A Superoxide dismutase mimic with high activity: Crystal structure, solution equilibrium and pulse radiolysis

Article abstract of DOI:10.1039/a909018e

A new binuclear copper(n) complex [Cu2(H_4tapt)]'10H2O, where tapt is W,Ar',A'",A'"'-tetra(2-aminopropyl)1,1,2,2-ethanetetraamide, has been synthesized and investigated in detail as a mimic of Superoxide dismutase. Crystal structure analysis shows that ea

Full text of DOI:10.1039/a909018e

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A superoxide dismutase mimic with high activity: crystal structure,
solution equilibrium and pulse radiolysis
Jian-Jun Zhang,ab Qin-Hui Luo,*ab De-Liang Long,b Jiu-Tong Chen,b Feng-Mei Lic and
An-Dong Liuc
a State Key Laboratory of Coordination Chemistry, Coordination Chemistry Institute,
Nanjing University, Nanjing 210093, P. R. China. E-mail: qhluo@jlonline.com
b Fuzhou State Key Laboratory of Structure Chemistry, Fuzhou 350002, P. R. China
c Institute of Low Energy Nuclear Physics, Beijing Normal University, Beijing 100875,
P. R. China
Received 15th November 1999, Accepted 18th April 2000
Published on the Web 30th May 2000
A new binuclear copper() complex [Cu2(HϪ4tapt)]ؒ10H2O, where tapt is N,NЈ,NЉ,Nٞ-tetra(2-aminopropyl)-
1,1,2,2-ethanetetraamide, has been synthesized and investigated in detail as a mimic of superoxide dismutase.
Crystal structure analysis shows that each copper atom is situated in a slightly distorted square-pyramidal
environment, with a water molecule at the apex. According to the result of pulse radiolysis, the complex has a
high rate constant toward O2Ϫ, kcat = 1.91 × 108 dm3 molϪ1 sϪ1 (pH 7.4). Potentiometric titration shows that the
ؒ
mimic exhibits high stability at physiological pH value (7.4). EPR and ES-MS spectra reveal intermolecular
interaction of two copper() nuclei.
We have synthesized a linear double-cavity ligand N,NЈ,NЉ,
Introduction
Nٞ-tetra(2-aminoethyl)-1,1,2,2-ethanetetraamide11 (taet)whose
structure is similar to that of bisdioxocyclam.24 Its binuclear
copper() complex [Cu2(HϪ4taet)] may effectively scavenge
reactive oxygen species in organisms.17 We modified its structure
Superoxide dismutase (SOD) catalyses the dismutation of
superoxide anion ( O2Ϫ) and plays an important role in the pro-
ؒ
tection of cells against oxygen toxicity.1 Superoxide anion is the
one-electron-reduction product of O2 in mammalian organisms
and the mediator of many diseases. It is involved in DNA dam-
age, lipid peroxidation, radiation injury, and vascular diseases,
etc.2 In a variety of scenarios, therapeutic dosage of SOD
enzyme has been promised,3 but SOD has many shortcomings,
such as short lifetime, high cost, tissue impermeability and
immunogenicity, etc. Therefore the stable, non-toxic metal
complexes (model compounds) which catalyse the dismutation
and obtained a new binuclear copper() complex [Cu2(HϪ4
-
tapt)]ؒ10H2O (see Fig. 5), where tapt denotes N,NЈ,NЉ,Nٞ-
tetra(2-aminopropyl)-1,1,2,2-ethanetetraamide. The complex
has a similar structure to that of the active site of CuZnSOD.
pH Titration and pulse radiolysis were used to investigate its
stability in aqueous solution and activity for superoxide anion
dismutation. As four methyl groups were introduced in the
backbone of the ligand, the new complex [Cu2(HϪ4tapt)]ؒ
10H2O has better stability than that of [Cu2(HϪ4taet)] in
aqueous solution, and also has high activity. In addition, its
electrochemical behaviour, electrospray mass spectrum and the
reaction mechanism for dismutation of superoxide anion have
been investigated.
of O2Ϫ offer considerable promise as SOD mimics for pharma-
ؒ
ceutical application and have attracted much attention.4
Although many SOD mimics have been reported, such as
copper() complexes of polypeptides,5 polydentate Schiff
bases,6 mixed ligands7 and imidazolate-bridged heterobinuclear
Cu–Zn complexes8 or binuclear copper complexes,9 complexes
of macrocyclic ligands,8b,10 and so on, only a few mimics which
are both thermodynamically stable and highly active in the
range of physiological pH were reported.9,11 Therefore search-
ing both highly stable and active complexes in aqueous solution
as SOD mimics for pharmaceutical application has been a
challenge for bioinorganic chemists.
Experimental
Materials
Tetraethyl 1,1,2,2-ethanetetracarboxylate was synthesized
according to the literature.18 The reagents used for the solution
studies were of reagent grade and the solutions prepared using
twice-distilled water. Carbonate-free NaOH for pH titration
was obtained by Powell’s method.19 A stock solution of
Cu(NO3)2 approximately 1.0 × 10Ϫ2 mol dmϪ3 for pH titration
was standardized by using Na2edta. The content of HCl in the
ligand was analysed by conductometric titration using a BSD-A
conductometer. In pulse radiolysis experiments all the reagents
were recrystallized twice in twice-distilled water. The solutions
for pulse radiolysis contain about 3.21 × 10Ϫ6–1.32 × 10Ϫ5 mol
dmϪ3 Cu(ClO4)2 and tapt, molar ratio of Cu:tapt 2:1.4, and 0.1
mol dmϪ3 HCO2Na, pH kept at 7.4 by using phosphate buffer.
