A novel imidazolate-bridged copper-zinc heterodinuclear complex as a Cu, Zn-SOD active site model

Article abstract of DOI:10.1039/a906226b

A novel imidazolate-bridged Cu(II)-Zn(II) heterodinuclear complex of an imidazole derivative containing two metal-binding groups has been synthesized, and its structure and properties have been clarified.

Full text of DOI:10.1039/a906226b

A novel imidazolate-bridged copper–zinc heterodinuclear complex as a Cu,
Zn–SOD active site model
Hideki Ohtsu,a† Yuichi Shimazaki,a Akira Odania and Osamu Yamauchi*ab
a
Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.
E-mail: b42215a@nucc.cc.nagoya-u.ac.jp
Research Center for Materials Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.
b
Received (in Cambridge, UK) 2nd August 1999, Accepted 25th October 1999
A novel imidazolate-bridged Cu(ii)–Zn(ii) heterodinuclear
complex of an imidazole derivative containing two metal-
binding groups has been synthesized, and its structure and
properties have been clarified.
has an imidazolate-bridged dinuclear structure. In line with the
previous observation,6d the copper and zinc sites are very
similar to each other and indistinguishable, because the
complex has a C2v-like axis and both metal sites are statistically
disordered. The metal ions are both in an approximately trigonal
bipyramidal geometry with four nitrogen atoms of bdpi and a
nitrogen atom of the solvent MeCN coordinated. The coordina-
tion site occupied by MeCN may be susceptible to ligand
substitution, providing a possible binding site for substrate
superoxide. The Cu(ii)–Zn(ii) distance of 6.197(2) Å in 1 is the
same as that of native Cu, Zn–SOD (6.2 Å).1
The electronic spectrum of an acetonitrile solution of 1 (1
mM) showed two broad d–d bands at 645 and 880 nm (e = 60
and 220 M21 cm21, respectively). This spectrum is very close
to that of [Cu(tpa)Cl]PF6 [tpa = tris(2-pyridylmethyl)amine],
indicating a trigonal bipyramidal structure at the copper site.8 In
addition, it exhibited an imidazolate-to-Cu(ii) charge transfer
(CT) band at 320 nm (e = 1500 M21 cm21). The intensities of
the d–d and CT bands for 1 were half as strong as those observed
for 2 at 645, 880 and 320 nm (e = 110, 420 and 3000 M21
cm21, respectively). The EPR spectrum of a solution of 1 (0.5
mM) in 1+1 MeCN–MeOH at 77 K (g// = 2.10, g4 = 2.24 and
|A4| = 12.0 mT) showed a line shape characteristic of
mononuclear trigonal bipyramidal Cu(ii) complexes,9 but was
very different from that of 2 having a single symmetrical
derivative (g = 2.13), which is similar to the spectrum of the
imidazolate-bridged Cu(ii)-glycylglycinate complex10 and
other imidazolate-bridged Cu(ii)–Cu(ii) dinuclear complexes.11
These results as well as the ESI-mass spectrum in MeCN
demonstrate that 1 in solution retains its imidazolate-bridged
Copper–zinc superoxide dismutase (Cu, Zn-SOD) contains an
imidazolate-bridged Cu–Zn heterodinuclear metal center in its
active site.1–3 This enzyme catalyzes the dismutation of highly
toxic superoxide anion produced as a byproduct to dioxygen
and hydrogen peroxide in a sequence of dispersion-controlled
reactions.4 Since the enzyme structure was determined by X-ray
analysis,1 the unique active site structure attracted much
attention, and a number of imidazolate-bridged complexes, such
as the Cu(ii)-Cu(ii) homodinuclear complexes by Lippard et
al.,5 have been reported as mimics of the metal site and provided
valuable insights into the structure and properties of the SOD
active site. In contrast to this, only a few imidazolate-bridged
Cu(ii)–Zn(ii) heterodinuclear model complexes have so far
been reported.6 Because such model complexes are often
constructed by self-assembly of one or two complex units by
incorporating a bridging imidazolate ring, the labile nature of
Cu(ii) and Zn(ii) complexes and dissociation of self-assembled
structures made it difficult to study in detail their structures in
solution and SOD functions. We here report the first imidazo-
late-bridged Cu(ii)–Zn(ii) heterodinuclear complex derived
from a new imidazole derivative, Hbdpi {Hbdpi = 4,5-bis[di(2-
pyridylmethyl)aminomethyl]imidazole}, which has two sets of
3N donor groups tethered to the 4 and 5 positions of the bridging
imidazole ring.
