SOD activities of the copper complexes with tripodal polypyridylamine ligands having a hydrogen bonding site

www.elsevier.com/locate/ica Inorganica Chimica Acta 324 (2001) 108– 116

SOD activities of the copper complexes with tripodal polypyridylamine ligands having a hydrogen bonding site Koichiro Jitsukawa a,*, Manabu Harata a, Hidekazu Arii a, Hiromu Sakurai b, Hideki Masuda a,* b

a Department of Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466 -8555, Japan Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical Uni6ersity, Misasagi, Yamashina-ku, Kyoto 607 -8414, Japan

Received 8 May 2001; accepted 13 June 2001

Abstract As a structural mimic of the Arg 141 residue near copper site in native bovine Cu,Zn– superoxide dismutase (Cu,Zn–SOD), four mononuclear copper complexes with tris(2-pyridylmethyl)amine derivatives having a hydrogen bonding site (pivalamido, neopentylamino, or amino groups) at the pyridine 6-position, [Cu(tnpa)(OH)]ClO4 (1), [Cu(tapa)Cl]ClO4 (2), [Cu(tapa)(OH)]ClO4 (3), and [Cu(bppa)](ClO4)2 (4), and two analogous dinuclear copper complexes, [Cu2(tppen)(H2O)2](ClO4)4 (5) and [Cu2(tppen)Cl4] (6), were prepared, and the correlation between the coordination structures of the copper complexes and their superoxide dismutation activities were examined. Their structures in both solution and solid states were characterized by electronic absorption and ESR spectroscopic (for all the complexes) and by X-ray analytical methods (for 1, 2, 5 and 6), respectively. The coordination geometries around the copper ions were determined to be five-coordinate trigonal bipyramidal for 1, 2 and 3 and to be an intermediate of five-coordinate trigonal bipyramidal and square pyramidal for 5 and 6 in both crystal and solution phases, in contrast to four-coordinate square planar structure for 4 reported previously. The cyclic voltammetry measurement of the mononuclear complexes 1–4 showed quasi-reversible redox potentials (Cu(II)/Cu(I) couple) in the range between − 330 mV (vs. NHE at pH 7; O2/O2’−) and +890 mV (vs. NHE at pH 7; O2’−/H2O2), which are in the range responsible for superoxide dismutation (SOD) reaction, although the dinuclear copper complexes, 5 and 6, gave only reduction potentials. The SOD activities of complexes 1, 2, and 3 were moderate and those of 5 and 6 were rather high, although 4 showed the lowest activity of all. Those of 1, 2 and 3 with a trigonal bipyramidal structure and hydrogen bonding interaction site are slightly high in comparison with that of the corresponding Cu(II) complex without hydrogen bonding site, [Cu(tpa)(H2O)](ClO4)2 (7). Relatively higher SOD activity observed in complexes 5 and 6 may be explained in terms of higher flexibility in the conformation and cooperativity by dinuclear copper ions. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Superoxide dismutase; Copper complexes; Tripodal amine ligand complexes

1. Introduction Metal –dioxygen systems in metallo-proteins and -enzymes are very important for their biological reactions. Many kinds of structural and functional model investigations and their mechanistic approaches have been carried out in order to elucidate the interaction between the metal and oxygen species [1]. Copper plays an important role as one of the essential metallic elements * Corresponding authors. Tel.: + 81-52-735-5240; fax: +81-52735-5247. E-mail address: [email protected] (K. Jitsukawa).

required for normal metabolic processes, especially oxidation and oxygenation reactions, because of its appropriate redox property [2]. Among metalloenzymes containing copper ions, superoxide dismutase (SOD) in mammalian cells, plasma and extracellular spaces can catalyze a very rapid two-step disproportionation of superoxide molecules to dioxygen and hydrogen peroxide [3]. An alternate process of reduction and oxidation, as shown in Eqs. (1) and (2), is essential for the enzyme reaction in the dismutation of superoxide anion. Therefore, the biological systems utilize transition metal ions, such as Cu, Fe, and Mn, as the redox-active metal center of the SOD enzymes [4]. The iron and man-