Copper() ion is the active center of CuZnSOD. It is co-
ordinated to four imidazole nitrogen atoms and one imidazole
serves as a bridging ligand between CuII and ZnII. In addition,
a water molecule is co-ordinated to copper atom at the apex,
forming a distorted square-pyramidal configuration.12 The
main function of ZnII is to stabilize the protein structure and it
has little effect on the activity of the enzyme. The structures of
dioxotetraamine ligands are similar to those of polypeptides
and they have the ability to stabilize high oxidation states of
metal ions. The hydrogen of the amide group is lost upon co-
ordination of divalent metal ions, and stable complexes may be
formed.13 Their transition metal complexes have been used as
models of enzymes,14 their macrocyclic copper() complexes
have antiinflammatory activity,15 and non-cyclic copper()
complexes have therapeutic effect for rheumatoid arthritis.16
Preparation of compounds
tapt and taptؒ4HClؒEtOHؒ0.5H2O. Tetraethyl 1,1,2,2-ethane-
tetracarboxylate (10 g, 0.03 mol ) was added to a 1,2-diamino-
DOI: 10.1039/a909018e
J. Chem. Soc., Dalton Trans., 2000, 1893–1900
This journal is © The Royal Society of Chemistry 2000
1893

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propane solution (100 cm3, 1.22 mol) at 0 ЊC. The resulting
solution was stirred for one week, then the remaining 1,2-
diaminopropane was distilled off under reduced pressure and
the ligand isolated, washed with diethyl ether and dried in a
vacuum desiccator. Yield 11.6 g (90%). The ligand tapt can be
used for the subsequent synthesis without further purification.
The very hygroscopic ligand was converted into the hydro-
chloride salt taptؒ4HClؒEtOHؒ0.5H2O for further studies. Thus
tapt (2 g, 4.04 mmol) was dissolved in 10 cm3 methanol, then
a dry stream of HCl was introduced. The resulting white pre-
cipitate was filtered off and recrystallized twice in MeOH–
EtOH (1:1), then dried over P2O5 in vacuum desiccator. Yield
1.17 g (40%) (Found: C, 38.41; H, 7.47; N, 17.52; HCl, 22.85%.
C20H49Cl4N8O5.5 requires: C, 38.04; H, 7.82; N, 17.74; HCl,
The equilibria in the system can be represented by eqn. (1)
qM ϩ pH ϩ rL
MqHpLr
(1)
βpqr = [MqHpLr]/[M]q[H]p[L]r
where q, p, and r denote the number of metal ions, hydrogen
ions and ligand molecules bound in the complex respectively.
A negative p value shows that hydrogen ion is released during
co-ordination. The values of βpqr were calculated by using the
program LEMIT21a which is based on the Newton–Raphson
and Gauss–Newton methods to minimize U in eqn. (2) where
expt
2
U = Σ (CHicalc Ϫ CHi
)
(2)
23.09%). νmax/cmϪ1(KBr) 3380m [ν(N–H)] and 1665s [ν(C᎐O)].
᎐
CHicalc and CHiexpt denote calculated and experimental values of
the Hϩ concentration at the ith point respectively.
1H NMR (500 MHz, D2O, 295 K): δ 3.63 (1 H, s, COCH2CO),
3.58 (4 H, d, CONHCH2), 3.51 (6 H, d, CH3) and 3.43 (2 H, q,
MeCHNH2).
Electrochemical measurements
[Cu2(H؊4tapt)]ؒ10H2O. To an aqueous solution (50 cm3) of
tapt (1 g, 2.32 mmol) was added 4.5 mmol of copper()
hydroxide suspended in 500 cm3 water. The resulting solution
was stirred at 50 ЊC for about 3 h, and filtered. The reddish
violet crystals were isolated after concentration of the solution
under reduced pressure. Yield 0.85 g (50%) (Found: C, 28.99;
H, 7.26; N, 14.98; H2O, 25.41%. C9H27CuN4O7 requires: C,
29.46; H, 7.42; N, 15.27; H2O, 24.55%). νmax/cmϪ1 (KBr) 3385m
The experiments were carried out on a PAR Model 273 poten-
tiostat coupled to a PAR Model 175 universal programmer. In
CV measurements a glassy carbon electrode was employed as
working electrode, a saturated calomel electrode (SCE) as refer-
ence electrode and platinum coil wire as auxiliary electrode.
Experiments were performed under a purified nitrogen atmos-
phere at 25 0.1 ЊC. The complex concentration was 1.0 × 10Ϫ3
mol dmϪ3 in 0.5 mol dmϪ3 sodium sulfate aqueous solution.
The solution was deaerated for ca. 15 min before applying the
voltage. The half wave potential E1/2 was calculated approxi-
mately from (Epa ϩ Epc)/2.
In controlled potential analysis a platinum web electrode
was employed as working electrode instead of glassy carbon.
Counter and reference electrodes were separated from the
working compartment by a glass frit and a salt bridge respec-
tively. The electrolysis time was 40 min. The concentration
of the electrolysis solution was 0.5 × 10Ϫ3 mol dmϪ3. Blank
corrections were made by deducting faradaic impurity, faradaic
residual and charging currents. The current–time function was
integrated over the time period of the experiment (40 min).
[ν(N–H)], 3700–2800s (hydrogen bonds) and 1600s [ν(C᎐O)].
᎐
λmax/nm (water) 532 (ε/dm3 molϪ1 cmϪ1 16.4) and 260.2 (4744).
Λm (water, 298 K) 8.3 S cm2 molϪ1. µ/µB (295 K, per Cu)
2.05. Single crystals suitable for X-ray structure analysis were
obtained by slow evaporation of the mother liquor for one
week.