Hbdpi was prepared from imidazole-4,5-dialdehyde7 and
bis(2-pyridylmethyl)amine by reductive aminomethylation
using sodium cyanoborohydride in methanol, and was charac-
terized by 1H and 13C NMR.‡ The heterodinuclear Cu(ii)–Zn(ii)
.
complex of bdpi, [CuZn(bdpi)(MeCN)2](ClO4)3 2MeCN 1, was
obtained as crystals by mixing Hbdpi and equimolar amounts of
Cu(ii) and Zn(ii) in methanol and subsequent addition of
triethylamine, and the composition of the dried sample of 1 was
determined by elemental analysis, ICP, and ESI-mass spectral
measurements. The corresponding Cu(ii)–Cu(ii) homodinu-
Fig. 1 X-Ray structure of complex 1 (perchlorate ions and the MeCN
molecule of crystallization are omitted for clarity, and the Cu and Zn sites
were so assigned that smaller R and Rw values could be obtained). Selected
bonds (Å) and angles (°): Cu(1)–N(1) 2.001(7), Cu(1)–N(3) 2.104(7),
Cu(1)–N(5) 2.074(7), Cu(1)–N(7) 2.105(8), Cu(1)–N((9) 2.004(9), Zn(1)–
N(2) 2.005(7), Zn(1)–N(4) 2.112(7), Zn(1)–N(6) 2.076(8), Zn(1)–N(8)
2.084(7), Zn(1)–N(10) 2.023(10), N(1)–Cu–N(3) 82.5(3), N(1)–Cu(1)–
N(5) 122.5(3), N(1)–Cu(1)–N(7) 118.3(3), N(3)–Cu(1)–N(9) 172.9(3),
N(2)–Zn(1)–N(4) 83.0(3), N(2)–Zn(1)–N(6) 120.3(3), N(2)–Zn(1)–N(8)
120.1(3), N(4)–Zn(1)–N(10) 174.8(3).
.
clear complex, [Cu2(bdpi)(MeCN)2](ClO4)3 2H2O 2 was pre-
pared in a similar manner using two equivalents of Cu(ii).
The molecular structure of complex 1 was determined by X-
ray analysis§ and is depicted in Fig. 1. As expected, the complex
† Present address: Department of Material and Life Science, Graduate
School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan.
Chem. Commun., 1999, 2393–2394
This journal is © The Royal Society of Chemistry 1999
2393

.
