Homo- and heterobinuclear Cu2+ and Zn2+ complexes of abiotic cyclic hexaazapyridinocyclophanes as SOD mimics

Article abstract of DOI:10.1039/c3dt51012c

The new receptor 3,7,11,15,19,23-hexaaza-1(2,6)-pyridinacyclotetracosaphane (L1) containing a complete sequence of propylenic chains has been synthesised. The acid-base behaviour and Cu²⁺ and Zn²⁺ coordination have been analysed by p

Full text of DOI:10.1039/c3dt51012c

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Homo- and heterobinuclear Cu2+ and Zn2+ complexes
of abiotic cyclic hexaazapyridinocyclophanes as SOD
mimics†
Cite this: Dalton Trans., 2013, 42, 11194
Raquel Belda,a Salvador Blasco,a Begoña Verdejo,*a Hermas R. Jiménez,b
Antonio Doménech-Carbó,c Conxa Soriano,a Julio Latorre,d Carmen Terencioe and
Enrique García-España*a
The new receptor 3,7,11,15,19,23-hexaaza-1(2,6)-pyridinacyclotetracosaphane (L1) containing a com-
plete sequence of propylenic chains has been synthesised. The acid–base behaviour and Cu2+ and Zn2+
coordination have been analysed by potentiometric measurements in 0.15 M NaClO4 for L1 and for the
related compounds 3,7,11,14,18,22-hexaaza-1(2,6)-pyridinacyclotricosaphane (L2), 3,7,10,13,16,20-
hexaaza-1(2,6)-pyridinacycloheneicosaphane (L3) and 3,7,10,12,15,19-hexaaza-1(2,6)-pyridinacycloicosa-
phane (L4). The crystal structure of [(CuH4L2)(H2O)(ClO4)](ClO4)5·3H2O shows an interesting combination
of a metal ion coordinated by the pyridine nitrogen atom and the adjacent amine groups of the chain,
and a perchlorate anion sitting at the middle of the site defined by the remaining four protonated
ammonium groups of L1. Paramagnetic NMR data suggest that imidazole coordinates to the Cu2+ ions as
a bridging ligand in a wide pH range. SOD activity for Cu2+–Cu2+ and Cu2+–Zn2+ complexes with L1–L4
have been measured by NBT assays at pH 7.4, obtaining some of the lowest values so far reported for
SOD mimics. SOD activity has also been checked by chemiluminescence assays using polymorphonuclear
leukocytes (PMNLs).
Received 17th April 2013,
Accepted 30th May 2013
DOI: 10.1039/c3dt51012c
www.rsc.org/dalton
molecular oxygen. Hydrogen peroxide is then removed by the
concerted action of catalase-type enzymes (CAT).1,2
Introduction
Human diseases such as myocardium infarction or Parkin-
While under normal conditions superoxide radicals are
son’s disease are associated with the presence of oxidative trapped by SODs present at cellular mitochondria, plasma or
agents such as hydrogen peroxide or superoxide anions extracellular spaces, the control of the free radical flow coming
formed as a consequence of aerobic metabolism. Protective from O2 chemistry can be problematic when excessive amounts
enzymes like superoxide dismutases (SODs), a family of oxido- of superoxide anion are produced. Therefore, the design and syn-
reductases containing Cu/Zn (SOD1 and SOD3) or Mn (SOD2) thesis of molecules able to mimic SOD activity is a research
at their active centers, act as an important antioxidant defence topic of current interest both from academic and pharmaceutical
catalyzing superoxide dismutation into hydrogen peroxide and points of view. For these reasons, since the pioneering work of
Lippard et al.3 with Cu–Cu and Cu–Cu–imidazole polyamine
complexes, a great deal of work has been done in this area.4
One recurrent feature in metal sites of enzymes is their
aInstituto de Ciencia Molecular, C/Catedrático José Beltrán no. 2, 46980 Paterna,
Valencia, Spain. E-mail: enrique.garcia-es@uv.es
coordinative unsaturation that permits the approach and
binding of the target substrate to the metal center. In this
respect, some polyazacyclophane receptors with different poly-
amine chains containing up to six nitrogen atoms and
different aromatic spacers, such as benzene, pyridine or
terpyridine, have been synthesized in our group during the last
few years.5 Although these receptors have been proved to be
able to bind simultaneously two metal ions, the low number of
nitrogen donors in the receptor prevents the coordination
spheres of the metal from being filled giving rise to unsatu-
rated coordination spheres. These arrangements are favourable
bDepartamento de Química Inorgánica, Universidad de Valencia, C/Dr Moliner 50,
46100 BurjassotValencia, Spain
cDepartamento de Química Analítica, Universidad de Valencia, C/Dr Moliner 50,
46100 BurjassotValencia, Spain
dInstituto de Materiales, C/Catedrático José Beltrán no. 2, 46980 Paterna, Valencia,
Spain
eDepartamento de Farmacología, Facultat de Farmacia, Universitat de València,
Av. Vicent Andrés Estellés s/n 46100, BurjassotValencia, Spain
†Electronic supplementary information (ESI) available. CCDC 934155. For
ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c3dt51012c
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are presented in Table 1. The distribution diagram for L1 is
shown in Fig. S1 of the ESI.†
These ligands follow the typical protonation behaviour
usually found for cyclic amines based on charge density con-
siderations and minimization of repulsions between
ammonium groups.7 In this respect, while the first five proto-
nation constants of receptor L1 are close to those previously
reported for L2,5a the value obtained for the last protonation
constant of L1 is slightly higher. This can be ascribed to the
presence of a further propylenic chain in L1, which increases
the separation between charged centres at this stage. The alter-
nate sequence of ethylenic and propylenic chains of L3 pro-
motes a decrease in basicity already in the fifth protonation
step.6 Similar reasoning explains the slightly higher fifth proto-
nation constant and the remarkable higher last protonation
constant of L1 in comparison with L4, which presents three
ethylenic central chains5a (see Table 1).
Cu2+ complexation
The equilibrium constants for the formation of Cu2+ com-
plexes of L1 determined by potentiometric titration are pres-
ented in Table 2. The values for the related pyridinophanes L2,
L3 and L4 have also been included for comparison.
L1 forms [CuHxL](2+x)+ mononuclear complexes with x = 0–4
as well as the binuclear species [Cu2L]4+ and [Cu2L(OH)]3+. The
distribution diagram shows that for a 1 : 1 Cu2+ : L molar ratio
only mononuclear species are present in solution throughout
the pH-range covered in the studies (2.5–11.0) (Fig. 1A).
Although deriving coordination numbers just from free energy
Chart 1
for the recognition and/or activation of additional guests as
exogen ligands in mixed complexes. An example of this behav-
iour is the recognition of imidazolate as a bridging ligand by a
binuclear complex of a terpyridinophane receptor.4g
Table 1 Stepwise protonation constants of L1–L4 determined at 298.1 0.1 K
in 0.15 M NaClO4
Within this context, here we report the synthesis and charac-
terization of the new receptor 3,7,11,15,19,23-hexaaza-1(2,6)-
pyridinacyclotetracosaphane (L1) (see Chart 1). L1 meets the
above mentioned characteristics being able to incorporate two
metal ions with coordinatively unsaturated coordination sites.