0020-1693/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 0 - 1 6 9 3 ( 0 1 ) 0 0 5 6 7 - 9

K. Jitsukawa et al. / Inorganica Chimica Acta 324 (2001) 108–116

ganese SODs show superoxide dismutation activity as a mononuclear complex, whereas SOD containing copper ion functions as a dinuclear complex of copper and zinc ions [5]. Thus, several mononuclear Mn(II) [6] and Fe(III) complexes [7] with macrocyclic ligands and Fe(II) complexes with polypyridine ligands [8] were designed and investigated as SOD mimics for the therapeutic agents [5]. E-Cu(II) +O2− “ E%-Cu(I) +O2

(1)

E%-Cu(I)+ O2− +2H+ “E-Cu(II) + H2O2

(2)

Using copper complexes, investigation for SOD model has been also reported. The structural and/or functional models have been performed as Cu,Zn–SOD model using homo and hetero dinuclear metal complexes containing one or two copper ions [9,10], because the native Cu,Zn– SOD enzyme contains an imidazolate-bridging Cu– Zn hetero dinuclear metal center, where the copper ion is coordinated by four imidazole nitrogens of histidine residues [11]. They showed moderate SOD activity, although they are rather low in comparison with the native Cu,Zn– SOD. The highest IC50 values obtained from the cytochrome c assay method using the xanthine oxidase reaction as a superoxide source [12] are 0.24 and 0.32 mM in the Zn – Cu hetero dinuclear complexes [9d]. The investiga-

Chart 1. Hydrogen bonding interaction site to stabilize superoxide adduct.

Chart 2. Tripodal polypyridylamine ligands.

109

tion using mononuclear copper complexes has also been performed. For example, mononuclear copper complex with diacetyl-di(N4-methylthio-semicarbazone) ligand has been reported as a SOD mimic, in which biological assay for ischemia-reperfusion-induced tissue damage demonstrated an effective value [13]. However, mononuclear copper complexes with macrocyclic or acyclic polyamine ligands showed less activity; their IC50 values were about 70–2000 mM [14], which are less SOD activities compared with the above artificial dinuclear ones [2,9,10]. Among of them, a Cu(II) complex with cimetidine ligand gave rather high IC50 value (4 mM), although its detailed structure was not fully characterized [15]. The crystal structure of native bovine Cu,Zn–SOD revealed that Arg 141 residue near the copper center, which is located in the cavity of the enzyme, is linked with water molecules through hydrogen bonds [11]. It has been considered to contribute to the transportation of superoxide anion for the active site through such hydrogen bonding systems. Hence, in design for SOD mimic using mononuclear copper complexes with tetracoordinate ligands, introduction of the hydrogen bonding moiety near outer coordination sphere of the copper site may be essential for SOD activity. Previously, by the use of the copper or iron complexes with tetra-coordinate tris(2-pyridylmethyl)amine derivatives bearing pivalamido or neopentylamino substitutents at the pyridine six-positions, we have demonstrated the importance of hydrogen bonding system for stable binding of active oxygen species, such as superoxide, hydroperoxide, alkylperoxide, and hydroxide, to metal ions [16–23]. For example, crystal structure of [Cu(bppa)(OOH)]ClO4 complex, which is stable for a month even at room temperature, revealed that the distances between the peroxo oxygen bound to the copper center and nitrogen atoms of two 6-pivalamido groups of BPPA were 2.78 and 2.79 A, , respectively [20]. At this stage, it is clear that the hydrogen bond induces the stabilization of peroxo species bound to the metal ion. Therefore, as illustrated in Chart 1, the tripodal polypyridine unit of the tris(2-pyridylmethyl)amine (TPA) has been designed as a structural model of tetra-coordinate His residues, and the 6-amino or 6neopentylamino group has been introduced as a hydrogen bonding site of guanidinium group in the Arg residue of the native bovine Cu,Zn–SOD. The following ligands were prepared as six-substituted tris(2pyridylmethyl)amine derivatives (Chart 2); tris(6peopentylamino-2-pyridylmethy)amine (TNPA), tris(6amino-2-pyridylmethy)amine (TAPA), bis(6-pivalamido-2-pyridylmethyl)(2-pyridylmethyl)amine (BPPA), and N,N,N%,N%-tetrakis(6-pivalamido-2-pyridylmethyl)ethylenediamine (TPPEN). Using these ligands, we prepared copper complexes and examined the correlation between coordination structures of the complexes and their SOD activities.