Pulse radiolysis
The apparatus consisted of a BF-5 linear electron accelerator
and an optical monitor system. The electron pulse had an
energy of 10 MeV, a beam current maximum of 150 mA and
pulse width of 2.6 µs. The analytical light source was a 500 W
xenon lamp powered by a voltage-current stabilizer. Dosimetry
Ϫ3
ؒ
used (SCN)2 formed in N2O-saturated 0.1 mol dm KSCN
solution, with ε = 7600 dm3 molϪ1 cmϪ1 at 475 nm.20 In this
work the dose was measured to be 30 krad. The solutions of
complex were placed in a Suprasil quartz cell (diameter 1 cm,
length 1.4 cm). Before the electron beam entered into the
reaction cell, superpure oxygen gas was bubbled into the solu-
Crystal structure determination
Data were collected on a Siemens Smart/CCD area-detector
diffractometer. Data reductions and cell refinements were per-
formed with Smart-CCD software.22a An absorption correction
by using SADABS22b software was applied, which resulted in
transmission coefficients ranging from 0.640 to 0.901. Structure
solution and refinement were carried out on a Silicon Graphics
Indy workstation using the SHELXTL program package.23
The structure was solved by direct methods. All non-hydrogen
atoms were located on the E map or successive Fourier differ-
ence syntheses. The structure was refined by full-matrix least
squares techniques on F2 with anisotropic thermal parameters
for all non-hydrogen atoms. Hydrogen atoms on carbons were
located geometrically and refined in riding mode, whereas those
of water molecules were initially found on Fourier difference
maps and then refined as restrained by using the DFIX
instruction.
Ϫ
ؒ
tions for 15 min. The optical density of O2 was recorded
immediately after one single electron pulse was injected into
Ϫ
ؒ
solutions. The molar absorption coefficient of O2 at 250 nm
is 2000 dm3 molϪ1 cmϪ1. The O2 concentration produced by
Ϫ
ؒ
one single electron pulse was calculated to be 7.2 × 10Ϫ5 mol
dmϪ3
.
pH Titration
Protonation constants of the ligand and stability constants of
the copper() complexes were determined by using a Corning
pH meter with a glass–silver–silver chloride combination elec-
trode with precision 0.001 pH. The electrode was standard-
ized by buffer solutions of potassium hydrogenphthalate and
sodium borate. Typical concentrations of the experimental
solutions of ligand were 0.002755 mol dmϪ3 , and the molar
ratio of taptؒ4HCl:Cu(NO3)2 :HNO3 was controlled to be
1:1:1 or 1:2:1. The experimental solutions were titrated with
carbonate-free 0.09406 mol dmϪ3 NaOH by using a micro-
syringe with precision 2 µl under a nitrogen atmosphere in
a sealed jacketed vessel at 25 ЊC.21 The ionic strength was kept
at 0.10 mol dmϪ3 with KNO3. For each system, at least two
titrations have been performed, each of them having no less
than 60 experimental points.
CCDC reference number 186/1940.
See http://www.rsc.org/suppdata/dt/a9/a909018e/ for crystal-
lographic files in .cif format.
Other physical measurements
Elemental analyses were performed using a Perkin-Elmer 240c
analytical instrument. The IR spectra were measured as a KBr
disc using a Nicolet 5DX FT-IR spectrophotometer. Thermo-
analysis of the complex was performed on a SDT 2960C TA
thermoanalyser under an argon atmosphere with a rate of
10 ЊC minϪ1. Proton NMR spectroscopy was performed on
1894
J. Chem. Soc., Dalton Trans., 2000, 1893–1900

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Fig. 1 The EPR spectra of the complex [Cu2(HϪ4tapt)]ؒ10H2O in
methanol (1.0 × 10Ϫ2 mol dmϪ3); (1) at 110 K; (2) at room temperature.
a Bruker AM-500 spectrometer. Magnetic susceptibility was
measured using a CHAN-2000 Faraday-type magnetometer.
X-Band EPR spectra were recorded on a Bruker ER 420 spec-
trometer operating at 110 K and room temperature in methanol
solution with frequency modulation, 110 kHz, UV-Vis spectra
by using a UV-3100 spectrometer. The electrospray mass spec-
trum was obtained on a Finnigan LCQ mass spectrograph, with
sample concentrations of about 1.0 mmol dmϪ3. The diluted
solutions were electrosprayed at a flow rate of 5 × 10Ϫ6 dm3
minϪ1 with a needle voltage of ϩ4.5 kV. The temperature of the
heated capillary in the interface was 200 ЊC and a fused silica
sprayer was used. The mobile phase was an aqueous solution of
methanol (1:1 v/v). The sample was run in the positive-ion
mode.
Fig. 2 The ES mass spectra of (a) [Cu2(HϪ4tapt)]ؒ10H2O in methanol
solution and (b) [Cu2(HϪ4taet)]ؒ10H2O in aqueous solution.
Results and discussion
Characterization of the complex
from g|| ϩ 2 g⊥ = 3 giso). The result is similar to that for a
13-membered analogue [Cu2(HϪ4bdt)], where bdt denotes
bis(1,4,7,10-tetraazacyclotridecane-11,13-dione),24 but is quite
different from its ethyl analogue [Cu2(HϪ4taet)] which shows no
interaction between the two copper() nuclei in aqueous solu-
tion.11 This may be due to the intermolecular polymerization
between two molecules of [Cu2(HϪ4tapt)]ؒ as demonstrated by
electrospray mass spectra.
The IR spectrum of ligand hydrochloride taptؒ4HClؒEtOHؒ
0.5H O shows that the C᎐O stretching vibration occurs at 1665
᎐
2
cmϪ1. Comparing with that of [Cu2(HϪ4tapt)]ؒ10H2O at 1600
cmϪ1, a shift of about 65 cmϪ1 towards lower wavenumber
means the conjugation of O–C–N is strengthened after the
deprotonation of the amide group. The strong and broad
absorption band at 2800–3700 cmϪ1 of the complex indi-
cates the formation of multi-hydrogen bonds involving
water molecules. The molar conductivity shows that the
complex is neutral, due to the deprotonation of four amide
groups. Based on the above mentioned data and elemental
analysis, the structure of the complex was proposed to be as in
Fig. 5(a).