With the aim to study the ability of the Cu2+ and Cu2+–Zn2+
complexes of ligands L1–L4 as SOD mimics, we have analysed
the Cu2+ and Zn2+ coordination behaviour of the new receptor,
along with the Zn2+ coordination chemistry of L2–L4 and the
formation of mixed Cu2+–Zn2+ complexes by L1.5a,6 These
studies have been conducted by means of potentiometric,
electrochemistry and paramagnetic NMR techniques. The SOD
activity of the complexes has been checked either by the nitro
blue tetraazolium (NBT) or by the chemiluminescence poly-
morphonuclear leukocytes (PMNL’s) in vitro methods. Some of
the IC50 values obtained rank among the lowest so far reported
in the literature for SOD mimics.
Entry
Reactiona
L1
L2c
L3d
L4c
1
2
3
4
5
6
L + H ⇄ LH
10.67(2)b
9.41(1)
8.24(5)
7.35(7)
6.98(9)
5.87(2)
10.67(1)
9.85(1)
8.60(1)
7.49(1)
7.12(1)
4.99(2)
9.82(2)
9.38(2)
8.28(2)
7.35(2)
5.83(3)
4.66(4)
10.04(2)
9.43(2)
8.45(2)
7.53(2)
5.89(2)
2.83(3)
HL + H ⇄ H2L
H2 L + H ⇄ H3L
H3L + H ⇄ H4L
H4L + H ⇄ H5L
H5 L + H ⇄ H6L
a Charges omitted. b Numbers in parentheses are standard deviations
in the last significant figure. c Taken from ref. 5a. d Taken from ref. 6.
Table 2 Logarithms of the equilibrium constants for the interaction of Cu2+
with L1–L4 determined at 298.1 0.1 K in 0.15 M NaClO4
Entry Reactiona
L1
L2c
L3d
L4c
1
2
3
4
5
6
7
Cu + L ⇄ CuL
CuL + H ⇄ CuHL
CuHL + H ⇄ CuH2L
CuH2L + H ⇄ CuH3L 6.13(2)
CuH3L + H ⇄ CuH4L 3.82(2)
Cu + CuL ⇄ Cu2L
Cu2L + H2O ⇄
Cu2L(OH) + H
17.22(6)b 18.34(3) 20.38(2)
19.29(3)
9.75(3)
7.44(2)
9.53(3) 8.24(2)
7.56(3) 7.35(2)
4.65(2) 3.07(2)
9.64(3)
6.71(2)
3.36(2)
4.69(2)
—
—
Results and discussion
Protonation studies
7.96(6) 11.69(3) 10.10(2)
10.92(3)
−7.21(6) −7.72(3) −7.37(2) −10.07(4)
a Charges omitted. b Numbers in parentheses are standard deviations
The protonation constants of receptor L1 calculated by pH-
metric titration along with those previously reported for L2–L4 in the last significant figure. c Taken from ref. 5a. d Taken from ref. 6.
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terms can be highly misleading, comparisons of the stability
The existence of complexes with high protonation degrees
constants of L1 with those of the related ligands L2–L4 pre- is supported by the preparation of crystals of a tetraprotonated
viously studied and with other systems suggest at least three as Cu2+ complex of L2 suitable for X-ray diffraction. As far as we
the number of nitrogen atoms tightly bound to Cu2+. In view know this constitutes the first X-ray structure solved for a hexa-
of previous structures and taking into account the UV-Vis spec- azacyclophane receptor with these characteristics. Crystals of
tral changes produced when varying the pH of a solution con- [CuH4L2(ClO4)(H2O)](ClO4)5·3H2O were obtained by slow evap-
taining Cu2+ and L1 in a 1 : 1 molar ratio it is reasonable to oration of solutions containing L2 and Cu(ClO4)2·6H2O in a
assume that one of such atoms would be the pyridine nitrogen 1 : 1 Cu2+ : L molar ratio at pH 4 (Fig. 2). The asymmetric unit
and the other two would come from the polyamine chain. contains [Cu(H4L2)(H2O)(ClO4)]5+ cations, perchlorate counter-
Similar to L2 (Fig. 1B), the absorption peaks associated with anions and lattice water molecules. The coordination geometry
the d–d band in the visible region and the pyridine band in of Cu2+ is an axially elongated square pyramidal (see Fig. 2)
the UV region reach their maximum intensity as soon as the with the equatorial positions occupied by the pyridine nitro-
first protonated complex [CuH4L1]6+ is formed, remaining con- gen atom (Cu–N1 1.909(7) Å), the benzylic nitrogen atoms (Cu–
stant above pH 4 (Fig. 1A). In agreement with previously N2 2.063(7) Å, Cu–N7 2.043(8) Å) and an oxygen atom of a
reported studies for L2–L4, the pyridine moiety should act as water molecule (Cu–O1 1.966(6) Å). The elongated axial posi-
an anchorage point for the metal.5a Therefore, several nitrogen tion is occupied in the solid state by an oxygen atom of a per-
atoms are either uncoordinated or weakly coordinated to the chlorate anion (Cu–O11 2.338(7) Å). The Cu2+ atom is placed
metal prone to bear protonation processes. This is reflected in 0.12 Å above the equatorial plane defined by the donor atoms.
the first three protonation constants of [CuL1]2+ (entries 2–4 in The plane defined by the ammonium groups and the co-
Table 2), which display values that are, in some cases, even ordinated benzylic nitrogens is almost normal to the plane of
higher than the stepwise protonation constants of the free the pyridine ring (84°) with a sort of L-shaped conformation.
receptor at the stages at which both reactions display the same
Table 3 presents a selection of bond distances and angles.
charges (for instance [CuL]2+ + H+ ⇄ [CuHL]3+, log K = 9.75(3) In agreement with the presence of highly protonated mono-
with H2L2+ + H+ ⇄ H3L3+, log K = 8.24(5), entries 3–5 in nuclear species inferred from the potentiometric studies
Table 1 and 2–4 in Table 2).
(Table 2), the four nitrogen atoms of the polyamine chain not
involved in the coordination to the metal remain protonated.
One perchlorate anion sits at this positively charged side of
the molecule. Apart from interacting electrostatically with all
the ammonium groups, this perchlorate anion forms a hydro-
gen bond with one of the ammonium groups (N3–H3C⋯O31
2.16 Å) and a bifurcated hydrogen bond with the coordinated
water molecule (O1–H1WB⋯O32 2.21 Å) and one of the coordi-
nating NH groups (N7–H7⋯O32 2.37 Å) (Fig. 3).
Fig. 1 Distribution diagram for the system (A) Cu2+–L1 and (B) Cu2+–L2; [Cu2+
]
= [L] = 10−3 M. UV-Vis spectroscopic parameters absorbance ( and ×) are over-
Fig. 2 ORTEP drawing of the cation [Cu(H4L2)(H2O)(ClO4)]5+. Thermal ellip-
●
●
●
laid. (A) (×) 300 nm, ( ) 600 nm; (B) (×) 305 nm, ( ) 590 nm.
soids are drawn at the 50% probability level.
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Table 3 Selected bond lengths (Å) and angles (°) for the compound
[(CuH4L2)(H2O)(ClO4)] (ClO4)5·3(H2O)
Selected distances/Å
Cu1–O1 1.966(6) O1–Cu1–O11
Selected angles/°
84.8(3) N1–Cu1–N7 82.5(3)
Cu1–O11 2.338(7) N7–Cu1–O11 111.6(3) O1–Cu1–N7 96.3(3)
Cu1–N1
Cu1–N2
Cu1–N7
1.909(7) N1–Cu1–O11
2.063(7) N2–Cu1–O11
2.043(8) O1–Cu1–N2
97.4(3) N1–Cu1–N2 82.7(3)
87.7(3)
98.0(3)
Another aspect that deserves to be pointed out is the lower
stability constant that [CuL1]2+ has in comparison with the
analogous complexes of L2–L4. Although all these receptors
have the same number of nitrogen atoms, the steric hindrance
afforded by the binding to the metal is minimized for L2–L4
due to the formation of five and six-membered chelate rings.
