004