Electrospray mass spectra
Electrospray mass spectrometry (ES-MS) is a new technique for
the characterization of unstable species and intermediates in
solution since it allows preexisting ions to be transferred to the
gas phase with minimum fragmentation.25 The ES mass spectra
of [Cu2(HϪ4tapt)]ؒ10H2O in methanol and [Cu2(HϪ4taet)]ؒ
10H2O in aqueous solution are shown in Fig. 2 (a) and (b). In
Fig. 2(a) the peak at m/z 555.1 is dominant and is assigned as
[Cu2(HϪ4tapt) ϩ Hϩ]ϩ. This may lose a Cu2ϩ ion and be further
The complex begins to lose weight at 66 ЊC, and has
lost 25.41% by 106 ЊC, corresponding to ten water molecules
(calc. 24.55%). Crystal structure analysis shows the ten water
molecules are composed of 2 co-ordinated and 8 crystal, but
there is only one peak in the DTA curve. This is quite different
from its analogue [Cu2(HϪ4taet)]ؒ10H2O,11 which has three
weight loss processes from 40 to 128 ЊC. This may be due to the
weak bonding of the ten water molecules in this complex. The
complex begins to decompose after 240 ЊC.
The magnetic susceptibility (χm) of the complex was meas-
ured to be 3.63 × 10Ϫ3 cm3 molϪ1, corresponding to a magnetic
moment of 2.05 µB per copper(), that is copper() has a single
electron configuration (d9).
The EPR spectra of the complex in methanol solution at
room temperature and 110 K are shown in Fig. 1. The g|| region
of Fig. 1 curve (1) displays a six-line set arising from hyperfine
interaction between the odd electron and two copper nuclei
(I = 3/2). This may involve partial overlapping of the g|| and g⊥
regions because the interaction of two copper() nuclei (I = 3/2)
may cause a seven-line set. From Fig. 1 curve (2), giso = 2.085
and Aiso = 4.5 mT = 43.82 × 10Ϫ4 cmϪ1 were obtained. Also the
frozen solution spectrum (Fig. 1 curve (1)) yielded A|| = 9.5
mT = 92.52 × 10Ϫ4 cmϪ1, g|| = 2.226 and g⊥ = 2.014 (calculated
protonated. It should be noted that
a dimer [(Cu2-
(HϪ4tapt))2 ϩ Hϩ]ϩ at m/z 1107.1 with a relatively high abun-
dance up to 42% and a trimer [(Cu2(HϪ4tapt))3 ϩ 2Hϩ]ϩ at m/z
831.5 formed through self-polymerization of the species
[Cu2(HϪ4tapt)] are observed. For [Cu2(HϪ4taet)]ؒ10H2O in
aqueous solution the situation is quite different. From Fig. 2(b)
and Table 1 we can see that the abundance of the dimer
[(Cu2(HϪ4taet))2 ϩ Hϩ]ϩ is negligible, that is to say the mono-
mer is dominant in the aqueous solution of [Cu2(HϪ4taet)]ؒ
10H2O as well as in its methanol solution. In order to demon-
strate the peak assignment is right, the isotopic distributions of
the peak at m/z 1107, including the relative abundance and the
position, were calculated by permutation and combination of
65Cu, 63Cu, 12C and 13C.26 The calculated and observed results
shown in Table 2 are in good agreement. This result further
supports that the dimer is formed through intermolecular
interactions. The intermolecular interaction of two copper()
nuclei may cause a seven-line set in EPR spectra, and has
been observed in many square planar macrocyclic binuclear
copper() complexes.27
J. Chem. Soc., Dalton Trans., 2000, 1893–1900
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Table 1 Peak assignment of the complexes in Fig. 2
[Cu2(HϪ4tapt)]ؒ10H2O
[Cu2(HϪ4taet)]ؒ10H2O
Peak (m/z)
Species
Peak (m/z)
Species
246.6(30)
277.1(7)
492.1(22)
523.3(4)
555.1(100)
570.1(10)
831.5(8)
1046.1(20)
1107.1(42)
[Cu2(HϪ4tapt) Ϫ Cu2ϩ ϩ 4Hϩ]2ϩ
[Cu2(HϪ4tapt) ϩ 2Hϩ]2ϩ
218.6(100)
249.1(10)
376.1(43)
436.1(80)
498.9(36)
934.1(8)
[Cu2(HϪ4taet) Ϫ Cu2ϩ ϩ 4Hϩ]2ϩ
[Cu2(HϪ4taet) ϩ 2Hϩ]2ϩ
[Cu2(HϪ4tapt) Ϫ Cu2ϩ ϩ 3Hϩ]ϩ
[(Cu2(HϪ4tapt))2 Ϫ Cu2ϩ ϩ 4Hϩ]2ϩ
[Cu2(HϪ4tapt) ϩ Hϩ]ϩ
[Cu2(HϪ4tapt) Ϫ 2Cu2ϩ ϩ 5Hϩ]ϩ
[Cu2(HϪ4taet) Ϫ Cu2ϩ ϩ 3Hϩ]ϩ
[Cu2(HϪ4taet) ϩ Hϩ]ϩ
[(Cu2(HϪ4tapt))2 ϩ CH3OH ϩ 2Hϩ]2ϩ
[(Cu2(HϪ4tapt))3 ϩ 2Hϩ]2ϩ
[(Cu2(HϪ4taet))2 Ϫ Cu2ϩ ϩ 3Hϩ]ϩ
[(Cu2(HϪ4taet))2 ϩ Hϩ]ϩ
995.1(4)
[(Cu2(HϪ4tapt))2 Ϫ Cu2ϩ ϩ 3Hϩ]ϩ
[(Cu2(HϪ4tapt))2 ϩ Hϩ]ϩ
Note: the relative abundance is given in parentheses.