In fact, an analysis of binding constant values with polyamine
ligands having different aliphatic chains previously reported
indicates that the less stable complexes are formed with
ligands having a sequence of just propylenic chains which give
rise to six-membered chelate rings, and the most stable ones
with ligands affording an alternate sequence of five- and six-
membered rings as occurs in the case of L3.8
Fig. 3 (A) Space-fill representation of [Cu(H4L2)(H2O)(ClO4)]5+
network in [Cu(H4L2)(H2O)(ClO4)]5+
. (B) H-bond
.
The distribution diagram calculated for a 2 : 1 Cu2+ : L
molar ratio shows that the binuclear species [Cu2L1]4+ and
[Cu2L1(OH)]3+ start to prevail in solution above pH 6, being
[Cu2L1(OH)]3+ predominant over pH 7.5 (Fig. S3†). The intro-
duction of a second metal ion in L1 is accompanied by an
important decrease in the stepwise formation constant in com-
parison with the hexaazapyridinophane ligands L2–L4 (entry 6
in Table 2), which can be again explained by the hydrocarbon
chain sequence of L1, which have a worse bite angle to Cu2+.
Analogous to L2 and L3, the high propensity of the [Cu2L1]4+
to hydrolyze (entry 7 in Table 2) suggests the formation of a
hydroxo bridge between the two metal centres.
mononuclear species with protonation degrees varying from 2
to −1 have been detected in all cases, while for L2 and L4 a tri-
protonated [ZnH3L]5+ species was additionally identified. As
expected, the values of the constants obtained are lower than
those obtained for the Cu2+ complexes and remain less
affected by the sequence of ethylenic and propylenic chains
present in the structure of L1–L4.8 As occurred for Cu2+, the
number of protonated species and high values of the protona-
tion constants (entries 2–4 in Table 4) suggest that such pro-
cesses involved uncoordinated nitrogen atoms.
For a 2 : 1 Zn2+ : L molar ratio, [Zn2L]4+, [Zn2L(OH)]3+ and
[Zn2L(OH)2]2+ species have been observed for L2 and L4.
However, for L1 only binuclear mono- and bihydroxylated
species were detected probably due to a lower stability of the
[Zn2L]4+ neutral binuclear species which readily hydrolyzes as
Zn2+ complexation
Table 4 presents the equilibrium constants determined for the
formation of Zn2+ complexes of L1 and its related pyridino-
phanes L2–L4. For a 1 : 1 Zn2+ : L molar ratio, [ZnHxL](2+x) shown in the distribution diagrams shown in Fig. S4.† Finally,
Table 4 Logarithms of the stability constants for the formation of Zn2+ complexes with the receptors L1–L4 determined at 298.1 0.1 K in 0.15 M NaClO4
Entry
Reactiona
L1
10.32(4)b
8.24(4)
6.99(3)
L2
10.76(4)
9.28(3)
6.65(6)
L3
L4
10.75(4)
9.33(3)
6.50(7)
1
2
3
4
Zn + L ⇄ ZnL
ZnL + H ⇄ ZnHL
ZnLH + H ⇄ ZnH2L
ZnLH2 + H ⇄ ZnH3L
10.80(3)
8.42(3)
7.11(2)
—
—
6.97(4)
7.15(5)
5
6
7
8
Zn + L + H2O ⇄ ZnL(OH) + H
ZnL + H2O ⇄ ZnL(OH) + H
2Zn + L ⇄ Zn2L
0.21(6)
−10.11(7)
—
0.005(5)
−10.75(7)
15.69(5)
4.93(6)
0.98(4)
−9.82(5)
14.47(7)
3.67(8)
—
−0.84(3)
—
—
−0.10(5)
−10.85(7)
15.57(7)
4.79(7)
ZnL + Zn ⇄ Zn2L
—
9
2Zn + L + H2O ⇄ Zn2L(OH) + H
2Zn + L + 2H2O ⇄ Zn2L(OH)2 + H
Zn2L + H2O ⇄ Zn2L(OH)
Zn2L(OH) + H2O ⇄ Zn2L(OH)2
6.36(6)
−2.03(1)
—
8.10(2)
8.09(2)
10
11
12
−1.80(3)
−7.59(5)
−9.90(3)
−1.80(3)
−7.48(5)
−9.89(4)
−8.39(6)
a Charges omitted. b Numbers in parentheses are standard deviations in the last significant figure.
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for L3 a constant for a monohydroxylated species could not be
derived from the emf data. Similar to Cu2+ the high values of
the first hydrolytic step of the binuclear complex could be sup-
porting the coordination of hydroxide as a bridging ligand
between the metal centers.
Cu2+–Zn2+ mixed complexes
Owing to the presence of these two metal ions in the CuZn–SOD
active site, heterobinuclear complexes Cu2+–Zn2+–L systems have
been studied. However, due to the complexity of these systems
(a high number of constants, overlapping equilibria, etc.; see the
Experimental section for a description of the procedure followed)
we have limited our studies to L1 and L2 (Table 5). As can be
seen in the distribution diagrams (see Fig. 4) for receptor L2,
Cu2+–Zn2+ mixed hydroxylated species [CuZnL(OH)]3+ and
[CuZnL(OH)2]2+ prevail in solution above pH 8. However, for
receptor L1 the predominant one is [CuZnL(OH)2]2+.
Paramagnetic 1H NMR studies
The electronic structure and the coordination characteristics
of the binuclear Cu2+–Cu2+ complexes with receptors L1 and
L2, and its interaction with the imidazole ligand have been
analyzed by means of paramagnetic nuclear magnetic reson-
ance. We have recorded the paramagnetic 1H NMR spectra,
measured the longitudinal and transversal relaxation times, T1
and T2, and analyzed the temperature dependence of the
hyperfine-shifted resonances.
1
The H NMR spectrum of the system Cu2L1 in D2O at pH
7.0 displays, in the downfield region, two well resolved isotro-
pically shifted signals (a, b). Additionally, this spectrum shows
four signals (c–f) shifted upfield (Fig. 5B). The hyperfine-
shifted resonances, linewidths at half-height, longitudinal
relaxation time values (T1) and assignments are included in
Table 6 and Scheme 1A. Although Cu2L1 shows a similar
pattern of chemical shifts with Cu2L2,5c it presents some
differences (Fig. S5†): Cu2L1 has a greater number of isotropic
shifted α-CH2 and β-CH2 protons and higher longitudinal
relaxation times (T1) than Cu2L2. This behaviour can be inter-
preted as being due to a change in the coordination site of the
copper ions and therefore in the characteristic of the spin-
coupled binuclear copper system.
Fig. 4 Distribution diagram for the ternary systems: (A) Cu2+–Zn2+–L1 and (B)
Cu2+–Zn2+–L2. [Cu2+] = [Zn2+] = [L] = 10−3 M.
The analysis of the 1H NMR spectrum of the ternary
complex Cu2L1–Im for a 1 : 2 molar ratio in D2O at pH 7.0
shows a new signal (g′, Fig. 5A). The broad signal g′ at
Table 5 Logarithms of the stability constants for the formation of Cu2+ and
Zn2+ complexes with the receptors L1 and L2 determined at 298.1 0.1 K in
0.15 M NaClO4
Fig. 5 400 MHz proton NMR spectra in D2O at 298 K of (A) Cu2L1–Im at pH
7.5 and (B) Cu2L1 at pH 7.0. In the downfield and upfield regions, the intensity
of the spectrum is multiplied by five or ten. The asterisks mark the residual
solvent and impurity signals (*, H2O; **, HDO).