K. Jitsukawa et al. / Inorganica Chimica Acta 324 (2001) 108–116

110

Table 1 Crystallographic data and refinement parameters for the complexes, 1, 2, 5 and 6

Empirical formula FW Color Crystal dimension (mm) Crystal system Space group Unit cell dimensions a (A, ) b(A, ) c (A, ) h (°) i (°) k (°) V (A, 3) Dcalc (g cm−3) Z F(000) v(Mo Ka) (cm−1) Radiation T (K) 2qmax (°) No. of reflection measured No. of reflection used [I\3.00|(I)] R Rw a

[Cu(tnpa)OH]ClO4 (1)

[Cu(tapa)Cl]ClO4 (2)

[Cu2(tppen)(H2O)2](ClO4)4 ·3CH3CN·2H2O (5)

[Cu2(tppen)Cl4]·2CH3OH (6)

C33H54N7O6CuCl 727.83 green 0.2×0.3×0.6

C18H21N7O4CuCl2 533.86 red 0.2×0.3×0.5

C52H81N13O24Cu2Cl4 1541.17 blue 0.2×0.2×0.5

C25H40N5O4Cu1Cl2 609.07 green 0.1×0.3×0.5

triclinic P1(

monoclinic P21/c

triclinic P1(

monoclinic C2/c

12.7287(8) 13.0500(7) 13.449(1) 100.636(5) 97.025(6) 119.579(4) 2012.5(2) 1.199 2 774 6.52 graphite monochromated Mo Ka (u= 0.71073 A, ) 294 52.6 8164

13.635(2) 11.965(1) 16.851(1)

16.251(5) 13.306(2) 27.244(3)

2572.0(4) 1.462 4 1092 10.93 graphite monochromated Mo Ka (u = 0.71073 A, ) 294 52.6 5234

12.277(2) 16.284(4) 18.663(3) 71.55(2) 74.12(1) 81.14(1) 3395(1) 1.467 2 1608 8.653 graphite monochromated Mo Ka (u =0.71073 A, ) 294 43.9 8549

5757(2) 1.331 8 2416 9.781 graphite monochromated Mo Ka (u =0.71073 A, ) 294 48.6 5070

3760

2957

4343 a

1668

0.099 0.141

0.096 0.062

0.078 0.197

0.078 0.087

110.676(8)

102.25(2)

No. of reflections used [I\0.50|(I)].

2. Experimental

2.1. Materials and measurement Reagents used for synthesis were of the highest available grade, and were employed without further purification. All solvents for spectroscopic measurements were purified by distillation before use. Electronic absorption spectra were recorded on JASCO UVIDEC-660. ESR spectra of frozen solution were recorded at 77 K on a JEOL RE-1X ESR spectrometer. 1H-NMR spectra were measured on a Varian VXR-300S or JEOL Lambda-500 spectrometer with TMS as an internal standard. Cyclic voltammetric measurements were performed at 25 °C under an argon atmosphere using a Bioanalytical System CV-27 voltammograph at a scan rate of 100 mV s − 1 in aqueous acetonitrile (20%) solution. A 3 mm diameter glassy-carbon working electrode, an Ag/AgCl reference electrode, and a Pt-wire counter electrode were used in a glass cell having a working compartment and in 0.1 M of nBu4BF4 solution as a supporting electrolyte. Electrochemical potentials were converted to the normal hydrogen electrode (NHE) scale by addition of 222 mV [24].