Table 2 The isotopic distribution of the [(Cu2(HϪ4tapt))2 ϩ Hϩ]ϩ
fragment at m/z 1107.1
Table 3 Equilibrium constants at 25 0.01 ЊC, I = 0.10 mol dmϪ3
(KNO3)
Peak position
Calculated
Relative abundance (%)
Reaction
log Ka
9.48 0.03
9.15 0.02
8.45 0.01
7.67 0.04
tapt ϩ Hϩ
[Htapt]ϩ
Observed
Calculated
Observed
[Htapt]ϩ ϩ Hϩ
[H2tapt]2ϩ ϩ Hϩ
[H3tapt]3ϩ ϩ Hϩ
[H2tapt]2ϩ
1105.6
1106.6
1107.6
1108.6
1109.6
1110.6
1111.6
1112.6
1113.6
1114.6
1105.3
1106.2
1107.1
1108.1
1109.1
1110.1
1111.1
1112.0
1113.2
1114.3
53
21
100
37
72
27
24
8
54
38
100
42
72
31
34
6
[H3tapt]3ϩ
[H4tapt]4ϩ
Cu2ϩ ϩ tapt ϩ Hϩ
Cu2ϩ ϩ tapt
[Cu(Htapt)]3ϩ
20.00 0.02
13.62 0.04
4.78 0.02
[Cu(tapt)]2ϩ
Cu2ϩ ϩ tapt
[Cu(HϪ1tapt)]ϩ ϩ Hϩ
[Cu(HϪ2tapt)] ϩ 2Hϩ
Cu2ϩ ϩ tapt
Ϫ4.70 0.03
4
4
2Cu2ϩ ϩ tapt
2Cu2ϩ ϩ tapt
2Cu2ϩ ϩ tapt
[Cu2tapt)]4ϩ
16.78 0.02
4.61 0.01
Ϫ9.66 0.03
1
2
[Cu2(HϪ2tapt)]2ϩ ϩ 2Hϩ
[Cu2(HϪ4tapt)] ϩ 4Hϩ
a The equilibrium constants were also checked by the program
BEST.21b The errors are in the range of 5%.
[Cu(tapt)]2ϩ and [Cu(HϪ2tapt)]. The best fitting of the titration
curve was obtained assuming the formation of species
[Cu(Htapt)]3ϩ, [Cu(tapt)]2ϩ, [Cu(HϪ1tapt)]ϩ, and [Cu(HϪ2tapt)].
The titration curve (3) has a long buffer region (a = 2–8),
and the pH increases abruptly at about a = 9, indicating the
formation of species [Cu2(HϪ4tapt)] which gradually becomes
predominant with increasing pH. Assuming [Cu2(tapt)]4ϩ
,
[Cu2(HϪ2tapt)]2ϩ, and [Cu2(HϪ4tapt)] are formed, the best
curve fitting was obtained. The values of log K are listed in
Table 3. Comparing the equilibrium constants of [Cu2-
(HϪ4tapt)] with those of its ethyl analogue [Cu2(HϪ4taet)] [Table
2 in ref. 11(b)], we may see that the electron repelling effect
caused by methyl groups may improve the stability of this
complex.
The distribution curves are shown in Fig. 4(a) and (b). In Fig.
4(b) only [Cu2(HϪ4tapt)] exists at pH > 8.0, and at physiological
pH (7.4), although [Cu2(HϪ4tapt)] coexists with [Cu2(HϪ2
-
Fig. 3 pH Titration curves, I = 0.10 mol dmϪ3 (KNO3), 25 ЊC, of (1)
[taptؒ4HCl] = [HNO3] = 0.001929 mol dmϪ3, (2) [taptؒ4HCl] = [Cu2ϩ] =
[HNO3] = 0.002912 mol dmϪ3 and (3) [taptؒ4HCl] = [HNO3] =
tapt)]2ϩ, the former still predominates absolutely. It is remark-
able that the percentage of free Cu2ϩ is less than 0.01% at pH
7.4. Comparing with its ethyl analogue [Cu2(HϪ4taet)],11b in its
solution the amount of free Cu2ϩ ion is 0.07% at pH 8.3. This
means that the complex has higher stability than its ethyl
analogue.
0.002755, [Cu2ϩ] = 0.005341 mol dmϪ3
.
pH Titration
The co-ordinating ability of the ligand toward Cu2ϩ in aqueous
solution has been studied by means of pH titration of a solu-
tion containing taptؒ4HCl, Cu(NO3)2 and HNO3 in a molar
ratio of 1:1:1 or 1:2:1. Fig. 3 show the titration curves of
the ligand(1) and of ligand plus Cu2ϩ with molar ratios of
Cu2ϩ :tapt of 1:1(2) and 2:1(3) respectively. The first inflexions
of the three curves are all at about a = 1 (a denotes the
number of moles of NaOH added per mol ligand), showing
the existence of 1 mol free acid in the solutions. The titration
curve (2) has two inflexions at about a = 5 and 7 after a short
buffer region (a = 2–4), indicating the formation of species
Crystal structure of the complex [Cu2(H؊4tapt)]ؒ10H2O
Details of the crystal data, intensity collection and refinement
are listed in Table 4. An ORTEP28 view of the complex is shown
in Fig. 5(b). Selected bond distances and angles are given in
Table 5. Each copper atom has a N4O donor set, with two
nitrogen atoms of the amido groups and two nitrogen atoms
of amino groups co-ordinated. The distances between amido
nitrogen atoms (N(1), N(3)) and CuII (about 1.94 Å) are
shorter than those between amino nitrogens (N(2), N(4)) (about
1896
J. Chem. Soc., Dalton Trans., 2000, 1893–1900