Entry Reactiona
L1
L2
1
2
3
4
Cu + Zn + L + H ⇄ CuZnHL
Cu + Zn + L ⇄ CuZnL
Cu + Zn + L + H2O ⇄ CuZnL(OH) + H
Cu + Zn + L + 2H2O ⇄ CuZnL(OH)2 + 2H
30.23(6)b
—
14.55(3)
6.24(3)
—
23.26(9)
18.57(2)
5.34(6)
20.0 ppm for the imidazolate ligand protons can be assigned
on the basis of the longitudinal relaxation times and of the
integrated protons (see Table 6 and Scheme 1A). These results
show the formation of the ternary complexes (Cu2L1–Im, 1 : 1)
a Charges omitted. b Numbers in parentheses are standard deviations
in the last significant figure.
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Table 6 1H NMR hyperfine-shifted resonances of Cu2L1, Cu2L1–Im complexes in D2O at 298 K and pH 7.0
Syst.
Sig.
δ (ppm)
No. of protons
10
Assign.
Temperature dependence
T1 (ms)
Δν1/2 (Hz)
T2a (ms)
Cu2L1
a
c
d
b
e
8.8
2.9
1.8
βCH2
Anti-Curie
Anti-Curie
Anti-Curie
Anti-Curie
Curie
3.8
9.1
8.5
<1
<1
<1
3.3
8.4
8.4
<1
<1
<1
230
160
132
∼370
1209
1279
179
102
96
∼324
1194
1313
3821
1.4
2.0
2.4
∼0.86
0.26
0.25
1.8
3.1
3.3
∼0.98
0.27
0.24
0.08
3
24
Hm,p-Py
αCH2
7.5
−2.0
−9.9
8.8
3.0
1.8
f
Curie
Cu2L1–Im
a′
c′
d′
b′
e′
f′
g′
10
βCH2
Anti-Curie
Anti-Curie
Anti-Curie
Anti-Curie
Curie
Curie
Curie
3
24
Hm,p-Py
αCH2
7.5
−1.8
−10.0
20.0
3
αCH–Im
<1
a Measured from the linewidth at half-height.
comparison with those of the dimer Cu2L2. The magnitude of
T1 values of the β-CH2 and Hm,p-Py protons follows the order
Cu2L2 < Cu2L2–Im (pH = 7.0). This ordering is in accordance
with the coordination of the imidazole ligands that implies
increases of magnetic exchange. These results indicate the
presence of at least one imidazolate ligand coordinated as a
bidentate ligand, Cu–N–N–Cu (see Scheme 1B).
On the other hand, variable temperature 1H NMR spectra of
ternary complexes were registered from 283 to 323 K. Isotropi-
cally shifted signals are temperature dependent and follow an
anti-Curie behaviour excepting some signals belonging to
α-CH2 protons of the macrocyclic receptor that show Curie be-
haviour; thus the paramagnetically shifted signals decrease
with increasing temperature. The existence of anti-Curie be-
haviour (see Tables 6 and S2†) is indicative of the spin-coupled
binuclear copper systems with antiferromagnetic coupling.4g,5c
Electrochemistry
Scheme 1 (A) Cu2L1–Im. (B) Cu2L2–Im.
One of the most important chemical aspects in order to mimic
the SOD function (catalysis of superoxide anion dismutation)
is the reduction potential for the different receptors at physio-
logical pH that has to be intermediate between −0.33 V (O2/
in which the imidazolate ligand is coordinated as a bidentate
bridge ligand, Cu–N–N–Cu. In addition, the values of the
proton linewidths at half-height are relatively smaller in com-
parison with the Cu2L1 system. This is in accordance with the
coordination of one imidazolate bridge ligand that implies
increases of the degree of spin-coupling in the binuclear
copper system, in a similar way to the previously reported
binuclear complex of a terpyridinophane receptor, which pres-
ents similar T1 (<1 ms) and Δν1/2 (5969 Hz) values in compari-
son with those of the Cu2L1–Im.4g
O2˙−) (redox potential relative to 1 mol of O2 dm−3, E0(O2(aq)
/
O2˙−) = −0.16 V) and 0.89 V (O2˙−/H2O2).9 In order to obtain
this information, the electrochemistry of the Cu2+–L and Cu2+–
Zn2+–L systems has been studied at different pH values with
concentrations at the millimolar level. For the binuclear
complex Cu2L1, above pH 7.0 where predominates the Cu2L1
(OH) species, CVs consist of two reduction peaks at −0.22 and
−0.48 V with anodic counterparts at −0.38 and 0.0 V. The first
cathodic peak can be attributed to the reduction of Cu2+ to
Cu+, as clearly shown by peak current function values of ca.
600 μA cm−2 V−1/2 s1/2 mM−1, corresponding to a one electron-
per-Cu transfer. This suggests that the first reductive step cor-
responds to an overall 2-electron reduction where the two
copper centres behave independently. The variation of the
peak potential and peak current with the potential scan rate
suggests that there is some complex pre-dissociation preceding
the electron transfer process. The anodic peak at less negative
When the spectrum of the ternary complex Cu2L2–Im for a
1 : 2 molar ratio is recorded in D2O at pH 7.0, the spectrum
shows
a similar pattern of chemical shifts as Cu2L2
(Fig. S5B†), but a new signal (e′) appears (Fig. S5A†). The
assignments of the isotropically shifted signals by means of
the integration of signals, and the longitudinal relaxation
times (T1) of the paramagnetic signals are reported here (see
Table S2†). Additionally, as can be observed in Table S2,† the
longitudinal relaxation times in the Cu2L2–Im are higher in
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potentials exhibits a typical high profile, characteristic of the weakly acidic pHs can be interpreted in terms of two succes-
stripping oxidation of deposits of copper metal, thus showing sive one-electron reductions of the Cu2+ centre whereas at high
that in the second electron transfer, metallic copper is formed. pH values, as well as upon addition of imidazole, the electro-
This voltammetry is essentially unchanged upon addition of chemical pathway can be explained by a direct two-electron
imidazole as can be seen in Fig. 6.
The CVs of the heterobinuclear complex CuZnL1 system communication between the metal centres is blocked.
display, at pH values between 6.0 and 8.5, two cathodic waves All the studied systems present a similar behaviour, display-
reduction of the copper centre, suggesting that the electronic
at −0.40 and −0.60 V followed by anodic counterparts at −0.38 ing a first quasi-reversible one-electron reduction step. Assum-
and 0.00 V (see Fig. 7A). This voltammetry, which can be ing that CumIIHxL and CumIHyL complexes are formed at a
described in terms of two successive one-electron reduction given pH, the measured midpeak potentials can be regarded
processes corresponding to the stepwise reduction of Cu2+ to as apparent formal electrode potentials for the CumIIHxL/
Cu+ and Cu0, moves to an apparently direct two-electron CumIHyL couple. Collected data in Table 7 reveal that there is
reduction in the presence of imidazol (Fig. 7B). At pH values no evident correlation between the SOD activities and the
higher than 8.5 (see Fig. 7C and 7D), where CuZnL1(OH)2 pre- apparent formal electrode potentials. However, a correlation
dominates, CVs show only one reversible couple at a midpoint can be established between the SOD activities and the equili-
potential of −0.45 V with a peak current function of 685 μA brium constant for the disproportionation process 2CumIHxL
cm−2 V−1/2 s1/2 mM−1. This couple is accompanied in the → CumIIHyL + (x − y)H+ + Cu° (charge of complexes omitted),
anodic region of CVs by additional weak oxidation peaks at Kdis, that can be calculated from voltammetric data as pre-
−0.15 and +0.55 V. This response is essentially unchanged viously described,10 considering that the first electron transfer
upon addition of imidazole. The response at neutral and step behaves reversibly. Assuming that a Cu+ complex of stoi-
chiometry CumIHyL is formed, its stability constant, βI, can be
calculated from the corresponding value for the CumIIHxL
complex, βII, determined from potentiometric data, and the
formal electrode potential of the Cu2+/Cu+ couple, E°(II/I),
determined from voltammetric data, by means of the relation-
ship (mV at 298 K)
log βI ¼ log βII þ m½E°ðII=IÞ ꢀ E°ðCu2þ=CuþÞꢁ=59 þ ðy ꢀ xÞpH
ð1Þ
where E°(Cu2+/Cu+) denotes the standard potential for the
Cu2+/Cu+ couple (equal to +159 mV vs. NHE) in
a
Fig. 6 CVs at GCE for: (A) 1.59 × 10−3 M Cu2L1; (B) id. plus 1.60 × 10−3 M imi-
dazole in 0.15 M NaClO4 at pH 7.0. Potential scan rate 50 mV s−1
.