Crystal structure analyses were performed by the use of an Enraf– Nonius CAD4-EXPRESS four-circle diffractometer. Single crystals of copper complexes suitable for the X-ray diffraction measurement were mounted on a glass capillary. The diffraction data were collected with graphite-monochromated Mo Ka radiation with the …–2q scan technique at room temperature. Crystal data and experimental details are listed in Table 1. The details for analyses are according to the method reported previously [19,21].

2.2. Measurement of SOD acti6ity The activities for disproportionation of superoxide anion were examined according to the method for the xanthine–xanthineoxidase system established by Fridovich et al. [4]. The assay was carried out at 25 °C in HCl-collidine buffer (pH 7.8) containing ferricytochrome c (10 mM), xanthine (50 mM), and xanthineoxidase (appropriate amount), and the change in absorbance at 550 nm was followed. The IC50 values are defined as the 50% inhibition concentration of the cytochrome reduction.

K. Jitsukawa et al. / Inorganica Chimica Acta 324 (2001) 108–116

111

preparation [17]. The structures of all ligands employed here were confirmed by the elemental and 1H NMR analyses.

2.4. Preparation of Cu(II) complexes

Fig. 1.

ORTEP

To a blue –green methanol (10 ml) solution containing TAPA (2 mmol, 67 mg) and Cu(ClO4)2·6H2O (2 mmol, 74 mg) was added KCl (2 mmol, 15 mg), where the solution color turned to light-red. Eighty-five milligrams (80% yield) of [Cu(tapa)Cl]ClO4 complex (2) was isolated as a single crystal after recrystallization in a mixed solvent of CH3CN and diethyl ether. The other complexes, [Cu(tnpa)OH]ClO4 (1), [Cu(tapa)OH]ClO4 (3), [Cu(bppa)]ClO4 (4), [Cu2(tppen)(H2O)2](ClO4)4 (5), and [Cu2(tppen)Cl4] (6) were also prepared according to the above method. Preparation and structural analyses of [Cu(bppa)](ClO4)2 complex (4) and [Cu(tpa)(H2O)](ClO4)2 (7) have been reported previously [21,25].

drawing of [Cu(tnpa)OH]+ (1).

3. Results and discussion

3.1. Preparation and characterization of copper complexes

Fig. 2.

ORTEP

drawing of [Cu(tapa)Cl]+ (2).

2.3. Synthesis of ligands Tripodal tetradentate ligands, TNPA and BPPA, were synthesized according to the method previously reported [21]. TAPA was obtained by hydrolysis of TPPA in an aqueous EtOH solution containing KOH. Dinucleating hexadentate ligand, TPPEN, was synthesized according to the method similar to the TPPA

Fig. 3.

ORTEP

Five mononuclear copper complexes, [Cu(tnpa)OH]ClO4 (1), [Cu(tapa)Cl]ClO4 complex (2), [Cu(tapa)OH]ClO4 (3), [Cu(bppa)]ClO4 (4), and [Cu(tpa)(H2O)](ClO4)2 (7) and two dinuclear copper complexes, [Cu2(tppen)(H2O)2](ClO4)4 (5) and [Cu2(tppen)Cl4] (6) were prepared and characterized as described above in order to investigate the correlation between their structures and the dismutation activity of superoxide ion. Some of these complexes were obtained as single crystals suitable for X-ray analysis except for the complex 3. Crystallographic data and refinement parameters for these complexes (1, 2, 5 and 6) are listed in Table 1, and the data for complex 4 are referred to the previous report [21].

drawing of [Cu2(tppen)(H2O)2]4 + (5).