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Table 4 Crystal data of complex [Cu2(HϪ4tapt)]ؒ10H2O
Table 5 Selected bond lengths (Å) and angles (Њ) for the complex
[Cu2(HϪ4tapt)]ؒ10H2O
Empirical formula
Formula weight
Crystal system
Space group
a/Å
b/Å
c/Å
α/Њ
C18H54Cu2N8O14
733.77
Cu–N(1)
Cu–N(4)
Cu–O(3)
O(1)–C(1)
N(1)–C(1)
N(2)–C(3)
N(3)–C(6)
C–C(1)
1.936(3)
Cu–N(3)
Cu–N(2)
1.950(3)
2.029(4)
6.9798(9)
1.269(4)
1.462(5)
1.308(5)
1.452(6)
1.531(5)
1.458(7)
1.508(6)
Triclinic
1.996(3)
2.564(4)
1.261(4)
1.311(5)
1.367(7)
1.463(5)
1.529(5)
1.563(7)
1.515(7)
1.528(6)
¯
P1
Cu ؒ ؒ ؒ Cu(#)
O(2)–C(5)
N(1)–C(2)
N(3)–C(5)
N(4)–C(7)
C–C(5)
8.4837(2)
8.7611(3)
11.12710(10)
86.308(2)
86.622(2)
78.476(2)
807.79(3), 1
293(2)
1.388
4045
β/Њ
γ/Њ
C–C(#)
C(3)–C(4)
C(7)–C(8)
C(2)–C(3)
C(6)–C(7)
U/Å3, Z
T/K
µ(Mo-Kα)/mmϪ1
Reflections collected
Independent reflections
Final R1, wR2 [I > 2σ]
(all data)
N(1)–Cu–N(3)
N(3)–Cu–N(4)
N(3)–Cu–N(2)
N(1)–Cu–O(3)
N(4)–Cu–O(3)
C(1)–N(1)–C(2)
C(2)–N(1)–Cu
C(5)–N(3)–C(6)
C(6)–N(3)–Cu
C(1)–C–C(5)
95.74(12)
84.13(13)
169.0(2)
103.34(13)
86.99(14)
116.8(3)
114.0(2)
117.5(3)
113.7(2)
118.1(3)
109.0(4)
117.5(3)
110.1(4)
116.0(5)
125.1(3)
118.2(3)
109.8(4)
112.9(4)
N(1)–Cu–N(4)
N(1)–Cu–N(2)
N(4)–Cu–N(2)
N(3)–Cu–O(3)
N(2)–Cu–O(3)
C(1)–N(1)–Cu
C(3)–N(2)–Cu
C(5)–N(3)–Cu
C(7)–N(4)–Cu
C(1)–C–C(#)
O(1)–C(1)–N(1)
N(1)–C(1)–C
N(2)–C(3)–C(4)
C(2)–C(3)–C(4)
O(2)–C(5)–C
169.6(2)
83.42(13)
94.72(14)
97.36(13)
93.5(2)
126.7(3)
110.0(3)
125.7(3)
108.5(3)
109.3(4)
124.1(3)
118.2(3)
119.1(5)
114.9(5)
116.6(3)
108.1(3)
113.6(4)
2733 (Rint = 0.0211)
0.0475, 0.1117
0.0567, 0.1172
C(5)–C–C(#)
O(1)–C(1)–C
C(3)–C(2)–N(1)
N(2)–C(3)–C(2)
O(2)–C(5)–N(3)
N(3)–C(5)–C
N(4)–C(7)–C(6)
C(6)–C(7)–C(8)
N(3)–C(6)–C(7)
N(4)–C(7)–C(8)
Fig. 4 Species distribution curves (a) for curve (2) in Fig. 3, (b) for
curve (3) in Fig. 3. Species: (a) (1) Cu2ϩ; (2) [Cu(Htapt)]3ϩ; (3)
[Cu(tapt)]2ϩ; (4) [Cu(HϪ1tapt)]ϩ; (5) [Cu(HϪ2tapt)]; (b) (1) Cu2ϩ; (2)
[Cu2(tapt)]4ϩ; (3) [Cu2(HϪ2tapt)]2ϩ; (4) [Cu2(HϪ4tapt)].
2.01 Å). This means the amido nitrogen atoms have stronger
co-ordinating ability. A slightly distorted square-pyramidal
configuration around the copper atom is completed by the
additional binding of one H2O molecule in axial position. This
structure has some similarities to that of CuZnSOD. The Cu–O
distance is about 2.6 Å which is longer than that of [Cu2(HϪ4
-
taet)]ؒ10H2O (2.56 Å),11 but shorter than that of CuZnSOD
(2.8 Å).29 The longer the Cu–O is the easier the water molecule
is detached from copper in solution; the vacant co-ordination
Fig. 5 (a) Chemical structure of the complex [Cu2(HϪ4tapt)]ؒ10H2O.
(b) An ORTEP view.
site can be occupied by O2Ϫ, thus the long Cu–O distance is
ؒ
beneficial to dismutation of O2Ϫ. The Cu–N distances fall in
ؒ
the range 1.94–2.03 Å, which are close to that of CuZnSOD
(2.06 Å). The copper ion deviates by about 0.2 Å from the least-
squares plane defined by N(1), N(2), N(3) and N(4) toward the
H2O molecule; this value is also close to that of CuZnSOD (0.3
Å). The two co-ordination planes are parallel with a distance
of 1.60 Å. The intramolecular Cu ؒ ؒ ؒ Cu distance is 6.9798 Å.
The intermolecular Cu ؒ ؒ ؒ Cu distance is 5.9655 Å. The angles
N–Cu–N in the complex are somewhat different from those in
CuZnSOD. In the complex N(2)–Cu–N(3) 169.0, N(1)–Cu–
N(4) 169.6Њ, but in CuZnSOD NHis-60–Cu–NHis-120 157, NHis-46
Cu–NHis-48 140Њ.29
–
In addition to the axial water molecule, each copper center
forms adducts with four water molecules. These ten water
molecules in the complex form twelve intra- or inter-molecular
hydrogen bonds with oxygen atoms of amido groups and
hydrogen atoms of amino groups respectively. The large net-
work of hydrogen bonds helps to stabilize the crystal. The bond
distances and angles are listed in Table 6.