Table 7 SOD activity for receptors L1–L4 obtained by the McCord–Fridovich
test and examples reported from the literature12
System
IC50/μM
kcat/106 M−1 s−1
E°/mV
log Kdis
Cu2L1
Cu2L2
Cu2L3
Cu2L4
CuZnL1
CuZnL2
CuZnL3
CuZnL4
Cu2L1Im
Cu2L2Im
Cu2L3Im
Cu2L4Im
CuZnL1Im
CuZnL2Im
CuZnL3Im
CuZnL4Im
Cu2(bpzbiap)Cl3
Cu2(bdpi)b
CuZn(bdpi)
Cu2Zn2SOD
0.11 (2)
1.2 (2)
0.22 (4)
0.24 (1)
0.5 (2)
0.22 (3)
2.2 (2)
1.7 (3)
0.25(5)
0.27(5)
0.24(6)
0.4(1)
0.36(9)
0.73(4)
4.9(2)
3.3(3)
0.25
27.00
2.37
13.50
12.50
5.94
13.0
1.34
1.75
11.88
11.00
12.37
7.42
8.25
4.07
0.65
0.90
11.9
81.2
108
−80
−150
−250
−250
−300
−380
−60
−3.72
1.53
−1.12
−0.03
0.73
−220
+90
−400
+80
−100
−210
−260
−460
−240
−390
−110
−230
+320
a
0.32
0.24
0.010(2)
430
Fig. 7 CV at GCE for: (A) 1.19 × 10−3 M CuZnL1, pH 7.4; (B) id. plus 1.20 ×
10−3 M imidazole, pH 7.4; (C) 1.19 × 10−3 M CuZnL1, pH 8.6; (D) id. pH 9.5. Elec-
a (Hbpzbiap) = 1,5-bis(1-pyrazolyl)-3-[bis(2-imidazolyl)methyl]azapentane.
b (Hbdpi) = 4,5-bis[di(2-pyridylmethyl)aminomethyl]imidazole.
trolyte: 0.15 M NaClO4 at the potential scan rate 50 mV s−1
.
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noncomplexing electrolyte. Then, the disproportionation con-
stant of the complex can be calculated as:
log Kdis ¼ log βII ꢀ 2 log βI þ m log KdisðCuþÞ þ log βx ð2Þ
Here, log Kdis(Cu+) = 6.24 at 298 K and βx represents the for-
mation constant for the HxL species. Combining voltammetric
and potentiometric data, we obtain the log Kdis values in
Table 7. In this table, the E°(II/I) values have been taken as the
onset potential for the first cathodic peak in the voltammo-
grams such as in Fig. 6 and 7.
As can be seen in Fig. 7, the voltammetric response of the
studied complexes is scarcely sensitive to the presence of imi-
dazole. This can be interpreted by the occurrence of an electro-
chemical reduction pathway involving relatively fast ligand
dissociation prior to the electron transfer step. The electro-
chemical pattern is quite similar for all the studied systems,
suggesting that a similar electrochemical reaction scheme is
operative. In the case of most binuclear complexes, the voltam-
metric profile clearly shows that the reduction of the metallic
centres is independent, i.e., that there is no electronic com-
munication between the metal units forming the complex.
Fig. 8 Antioxidant activity in leukocytes.
Fig. 8 depicts the results obtained from the luminescence
inhibition measurements at two different concentrations. As
can be seen all Cu2+ binuclear systems studied clearly inhibit
luminescence, generated by stimulation of PMNLs with 12-O-
tetradecanoylphorbol 13-acetate (TPA), which supports the
antioxidant properties of these systems. Interestingly, in agree-
ment with the NBT method, the lowest inhibition percentage
of the luminescence is observed for Cu2L2 (see Fig. 8).
Superoxide dismutase activity
NBT (nitro blue tetrazolium) assays11 allow calculating the IC50
values for Cu2LIm and CuZnLIm systems for receptors L1–L4
at physiological pH. The analysis of these experiments shows
that all homobinuclear Cu2LIm and heterobinuclear CuZnLIm
Conclusions
−
species exhibit a remarkable catalytic activity towards O2 dis-
mutation in comparison with the literature.12 The high activity
observed for many of these systems can be ascribed probably
to the flexibility of their chains that would permit molecular
re-arrangements along the catalytic pathway.13 Interestingly,
the lowest kcat is observed for the complex showing the highest
stepwise stability constant for the formation of the binuclear
copper complex and the largest dismutation constant (Cu2L2,
see Table 7). The substitution of one Cu2+ by a Zn2+ metal ion
yields a pronounced decrease in activity, except in the case of
Cu2L2 for which a 6-fold increase is observed. On the other
hand, addition of imidazole to the binuclear complexes does
not produce any significant change except for Cu2L2.
IC50 values obtained for copper complexes with different hexa-
azapyridinophane receptors show some of the lowest values
reported in the literature for Cu–SODs mimics. Through
different techniques, this behaviour can be related with the
coordination modes of the Cu2+ ions and with the flexibility of
their aliphatic chains that would permit the molecular re-
arrangements along the catalytic pathway. Although paramag-
netic 1H NMR studies suggest that imidazole bridges the metal
centres in its anionic imidazolate form, this seems to have no
particular role in the catalytic activity. The lowest kcat is
observed for the complex showing the highest stepwise stabi-
lity constant for the formation of the binuclear Cu2+ complex
and the largest dismutation constant. The studies performed
hold promise for the use of copper complexes of these ligands,
with the possible exception of Cu2L2, as antioxidant agents in
more complex fish and murine models. We will explore these
possibilities shortly.
Chemiluminescence in PNML SOD activity assays
Additionally to the McCord–Fridovich test, the SOD activity of
Cu2+ binuclear systems (with or without imidazole) has also
been complemented with luminescence inhibition studies on
polymorphonuclear human leukocytes (PNMLs).
−
PNMLs are able to generate great quantities of O2 in the
Experimental
Synthesis of L1
NADPH-oxidase system14 as a part of their role in the immune
−
system. This O2 production can be measured using luminol
chemiluminescence assays. The presence of free radical sca- Ligand L1 was prepared following a reported procedure (see
vengers inhibits luminescence, and in this sense a major inhi- Scheme S1†).6 All reagents and chemicals were obtained from
bition can be associated with a major antioxidant ability. commercial sources and used as received. Solvents used for
Trypan blue dye exclusion assay results show that all com- the chemical synthesis were of analytical grade and used
plexes are nontoxic (data not shown).
without further purification.