using Hbdpi and 2 equiv. of Cu(ClO4)2 6H2O. Elemental analysis and ICP
Cu(ii)–Zn(ii) heterodinuclear structure and is not a 1:1 mixture
of Cu(ii)–Cu(ii) (2) and Zn(ii)–Zn(ii) complexes. Whereas the
cyclic voltammogram of 2 in MeCN exhibited two reversible
redox waves at E1/2 20.09 and +0.19 V (vs. NHE), 1 showed
only one reversible Cu(i)/Cu(ii) redox couple at E1/2 +0.19 V
(vs. NHE), confirming that it has one copper ion site in the
molecule. In this connection, an imidazolate-bridged asym-
metric dinuclear Cu(ii) complex showed one broad peak in the
anodic and the cathodic wave,12 and several mononuclear
trigonal bipyramidal Cu(ii) complexes of tripodal ligands
containing pyridine and imidazole donors have been reported to
have E1/2 values between 20.187 and 20.290 V vs. NHE.13 The
positive shift of the E1/2 values of 1 as compared with other
mononuclear Cu(ii) complexes may therefore be attributed to
the electronic effect of the imidazolate-bound Zn(ii), which
could be an important factor for the efficient catalytic reaction
of Cu, Zn–SOD. The SOD activities of 1 and 2 have been
investigated by the cytochrome c assay14 using the xanthine
oxidase reaction as the source of superoxide. The IC50 values
for 1 and 2 were determined to be 0.24 and 0.32 mM [vs. Cu(ii)
ion], respectively, which are higher than the value reported for
native Cu, Zn–SOD (0.04 mM)15 but comparable with the values
of some structurally established model complexes.6d,12,15 In
addition, the IC50 value for [Cu(tpa)(MeCN)](ClO4)2 was
determined to be 1.4 mM. The difference in IC50 among 1, 2 and
[Cu(tpa)(MeCN)](ClO4)2 may be ascribed to the imidazolate-
bridge and zinc ion. The effect of zinc ion was recently reported
for a Cu–Zn heterodinuclear complex prepared in situ, which
was not structurally established.16
measurement were performed for the dried sample. Anal. Calc. for
[Cu2(bdpi)](ClO4)3·MeCN (C31H32N9O12Cl3Cu2): C, 38.94; H, 3.37; N,
13.18; Cu, 13.29. Found: C, 38.95; H, 3.32; N, 13.35; Cu, 13.27%. Effective
magnetic moment meff = 3.46 mB (300 K).
.
§
Crystal data:
1
(green): [CuZn(bdpi)(MeCN)2](ClO4)3 2MeCN,
¯
C37H41N12O13Cl3CuZn, M = 1083.09, triclinic, space group P1, a =
14.447(1), b 14.561(1), c 12.214(1) Å, a 108.002(7), b
106.920(7), g = 89.069(7)°, V = 2330.3(4) Å3, Z = 2, Dc = 1.508 g cm23
5067 reflections collected, 4821 independent reflections, 3209 reflections
used, 596 variables, R = 0.076, Rw = 0.110 [I > 2.00s(I)]. The crystal was
poorly diffracting. Rigaku AFC-5R four-circle automated diffractometer
=
=
=
=
,
with graphite monochromated Cu-Ka radiation, l
= 1.54178 Å, and
rotating anode generator. The structures were solved by the heavy-atom
method and refined anisotropically for non-hydrogen atoms by full-matrix
least-squares calculations. Hydrogen atoms for the structure were located
from difference Fourier maps, and their parameters were isotropically
refined. All the calculations were performed by using the teXsan
crystallographic software package from the Molecular Structure Corpora-
tion. CCDC 182/1462. See http://www.rsc.org./suppdata/cc/1999/2393/ for
crystallographic files in .cif format.
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In conclusion, the new imidazole derivative containing two
metal-binding groups incorporated Cu(ii) and Zn(ii) to form a
heterodinuclear SOD model complex which was stable in
solution, and the SOD activity measurements exhibited the
effects of the imidazolate bridge and zinc ion on the SOD
activity. Studies on the details of the SOD function and
mechanism are now under way.
We are grateful to Prof. Wasuke Mori, Kanagawa University,
for measurements of the magnetic susceptibility, Professor
Hiromu Sakurai, Kyoto Pharmaceutical University, for meas-
urements of the SOD activity and Sachiyo Nomura, Institute for
Molecular Science, for ICP measurements. This work was
supported by Grants-in-Aid for Scientific Research to A. O.
(No. 07CE2003(COE)) and O. Y. (Nos. 09304062 and
09045032) from the Ministry of Education, Science, Sports and
Culture of Japan, to which our thanks are due.