J. Chem. Soc., Dalton Trans., 2000, 1893–1900
1897

View Article Online
Table 6 Selected bond lengths (Å) and angles (Њ) of hydrogen bonds
for the complex [Cu2(HϪ4tapt)]ؒ10H2O
Hydrogen bond
D–H
D ؒ ؒ ؒ A
D–H ؒ ؒ ؒ A
O(3)–H(3D) ؒ ؒ ؒ O(4)
O(3)–H(3C) ؒ ؒ ؒ O(7A)
O(4)–H(4G) ؒ ؒ ؒ O(5)
O(4)–H(4F) ؒ ؒ ؒ O(6)
O(5)–H(5B) ؒ ؒ ؒ O(2)
O(5)–H(5A) ؒ ؒ ؒ O(3A)
O(6)–H(6C) ؒ ؒ ؒ O(2)
O(6)–H(6D) ؒ ؒ ؒ O(7)
O(7)–H(7B) ؒ ؒ ؒ O(1)
O(7)–H(7C) ؒ ؒ ؒ O(1)
N(4)–H(4E) ؒ ؒ ؒ O(2)
N(4)–H(4A) ؒ ؒ ؒ O(4)
0.83
0.84
0.83
0.83
0.84
0.86
0.84
0.82
0.83
0.85
0.90
0.90
2.796(6)
2.773(5)
2.755(6)
2.779(5)
2.714(4)
2.778(5)
2.743(4)
2.913(5)
2.731(4)
2.785(4)
3.159(5)
3.132(5)
158
156
169
159
162
173
171
165
172
171
158
154
Ϫ
ؒ
Fig. 7 Typical kinetic plot describing the decay of absorbance of O2
at 250 nm in an O2-saturated solution containing 1.32 × 10Ϫ5 mol dmϪ3
Cu(ClO4)2, 9.12 × 10Ϫ6 mol dmϪ3 ligand and 0.10 mol dmϪ3 sodium
Ϫ
Ϫ
ؒ
ؒ
formate at pH 7.4. Inset: plot of ln[ O2 ]0 Ϫ ln[ O2
] vs. t. The broken
line denotes the experimental data (355 points weretrecorded) and the
smooth curve denotes the fitted line.
E1/2 = Ep ϩ (Epul/2)
(3)
Considering the redox potential of O2Ϫ in neutral solution,33
we think that the CuII in the complex is able to be reduced but
ؒ
not oxidized by O2Ϫ. This gives a clue to the mechanism of
ؒ
Ϫ
ؒ
catalytical dismutation of O2 by the complex in the pulse
radiolysis study.
Pulse radiolysis
Fig. 6 Differential-pulse voltammetry profile of a solution containing
1 × 10Ϫ3 mol dmϪ3 complex (initial E = 0.2 V, final E = Ϫ0.6 V, incre-
ment in E = 0.04 V, amplitude = 0.05 V, pulse width = 0.06 s, sample
width = 0.02 s, pulse period = 0.2 s).
When O2-saturated solutions containing formate are radiol-
ؒ
ysed, the following species are primarily formed: H2O → OH,
H, H2, eaqϪ, H2O2, H3Oϩ. At sufficiently high concentration,
ؒ
ؒ
Ϫ
ؒ
the formate anion transforms these free radicals into O2
within the period of the pulse. It is necessary to ensure the
absence of trace metal ions, because they would make a con-
Electrochemical properties of the dicopper(II) complex
During scanning from 0 to ϩ1.0 V, the CV diagram of
[Cu2(HϪ4tapt)] displays one pair of peaks, the ratio of anodic
and cathodic peak currents ipa/ipc being close to unity. In the
same range of potential scan rates (v), the ip :v1/2 ratio is kept
constant. The peak separation between the anodic and cathodic
peaks ∆E at the scan rate 50 mV sϪ1 is 30 mV and increases with
increasing scan rate (119 mV at 300 mV sϪ1). These features
are indicative of a quasi-reversible CuIICuII–CuIIICuIII redox
process.
Ϫ
ؒ
tribution to O2 dismutation and lead to incorrect results. A
blank solution at pH 7 was radiolyzed and the second-order
rate constant for dismutation of O2 ks = 3.9 × 105 dm3 molϪ1
Ϫ
ؒ
s
Ϫ1 (pH 7.0) was obtained. The result is in agreement with that
reported by Bielski and Allen34 (ks = 4.0 × 105 dm3 molϪ1 sϪ1
,
pH 7.0). Therefore it is confirmed that the impurities existing in
the tested solution can be ignored.
Fig. 7 shows the decay curve of O2Ϫ vs. time t in the presence
ؒ
Ϫ
Ϫ
]
ؒ
ؒ
of complex recorded at 250 nm. Plotting ln[ O2 ]0 Ϫ ln[ O2
An electron transfer number n = 2.27 is obtained by controlled
potential electrolysis at ϩ0.9 V. It shows that two electrons per
molecule were transferred corresponding to the production of
a CuIIICuIII species during the positive scan and of a CuIICuII
species in the reverse scan. The difference between the cathodic
half-peak potential and cathodic peak potential Ep/2 Ϫ Epc is
about 65 mV corresponding to the separation of two redox
peak E2Њ Ϫ E1Њ (about 38 mV).30 This also shows that the wave
is due to the sequential transfer of two electrons in which
the potential for the addition of the second electron is about 38
mV more negative than the first. The half-wave potential E1/2
was calculated approximately to be ϩ0.684 0.005 V (SCE) or
t
vs. time t, where [ O2 ]0 and [ O2Ϫ]t denote the concentration of
Ϫ
ؒ
ؒ
O2Ϫ at initial stage and time t respectively, gives a straight line
ؒ
(Fig. 7 inset) which indicates that in the presence of the com-
plex the decay of O2 followed pseudo-first-order kinetics,
eqn. (4) where kobs represents the pseudo-first-order rate
Ϫ
ؒ
Ϫd[ O2Ϫ]/dt = kobs[ O2
]
(4)
Ϫ
ؒ
ؒ
constant which can be obtained from fitting the decay curve of
the concentration of O2Ϫ. Integrating eqn. (4) and fitting the
ؒ
Ϫ
ؒ
O2 decay curve, the square sum of error U in eqn. (5) was
0.926 V (NHE) at 50 mV sϪ1
.