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5,9,13-Tetra(p-tolylsulfonyl)1,5,9,13-tetrazatridecane (1)
(Yield: 89%) NMR (CDCl3): δH (ppm): 1.78–1.91 (m, 10H), 2.38
(s, 9H), 2.41 (s, 9H), 2.95–2.93 (m, 4H), 3.12–3.10 (m, 16H),
5.44 (t, 2H, J = 6 Hz), 7.31–7.23 (m, 12H), 7.71–7.61 (m, 12H)
δC (ppm): 143.8, 137.0, 135.6, 130.1, 129.9, 127.3, 47.7, 47.6,
47.5, 40.4, 29.6, 29.2, 21.7.
N,N′-Bis(3-aminopropyl)-1,3-propanediamine (10.0 g,
53.1 mmol) dissolved in 400 cm3 of THF and K2CO3 (53.6 g,
387.7 mmol) dissolved in 100 cm3 of water were placed in a
three-necked round-bottom flask provided with mechanical
stirring. Then, a solution of p-tolylsulfonyl chloride (55.4 g,
290.8 mmol) in 100 cm3 of THF was added dropwise over 1 h.
The solution was kept under stirring for one day, the organic
1,5,9,13,17,21-Hexakis(p-tolylsulfonyl)-3,7,11,15,19,23-
hexaaza-1(2,6)-pyridina cyclotetracosaphane (5)
phase was separated and vacuum evaporated to dryness. The 4 (0.9 g, 0.7 mmol) and K2CO3 (0.97 g, 7.0 mmol) were sus-
residue was suspended in ethanol and refluxed for two hours. pended in dried CH3CN (250 cm3). To this mixture, 2,6-bis(bro-
Then, it was filtered and washed exhaustively with ethanol. momethyl)pyridine (0.2 g, 2.6 mmol) in 150 cm3 was added
(Yield: 89%) NMR (CDCl3): δH (ppm): 1.84 (m, 6H), 2.41 (s, dropwise over 2 h. The suspension was refluxed for further
6H), 2.43 (s, 6H), 2.98 (d, 4H), 3.09–3.16 (m, 8H), 5.39 (t, 2H, 20 h and filtered off. The solution was vacuum evaporated to
J = 6 Hz), 7.28–7.32 (m, 8H), 7.63 (d, 4H, J = 8 Hz), 7.73 (d, 4H, dryness and the residue suspended in refluxing ethanol. Then,
J = 8 Hz).
it was filtered to give a white solid. (Yield: 98%) NMR (CDCl3):
δH (ppm): 1.94–1.66 (m, 10H), 2.38 (s, 6H), 2.39 (s, 6H), 2.40
(s, 6H), 3.23–2.94 (m, 20H), 4.30 (s, 4H), 7.36–7.27 (m, 12H),
7.65–7.54 (m, 15H). δC (ppm): 156.7, 143.6, 130.1, 130.0,
127.5, 127.4, 127.4, 47.8, 47.7, 29.9, 28.9, 21.7. MS m/z (FAB)
1327(M)+.
1,19-Phthalimido-4,8,12,16-tetrakis(p-tolylsulfonyl)-4,8,12,16-
tetraazanonadecane (2)
The tosylated amine 1 (18.0 g, 22.3 mmol), K2CO3 (32.2 g,
254.5 mmol) and N-(3-bromopropyl) phthalimide (17.0 g,
63.6 mmol) were suspended in 300 cm3 refluxing CH3CN.
After refluxing for 48 h, the mixture was filtered off. The solu-
tion was vacuum evaporated to dryness and the residue sus-
pended in refluxing ethanol. Then it was filtered to give 2 as a
white solid. (Yield: 46%) NMR (CDCl3): δH (ppm): 1.86–1.81
(m, 10H), 2.37 (s, 6H), 2.39 (s, 6H), 3.13–3.09 (m, 16H), 3.64 (t,
4H, J = 7 Hz), 7.29–7.22 (m, 8H), 7.64–7.58 (m, 8H), 7.69–7.66
(m, 4H), 7.80–7.78 (m, 4H). δC (ppm): 21.9, 47.1, 123.64,
127.55, 130.20, 132.44, 134.35, 168.50.
3,7,11,15,19,23-Hexaaza-1(2,6)-pyridina cyclotetracosaphane
(L1·6HBr·3H2O)
5 (0.33 g, 0.2 mmol) and phenol (1.6 g, 17.1 mmol) were sus-
pended in HBr–AcOH 33% (250 cm3). The mixture was stirred
at 90 °C for 24 h and then cooled. The resulting residue was fil-
tered off and washed several times with CH2Cl2 to give the
product L1 in a salt form. (Yield: 62%) NMR (CDCl3): δH
(ppm): 2.38–2.16 (m, 10H), 3.35–3.26 (m, 20H), 4.49 (s, 4H),
7.46 (d, J = 8 Hz, 2H), 7.93 (t, J = 8 Hz, 1H). δC (ppm): 150.8,
139.6, 123.1, 51.2, 44.9, 44.6, 44.1, 23.1, 22.6, 21.7. MS m/z
(ESI) 406.4 ([M + H]+). Anal. Calcd for C22H55N7Br6O3: C, 27.9;
H, 5.9; N, 10.4: Found: C, 27.8, H, 5.7, N, 10.2.
4,8,12,16-Tetrakis(p-tolylsulfonyl)-1,5,9,13,17,21-
hexaazaheneicosane (3)
A mixture of compound 2 (8.0 g, 6.7 mmol) and hydrazine
monohydrate 85% (32.9 cm3, 60.3 mmol) in THF (300 cm3)
was refluxed for 24 h, then cooled and the resulting solid fil-
tered off. After adding 50 cm3 of water, the solution was
vacuum evaporated to dryness, to give a solid residue. The
solid was dissolved in CH2Cl2 and the solution was dried with
anhydrous Na2SO4. Then, it was vacuum evaporated to dryness
to obtain 3. (Yield: 76%) NMR (CDCl3): δH (ppm): 1.66–1.90
(m, 10H), 2.18 (s, 1H), 2.38 (s, 6H), 2.40 (s, 6H), 2.73 (t, 4H, J =
7 Hz), 3.11–3.13 (m, 16H), 7.25–7.30 (m, 8H), 7.62–7.70 (m,
8H) δC (ppm): 21.7, 31.8, 38.9, 47.0, 47.2, 127.3, 130.0, 130.2,
135.9, 143.6, 143.7.
EMF measurements
The potentiometric titrations were carried out at 298.1 0.1 K
using 0.15 M NaClO4 as a supporting electrolyte. The experi-
mental set-up (burette, potentiometer, cell, stirrer, microcom-
puter, etc.) has been fully described elsewhere.15 The EMF data
were acquired with the computer program PASAT.16 The refer-
ence electrode was a Ag/AgCl electrode in a saturated KCl solu-
tion. The glass electrode was calibrated as a hydrogen-ion
concentration probe by titration of previously standardized
amounts of HCl with CO2-free NaOH solutions and the equi-
valent point determined by Gran’s method,17 which gives the
1,5,9,13,17,21-Hexakis(p-tolylsulfonyl)-1,5,9,13,17,21-
hexaazaheneicosane (4)
standard potential, E°′, and the ionic product of water [pKw
=
13.73(1)]. The computer program HYPERQUAD was used to
3 (10 g, 10.8 mmol) dissolved in 400 cm3 of THF and K2CO3 calculate the protonation and stability constants.18 The pH
(8.1 g, 58.8 mmol) dissolved in 100 cm3 of water were placed range investigated was 2.5–11.0 and the concentration of the
in a three-necked round bottom flask provided with mechan- metal ions and of the ligands ranged from 1 × 10−3 to 5 ×
ical stirring. Then a solution of p-tolylsulfonyl chloride (5.5 g, 10−3 M with M/L molar ratios varying from 2 : 1 to 1 : 2. When
29.4 mmol) in THF was added dropwise over 1 h. The solution more than one model fits the experimental data, the most
was kept under stirring for one day, and the organic phase was reliable chemical model was chosen by performing F tests at
separated and vacuum evaporated to dryness. The residue was the 0.05 confidence level.19,20 More details of data treatment
suspended in ethanol and refluxed exhaustively with ethanol. are included in the ESI.†
11202 | Dalton Trans., 2013, 42, 11194–11204
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UV-Vis measurements
performed by means of the program SHELXL-97.24 Crystal
data, data collection parameters, and results of the analysis are
listed in Table S1.† Due to the low reflectivity exhibited by the
crystal, in the data collection a θ of 22.78° was achieved.