K. Jitsukawa et al. / Inorganica Chimica Acta 324 (2001) 108–116

112

Fig. 4.

ORTEP

drawing of [Cu2(tppen)Cl4] (6).

The crystal structures of the complexes obtained here are shown in Figs. 1–4. The complex 1 was a trigonal bipyramidal structure, in which the bond lengths between the central copper and three pyridine nitrogen atoms were 2.120(6), 2.135(7) and 2.110(6) A, , and the three N(pyridine)–Cu –N(pyridine) angles were 112.4(2), 125.1(2) and 115.6(3)°, respectively. In this complex, the hydroxide ligand surrounded by three neopentylamino groups of tnpa ligand was located at the axial position of the copper, in which the bond length between copper and oxygen atom was 1.895(4) A, . Tertiary amino nitrogen of tnpa was located at another axial position as shown in Fig. 1. Selected bond lengths and angles of the complexes are listed in Table 2. The ~ value as a structural index parameter, which is calculated from the angles around metal center in the crystal [26], indicates that [Cu(tnpa)(OH)]ClO4 (1, ~= 0.89) complex is almost trigonal bipyramidal. In the solution phase as well as crystal one, the electronic absorption spectrum of 1 in acetonitrile showed two broad d–d bands at 691 (m= 158) and 870 nm (m= 239) that is characteristic to a trigonal bipiramidal structure at the copper center.

Table 2 Selected bond lengths (A, ) and bond angles (°) for the complexes, 1, 2, 5 and 6 [Cu(tnpa)OH]+ (1)

[Cu2(tppen)(H2O)2]4+ (5)

[Cu(tapa)Cl]+ (2)

[Cu2(tppen)Cl4] (6)

Bond lengths Cu–O(1H) Cu–N(1) Cu–N(2A) Cu–N(2B) Cu–N(2C)

1.895(4) 2.003(6) 2.120(6) 2.135(7) 2.110(6)

Cu(1)–Cl Cu(1)–N(1) Cu(1)–N(2A) Cu(1)–N(2B) Cu(1)–N(2C)

2.261(3) 1.986(7) 2.184(9) 2.119(9) 2.165(8)

Cu(1)–O(1W) Cu(1)–O(1A) Cu(1)–N(1) Cu(1)–N(1A) Cu(1)–N(1B) Cu(2)–O(2W) Cu(2)–O(1C) Cu(2)–N(1C) Cu(2)–N(1D) Cu(2)–N(2)

2.158(8) 1.945(9) 2.016(9) 1.968(9) 2.01(1) 2.147(9) 1.919(8) 1.97(1) 1.99(1) 2.02(1)

Cu(1)–Cl(1) Cu(1)–Cl(2) Cu(1)–N(1) Cu(1)–N(2B) Cu(1)–N(2A)

2.391(4) 2.281(5) 2.09(1) 2.10(1) 2.09(1)

Bond angles O(1H)–Cu–N(1) O(1H)–Cu–N(2A) O(1H)–Cu–N(2B) O(1H)–Cu–N(2C) N(1)–Cu–N(2A) N(1)–Cu–N(2B) N(1)–Cu–N(2C) N(2A)–Cu–N(2B) N(2A)–Cu–N(2C) N(2B)–Cu–N(2C)

178.1(2) 97.9(2) 99.0(2) 99.6(4) 80.3(2) 82.1(2) 81.5(2) 112.4(2) 125.1(2) 115.6(3)

Cl–Cu(1)–N(1) Cl–Cu(1)–N(2A) Cl–Cu(1)–N(2B) Cl–Cu(1)–N(2C) N(1)–Cu(1)–N(2A) N(1)–Cu(1)–N(2B) N(1)–Cu(1)–N(2C) N(2A)–Cu(1)–N(2B) N(2A)–Cu(1)–N(2C) N(2B)–Cu(1)–N(2C)