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Notes and references
9 M. Duggan, N. Ray, B. Hathaway, G. Tomlinson, P. Brint and K. Pelin,
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‡ Experimental: imidazole-4,5-dialdehyde (1.0 g, 8 mmol) was dissolved in
methanol (100 ml), to which bis(2-pyridylmethyl)amine (3.2 g, 16 mmol)
and a small amount of acetic acid were added. Sodium cyanotrihydroborate
(1.0 g, 15.5 mmol) was then added dropwise to the mixture, and after the
resulting solution had been stirred for three days at room temperature, it was
acidified with concentrated HCl and concentrated almost to dryness under
reduced pressure. The residue was dissolved in a saturated aqueous solution
of Na2CO3 (50 ml) and extracted with three 50 ml portions of CHCl3. The
combined extracts were dried over Na2SO4 and after removal of the solvent
gave a brown oily product, which was purified by silica gel column
chromatography with CHCl3–MeOH as eluent to give Hbdpi (3.16 g,
80.0%); 1H NMR (CDCl3, 400 MHz) d 3.65 (s, 4H), 3.75 (s, 8H), 7.12 (m,
4H), 7.47 (dt, 4H), 7.61 (td, 4H), 7.66 (s, 1H), 8.51 (dq, 4H). 13C NMR
(CDCl3; 100 MHz) d 48.2 (CH2), 59.2 (CH2), 121.7 (Py), 122.0 (Im), 123.2
(Py), 133.8 (Im), 136.2 (Py), 148.4 (Py), 158.9 (Py). Complex 1 was
prepared by mixing Hbdpi (0.50 g, 1.0 mmol) with a MeOH solution of
10 Y. Nakao, W. Mori, T. Sakurai and A. Nakahara, Inorg. Chim. Acta,
1981, 55, 103.
11 M. S. Haddad and D. N. Hendrickson, Inorg. Chem., 1978, 17, 2622.
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Superoxide anion was generated in situ by the xanthine–xanthine
oxidase system and detected spectrophotometrically by ferricytochrome
c reduction. The SOD activity was assayed in a g-collidine buffer (50
mM, pH 7.77, 25.0 °C) containing 10 mM ferricytochrome c, 50 mM
xanthine and an appropriate amount of xanthine oxidase to cause a
change of absorbance (DA550 = 0.025 min21). The IC50 value is defined
as the 50% inhibition concentration of cytochrome c reduction. The
inhibitory effect of Cu(ii) ion and its complexes used in this study on
xanthine oxidase was checked prior to the assay by using the absorbance
at the 295 nm peak due to uric acid at pH 7.77 (N. Cotell, J. L. Bernier,
J. P. Henichart, J. P. Catteau, E. Gaydou and J. C. Wallet, Free Rad.
Biol. Med., 1992, 13, 221; W. S. Chang, Y. J. Lee, F. J. Lu and H. C.
Chiang, Anticancer Res., 1993, 13, 2165). They did not show any
inhibitory effect under the experimental conditions used.
.
.
Cu(ClO4)2 6H2O (0.370 g, 1.0 mmol) and Zn(ClO4)2 6H2O (0.372 g, 1.0
mmol) and adding NEt3 (138.6 ml, 1.0 mmol). After filtration, the filtrate
was allowed to evaporate slowly in the open air to give [CuZn(bdpi)-
.
(MeCN)2](ClO4)3 2MeCN 1 as green crystals in 67.0% yield. Elemental
analysis and ICP measurement were performed for the dried sample which
lost the coordinated MeCN. The ratio of Cu(ii) to Zn(ii) was determined by
ICP (SEIKODENKO SPS-700) with the use of the standard solutions (1000
ppm) of Cu(ii) and Zn(ii) obtained from Nacalai Tesque. Anal. Calc. for
[CuZn(bdpi)](ClO4)3·0.25MeCN·3H2O (C29.5H35.75N8.25Cl3O15CuZn): C,
36.11; H, 3.67; N, 11.78; Cu, 6.48; Zn, 6.66. Found: C, 35.88; H, 3.56; N,
11.77; Cu, 6.46; Zn, 6.65%. ESI-mass (MeCN): m/z 816 [(M 2 ClO4)+].
15 U. Weser, L. M. Schubotz and E. Lengfelder, J. Mol. Catal., 1981, 13,
249.
16 S. Kawabata, T. Soma and K. Ichikawa, Chem. Lett., 1997, 1199.
.
[Cu2(bdpi)(MeCN)2](ClO4)3 2H2O 2 was prepared in a similar manner by
Communication 9/06226B
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Chem. Commun., 1999, 2393–2394