However the final refinement yields no ambiguity in the atom
assignation and the final refinement parameters are within
reasonable values. Molecular plots were produced with either
the program ORTEP25 or Mercury.26 Crystallographic data have
been deposited as CCDC 934155.
The solvents used were of spectroscopic or equivalent grade.
Water was twice distilled and passed through a Millipore
apparatus. All solutions were prepared 0.15 M in NaClO4. The
pH values were measured with a Mettler-Toledo MP-120 pH-
meter and adjustments of the hydrogen ion concentration of
the solutions were made with diluted HCl and NaOH solu-
tions. UV-Vis absorption spectra were recorded on an Agilent
8453 spectroscopy system.
McCord–Fridovich SOD activity assays
Paramagnetic NMR spectroscopy
SOD-like activity was determined by using the nitro blue tetra-
zolium (NBT) reduction method.27–29 The assay was carried
out in an HEPES buffer 50 mM at pH = 7.4 and at room temp-
erature. The concentration of NBT was 50 mM and the super-
oxide was generated with the xanthine oxidase–xanthine
system.
The paramagnetic NMR measurements were acquired on a
Bruker Avance400 spectrometer operating at 399.91 MHz. One-
dimensional spectra were recorded in a D2O solvent with pre-
saturation of the H2O signal during part of the relaxation delay
to eliminate the H2O signal.21 Relaxation delay times of
50–200 ms, 30–75 kHz spectral width range and acquisition
times of 60–200 ms were used. 1D spectra were processed
using exponential line-broadening weighting functions as apo-
dization with values of 10–30 Hz. Chemical shifts were refer-
enced to residual solvent protons of D2O resonating at
4.76 ppm (298 K) relative to TSP. Sample concentrations for
paramagnetic 1H NMR were 3 mM in Cu2L1 or Cu2L2 com-
plexes. The longitudinal relaxation times of the hyperfine
shifted resonances were determined using the inversion recov-
ery pulse sequence (d1–180°–τ–90°–acq, where d1 is the relax-
ation delay and acq the acquisition time), 15 values of τ were
selected between 0.4 ms and 200 ms.22 (d1 + acq) values were
at least five times the longest expected T1 ranging from 100 to
400 ms, and the number of scans was 16 000. The T1 values
were calculated from the inversion-recovery equation. Transver-
sal relaxation times were obtained measuring the line broaden-
ing of the isotropically shifted signals at half-height through
Cellular toxicity assays
The possible cytotoxicity of the complexes herewith tested was
determined by means of the trypan blue dye exclusion assay.30
Several aliquots of PMNL suspension were exposed to 1 μM of
a solution of the complex tested. After 15 minutes of incu-
bation, the dye was applied and the viable cells were counted
and compared with a blank that contained the buffer used.
Chemiluminescence in PNML SOD activity assays
The SOD-like activity of the complexes was tested by measuring
the inhibition of the luminescence of PMNLs.31,32 The lumino-
genic agent was luminol 40 μM and TPA 1 μM was used as a
leukocyte-activating agent. The method for the purification of
PMNLs from human blood has been described elsewhere.33
the equation T2−1 = πΔν1/2
.
Acknowledgements
Electrochemistry
Financial support from the Spanish Ministry of Science and
Innovation (CONSOLIDER INGENIO 2010 CSD2010-00065 Pro-
jects), FEDER funds and Generalitat Valenciana PROMETEO
2011/008 is gratefully acknowledged. R. B. thanks MCYT of
Spain for a Ph.D. grant. B. V. thanks MINECO for a Juan de la
Cierva grant.
The electrochemistry of the different CuII–L and CuII–ZnII–L
systems has been studied by cyclic voltammetry in the
3.4–10.0 pH range at concentrations ca. 1.0 × 10−3 M. 0.15 M
NaClO4 was used as a supporting electrolyte, and the pH
adjusted to the required value by adding appropriate amounts
of HClO4 and/or NaOH. Voltammograms were obtained in a
three-electrode cell under an argon atmosphere with glassy
carbon working electrodes, a platinum wire auxiliary electrode
and a AgCl (3 M NaCl)/Ag reference electrode, using a CH I660
equipment.
References
1 (a) I. Fridovich, Science, 1978, 201, 875–880; (b) I. Fridovich,
Annu. Rev. Biochem., 1995, 64, 97–112; (c) E. Bienvenue,
S. Choua, M.-A. Lobo-Recio, C. Marzin, P. Pacheco, P. Seta
and G. Tarrago, J. Inorg. Biochem., 1995, 57, 157–168;
(d) J. M. McCord and I. Fridovich, J. Biol. Chem., 1969, 244,
6049–6055; (e) J. S. Valentine, D. L. Wertz, T. J. Lyons,
L.-L. Liou, J. J. Goto and E. B. Gralla, Curr. Opin. Chem.
Biol., 1998, 2, 253–262.
Crystallographic analysis
Slow evaporation of vessels containing either L2 along with
Cu(ClO4)2 in a NaClO4 0.15 M water solution yielded blue
crystals suitable for X-ray diffraction. Analysis of single
crystals of the ligand was carried out with an Enraf-Nonius
KAPPA CCD single-crystal diffractometer (λ = 0.71073 Å). The
structure was solved by the Patterson method using the
program SHELXS-86.23 Isotropic least-squares refinement was
2 C. A. Salata, M.-T. Youinou and C. J. Burrows, Inorg. Chem.,
1991, 30, 3454–3461.
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3 (a) G. Kolks, C. R. Frihart, H. N. Rabinowitz and 11 M. M. Tarpey and I. Fridovich, Circ. Res., 2001, 89, 224–236.
S. J. Lippard, J. Am. Chem. Soc., 1976, 98, 5720–5721; 12 H. Ohtsu, Y. Shimazaki, A. Odani, O. Yamauchi, W. Mori,
(b) C.-L. O’Young, J. C. Dewan, H. R. Lilienthal and
S. J. Lippard, J. Am. Chem. Soc., 1978, 100, 7291–7300;
S. Itoh and S. Fukuzumi, J. Am. Chem. Soc., 2000, 122,
5733–5741.
(c) P. K. Coughlin, J. C. Dewan, S. J. Lippard, E.-I. Watanabe 13 A. Maroz, G. F. Kelso, R. A. J. Smith, D. C. Ware and
and J.-M. Lehn, J. Am. Chem. Soc., 1979, 101, 265–266; R. F. Anderson, J. Phys. Chem. A, 2008, 112, 4929–4935.