178.5(3) 100.3(3) 100.3(2) 99.0(2) 79.3(4) 81.2(3) 80.0(3) 114.9(3) 119.2(3) 117.4(3)

O(1A)–Cu(1)–O(1W) O(1A)–Cu(1)–N(1) O(1A)–Cu(1)–N(1A) O(1A)–Cu(1)–N(1B) O(1W)–Cu(1)–N(1) O(1W)–Cu(1)–N(1A) O(1W)–Cu(1)–N(1B) N(1)–Cu(1)–N(1A) N(1)–Cu(1)–N(1B) N(1A)–Cu(1)–N(1B) O(1C)–Cu(2)–O(2W) O(1C)–Cu(2)–N(1C) O(1C)–Cu(2)–N(1D) O(1C)–Cu(2)–N(2) O(2W)–Cu(2)–N(1C) O(2W)–Cu(2)–N(1D) O(2W)–Cu(2)–N(2) N(1C)–Cu(2)–N(1D) N(1C)–Cu(2)–N(2) N(1D)–Cu(2)–N(2)

88.8(4) 170.4(4) 89.5(4) 97.9(4) 100.6(4) 117.2(4) 103.6(4) 84.7(4) 81.7(4) 138.7(4) 87.7(3) 90.6(4) 95.6(4) 169.0(4) 115.8(4) 102.3(4) 103.3(4) 141.6(4) 84.9(4) 81.7(4)

Cl(1)–Cu(1)–Cl(2) Cl(1)–Cu(1)–N(1) Cl(1)–Cu(1)–N(2B) Cl(1)–Cu(1)–N(2A) Cl(2)–Cu(1)–N(1) Cl(2)–Cu(1)–N(2B) Cl(2)–Cu(1)–N(2A) N(1)–Cu(1)–N(2B) N(1)–Cu(1)–N(2A) N(2B)–Cu(1)–N(2A)

108.3(1) 115.6(3) 96.2(3) 91.6(3) 136.1(3) 93.5(3) 95.9(3) 81.6(4) 83.7(4) 165.2(4)

K. Jitsukawa et al. / Inorganica Chimica Acta 324 (2001) 108–116

In the crystal of 1, notably, all the NH bond vectors of three neopentylamino groups were directed toward the hydroxyl oxygen atom and the distances between hydroxyl oxygen and amino nitrogen atoms, 2.80, 2.82 and 2.70 A, , were in the range of hydrogen bond distances. Previously, in the crystal structure of [Cu(bppa)OOH]ClO4, we reported that the 6-pivalamido groups of the tripodal polypyridine ligand acted as a hydrogen-bonding moiety to stabilize the hydroperoxide anion, in which the hydrogen bond distances between the amino nitrogens and the hydroperoxide oxygen bound to copper were 2.78 and 2.79 A, [20]. Stabilization of hydroxide ligand bound to coper center was also realized in the Co– OH complex with the tripodal C3symmetric hydrogen bonding cavity formed by urea derivatives, in which the distances between O(hydroxide) and N(urea) were 2.680 and 2.748 A, [27]. Hence, the amino groups of the tripodal ligands in 1 might stabilize oxygen species coordinating to the copper center through the hydrogen bonds. In order to increase the hydrogen-bonding ability, we prepared TAPA ligand containing 6-amino group instead of 6-pivalamido or 6-neopentylamino groups. The crystal structure of 2 (Fig. 2) and the selected bond lengths and angles around copper ion (Table 2) indicate that 2 has a trigonal bipyramidal structure with three pyridine nitrogens of TAPA in the equatorial plane and the tertiary amine nitrogen and chloride in the two axial positions. The bond lengths between the central copper and three N(pyridine) atoms in 2 were 2.184(9), 2.119(9) and 2.165(8) A, and the three N(pyridine)–Cu – N(pyridine) angles were 114.9(3), 119.2(3) and 117.4(3)°, respectively. A larger ~ value (~= 0.99) of 2 compared with 1 (~= 0.89) indicates that 2 is a typical trigonal bipyramidal structure. The chloride ligand located at the axial position of the copper was hydrogen-bonded with three amino groups of TAPA ligand with the distances of  2.57 A, . Unfortunately, we could not obtain the crystals of 3 containing hydoxide ligand, but judging from their absorption spectra and ESR parameter in solution phase, the solution structures of 2 and 3 are quite similar to each other. The electronic spectra of the copper complexes in acetonitrile solution showed two broad d–d bands at 770 (sh, m =113) and 955 nm (m= 281) for 2 and 663 (sh, m =135) and 798 nm (m =178) for 3, respectively, indicating that the two copper(II) complexes with TAPA ligand have a trigonal bipyramidal structure in solution phase. The frozen ESR spectrum measured for an aqueous solution of complex 2 also exhibited the characteristic pattern for a trigonal bipyramidal geometry (g =2.00, A =92 G and gÞ = 2.20, AÞ = 76 G) [28], in agreement with the X-ray structure shown in Fig. 2. That of 3 gave almost similar spectral pattern to 2 in spite of a mixture of several species more than two. Conclusively, the mononuclear