Ϫ
calc
Ϫ expt 2
ؒ
ؒ
U = Σ([ O2
]
i
Ϫ [ O2
]
)
(5)
During scanning from 0 to Ϫ0.75 V the CV diagram of the
complex shows another pair of broad peaks. Differential-pulse
voltammetry was used to determine its E1/2 value. From the
differential-pulse voltammogram (Fig. 6) we obtained Ep =
Ϫ0.088 V, and proved that the electrode process was also a
sequential two-electron-transfer quasi-reversible process CuII-
CuII–CuICuI,31 and that the two copper centers have close redox
potentials. The E1/2 value was calculated approximately by using
eqn. (3),32 where Epul/2 denotes the pulse amplitude (50 mV),
and the E1/2 value is Ϫ0.063 V (SCE) or 0.179 V (NHE).
i
obtained: where i denotes the number of experimental points,
Ϫ
calc
Ϫ expt
ؒ
ؒ
[ O2
]
and [ O2
]
the calculated and experimental values
i
for the concentration of O2Ϫ respectively. Fitting a total of 355
data points, we obtained kobs = 1006 sϪ1 at the initial concen-
tration of complex [Cu2II(HϪ4tapt)]0 = 6.6 × 10Ϫ6 mol dmϪ3 (pH
7.4) with log U = Ϫ11.09.
ؒ
The catalytic mechanism of CuZnSOD and some low
molecular weight mimics has been shown to involve alternate
1898
J. Chem. Soc., Dalton Trans., 2000, 1893–1900

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Table 7 The rate constants of O2Ϫ dismutation catalysed by mimics of CuZnSOD and their equilibrium constants in aqueous solution
ؒ
Complexa
pH
kcat/dm3 molϪ1 sϪ1
log Kb
Reference
1
2
[Cu2(HϪ4tapt)]
[Cu2(HϪ4taet)]
7.4
7.8
8.3
7.8
7.0
7.8
7.0
4.8–9.0
1.9 × 108
2.8 × 108
1.6 × 108
4.7 × 106
7.15 × 106
1.2 × 106
1.9 × 106
2.4 × 109
Ϫ9.66
Ϫ15.22
[11(b)]
3
4
5
[Cu(HϪ4bdt)]
[Cu(HϪ2dt)]
[Cu(HϪ2npt)]
Ϫ7.09
Ϫ2.22
Ϫ2.96
35(a)
35(b)
37(b)
6
CuZnSOD
36
a dt denotes 1,4,7,10-tetraazacyclotridecane-11,13-dione, npt 12-(4-nitrobenzyl)-1,4,7,10-tetraazacyclotridecane-11,13-dione. b nCu2ϩ ϩ L
[Cun(HϪ2nL)] ϩ 2nHϩ (L denotes ligand, n = 1 or 2); K = [Cun(HϪ2nL)][H]2n/([Cu]n[L]).
reduction and reoxidation copper(), eqns. (6) and (7). In
II
Ϫ
Cu ϩ O2 → CuI ϩ O2
(6)
(7)
ؒ
2Hϩ
I
Ϫ
CuII ϩ H2O2
ؒ
Cu ϩ O2
radiolysis experiments the initial concentrations of complex
Ϫ
were 3.21 × 10Ϫ6–1.32 × 10Ϫ5 mol dmϪ3 and that of O2 was
ؒ
7.2 × 10Ϫ5 mol dmϪ3. The reaction kinetics of O2Ϫ dismutation
ؒ
was investigated at [Cu2II(HϪ4tapt)]0 < [ O2Ϫ]0. Under an excess
ؒ
Ϫ
Ϫ
ؒ
ؒ
of O2 , the complex catalysed dismutation of O2 and did
Ϫ 33
ؒ
not oxidize O2
.
The mechanism can be described as in
eqns. (8) and (9). Assuming a steady state for either [CuIICuII-
Fig. 8 Plot of pseudo-second-order rate constant of decay of O2Ϫ vs.
ؒ
k1
the initial concentration of [CuII2(HϪ4tapt)].
Ϫ
[CuIICuII(HϪ4tapt)] ϩ O2
ؒ
[CuICuII(HϪ4tapt)]Ϫ ϩ O2 (8)
is similar to that of the protein chain of CuZnSOD, so that
their configurations can be changed easily during dismutation
k2
Ϫ
Ϫ
[CuIICuI(HϪ4tapt)] ϩ O2
ؒ
Ϫ 6a
ؒ
Hϩ
of O2
.
Therefore they have high activity. In the com-
plex [Cu2(HϪ4tapt)] the introduction of a methyl group may
increase the thermodynamic stability and the permeability for
cells.
[CuIICuII(HϪ4tapt)] ϩ H2O2 (9)
(HϪ4tapt)] or [CuIICuI(HϪ4tapt)]Ϫ respectively, the initial con-
centration of complex [Cu2II(HϪ4tapt)]0 = [CuIICuI(HϪ4tapt)Ϫ] ϩ
[CuIICuII(HϪ4tapt)] and eqns. (10) and (11) are applicable. The
Acknowledgements
Ϫd[ O2Ϫ]/dt = (2k1k2/(k1 ϩ k2))[Cu2II(HϪ4tapt)]0[ O2Ϫ] =
ؒ
ؒ
This project was supported by the Natural Science Foundation
of China and the Foundation of Department of Education of
China.
kcat[Cu2II(HϪ4tapt)]0[ O2
]
(10)
(11)
Ϫ
ؒ
kobs = kcat[Cu2II(HϪ4tapt)]0
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line obtained goes through the origin with R = 0.993. So the
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constant together with results for some mimics and CuZnSOD
are listed in Table 7 for comparison.
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