(d) G. Kolks, C. R. Frihart, P. K. Coughlin and S. J. Lippard, 14 C. Dahlgren and A. Karlsson, J. Immunol. Methods, 1999,
Inorg. Chem., 1981, 20, 2933–2940; (e) G. Kolks, 232, 3–14.
S. J. Lippard, J. V. Waszczak and H. R. Lilienthal, J. Am. 15 E. García-España, M. J. Ballester, F. Lloret, J. M. Moratal,
Chem. Soc., 1982, 104, 717–725; (f) P. K. Coughlin,
A. E. Martin, J. C. Dewan, E.-I. Watanabe, J. E. Bulkowski,
J. Faus and A. Bianchi, J. Chem. Soc., Dalton Trans., 1988,
101–104.
J.-M. Lehn and S. J. Lippard, Inorg. Chem., 1984, 23, 1004– 16 M. Fontanelli and M. Micheloni, Proceedings of the
1009; (g) P. K. Coughlin and S. J. Lippard, Inorg. Chem.,
1984, 23, 1446–1451.
4 (a) M. G. B. Drew, C. Cairns, A. Lavery and S. M. Nelson,
J. Chem. Soc., Chem. Commun., 1980, 1122–1125;
I Spanish–Italian Congress on Thermodynamics of Metal Com-
plexes, Peñíscola, Castellón 1990. Program for the Automatic
Control of the Microburette and the Acquisition of the Elec-
tromotive Force Readings, Diputación de Castellón, 1990.
(b) M. G. B. Drew, M. McCann and S. M. Nelson, J. Chem. 17 (a) G. Gran, Analyst, 1952, 77, 661–671; (b) F. J. C. Rossotti
Soc., Dalton Trans., 1981, 1868–1878; (c) J.-L. Pierre, and H. Rossoti, J. Chem. Educ., 1965, 42, 375–378.
P. Chautemps, S. Refaif, C. Beguin, A. El Marzouki, 18 P. Gans, A. Sabatini and A. Vacca, Talanta, 1996, 43, 1739–
G. Serratrice, E. Saint-Aman and P. Rey, J. Am. Chem. Soc., 1753.
1995, 117, 1965–1973; (d) G. Tabbì, W. L. Driessen, 19 W. C. Hamilton, Statistics in Physical Chemistry, The Roland
J. Reedijk, R. P. Bonomo, N. Veldman and A. L. Spek, Inorg. Press Co., New York, 1964.
Chem., 1997, 36, 1168–1175; (e) D. Li, S. Li, D. Yang, J. Yu, 20 L. Bologni, A. Sabatini and A. Vacca, Inorg. Chim. Acta,
J. Huang, Y. Li and W. Tang, Inorg. Chem., 2003, 42, 6071– 1983, 69, 71–75.
6080; (f) Y.-H. Zhou, H. Fu, W.-X. Zhao, W.-L. Chen, C.-Y. Su, 21 T. Inubushi and E. D. Becker, J. Magn. Reson., 1983, 51,
H. Sun, L.-N. Ji and Z.-W. Mao, Inorg. Chem., 2007, 46, 734– 128–133.
739; (g) B. Verdejo, S. Blasco, E. García-España, F. Lloret, 22 R. L. Vold, J. S. Waugh, M. P. Klein and D. P. Phelps,
P. Gaviña, C. Soriano, S. Tatay, H. R. Jiménez, A. Doménech
and J. Latorre, Dalton Trans., 2007, 4726–4737.
5 (a) J. Aguilar, M. G. Basallote, L. Gil, J. C. Hernández,
M. A. Máñez, E. García-España, C. Soriano and B. Verdejo,
Dalton Trans., 2004, 94–103; (b) E. García-España, P. Gaviña,
J. Chem. Phys., 1968, 48, 3831–3832.
23 G. M. Sheldrick, C. Kruger and R. Goddard, Crystallographic
Computing 3: Data Collection, Structure Determination, Pro-
teins and Database, Clarendon Press, Oxford, UK, 1985,
p. 175.
J. Latorre, C. Soriano and B. Verdejo, J. Am. Chem. Soc., 2004, 24 G. M. Sheldrick, SHELXL-93: Program for Crystal Structure
126, 5082–5083; (c) B. Verdejo, J. Aguilar, A. Doménech,
C. Miranda, P. Navarro, H. R. Jiménez, C. Soriano and
E. García-España, Chem. Commun., 2005, 3086–3088.
Refinement, Institüt für Anorganische Chemie der Universi-
tät, Göttingen, Germany, 1993.
25 C. K. Johnson, ORTEP, Report ORNL-3794, Oak Ridge
National Laboratory, Oak Ridge, TN, 1971.
6 M. G. Basallote, A. Doménech, A. Ferrer, E. García-España,
J. M. Llinares, M. A. Máñez, C. Soriano and B. Verdejo, 26 C. F. Macrae, P. R. Edgington, P. McCabe, E. Pidcock,
Inorg. Chim. Acta, 2006, 359, 2004–2014.
G. P. Shields, R. Taylor, M. Towler and J. Van de Streek,
7 (a) J. E. Sarnesky, H. L. Surprenant, F. K. Molen and
J. Appl. Crystallogr., 2006, 39, 453–457.
C. N. Reilley, Anal. Chem., 1975, 47, 2116–2124; 27 L. W. Oberley and D. R. Spitz, Handbook of Methods of
(b) S. P. Dagnall, D. N. Hague and M. E. McAdam, J. Chem.
Soc., Perkin Trans. 2, 1984, 435–440; (c) S. P. Dagnall,
Oxygen Radicals Research, ed. R. A. Grenwald, CRC Press,
Boca Raton, USA, 1986, p. 218.
D. N. Hague and M. E. McAdam, J. Chem. Soc., Perkin Trans. 28 C. Beauchamp and I. Fridovich, Anal. Biochem., 1971, 44,
2, 1984, 1111–1114; (d) D. N. Hague and A. D. Moreton, 276–287.
J. Chem. Soc., Perkin Trans. 2, 1994, 265–270; (e) C. Frassineti, 29 J. Y. Zhou and P. Prognon, J. Pharm. Biomed. Anal., 2006,
S. Ghelli, P. Gans, A. Sabatini, M. S. Moruzzi and A. Vacca,
Anal. Biochem., 1995, 231, 374–382.
8 (a) T. G. Fawcett, S. M. Rudich, B. H. Toby, R. A. Lalancette,
40, 1143–1148.
30 E. Knoll-Köhler and J. Stiebel, Eur. J. Oral Sci., 2002, 110,
296–301.
J. A. Potenza and H. J. Schugar, Inorg. Chem., 1980, 19, 31 I. Ginsburg, R. Misgav, D. F. Gibbs, J. Varani and R. Kohen,
940–945; (b) G. Marongiu, E. C. Lingafelter and P. Paoletti,
Inorg. Chem., 1969, 8, 2763–2767.
9 P. M. Wood, Biochem. J., 1988, 253, 287–289.
Inflammation, 1993, 17, 227–243.
32 A. Halstensen, B. Haneberg, J. Glette, S. Sandberg and
C. O. Solberg, J. Immunol. Methods, 1986, 88, 121–128.
10 A. Doménech, E. García-España, V. Marcelino, B. Altava, 33 M. C. Terencio, M. L. Ferrándiz, I. Posadas, E. Roig, S. De
S. V. Luis, J. F. Miravet, A. Bianchi and L. Ferrini, Inorg.
Chim. Acta, 1996, 252, 123–129.
Rosa, A. De Giulio, M. Payá and M. J. Alcaraz, Naunyn-
Schmiedeberg’s Arch. Pharmacol., 1998, 357, 565–572.
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