113

copper complexes with tripodal polypyridine ligands (tnpa and tapa) have substantially the five-coordinate trigonal bipyramidal structure in both crystal and solution phases. The mononuclear copper(II) complexes with tripodal polypyridine ligands formed five-coordinate trigonal bipyramidal structure as mentioned above, but the structure of complex 4 with bppa ligand reported previously was a square planar [21]. In the crystal structure of 4, one of three pyridine nitrogen atoms of bppa was apart from the central metal ion with the distance of 2.7 A, , although the others coordinated to the copper ion with the distances of 1.957(4) and 1.925(4) A, . In addition, the electronic spectrum of 4 showed a broad band at 734 nm (m= 155), suggesting that 4 forms a square planar geometry also in solution phase. On the basis of the clear ESR spectral parameters (g = 2.22, A =182 G and gÞ = 2.06), coordination geometry of 4 was judged to be square planar in aqueous phase. On the other hand, the crystal structure of 5 (Fig. 3) revealed that the coordination structures around the copper ions are in an intermediate of trigonal bipyramidal and square pyramidal geometries on the basis of their bond lengths and angles listed in Table 2. In 5 each copper ion was coordinated by one tertiary amine nitrogen, two pyridine nitrogens, and one pivalamido oxygen of the TPPEN ligand and a water molecule. As shown in Fig. 4, the structure of the complex 6, where each copper ion is coordinated by one tertiary amine nitrogen and two pyridine nitrogens of tppen and two chloride ions, revealed the intermediate geometry. These findings are also evidenced from the intermediate ~ values between typical trigonal-bipyramidal (~=1) and square-pyramidal structures (~= 0); ~=0.53, 0.46 for 5 and ~= 0.49 for 6, respectively. The electronic absorption spectra of the dinuclear copper complexes, 5 and 6, exhibited broad bands with shoulder peaks at the longer wavelength side in methanol or aqueous acetonitrile solution; 650 nm (m= 200) for 5 and 780 nm (m= 378) for 6, respectively. These findings suggest that the coordination structures of 5 and 6 are square pyramidal. Furthermore, the coordination geometry of 5 in aqueous solution was also judged to be square pyramidal on the basis of its ESR parameter (g = 2.41, A = 120 G and gÞ = 2.08). Incidentally, antiferromagnetic spin–spin coupling interaction was not observed in both 5 and 6, indicating that metal–metal interaction through the bridging ligand such as hydroxyl group is not present in these dinuclear complexes. Structural characterizations for the copper complexes as mentioned above are summarized in Table 3.

3.2. Electrochemical properties The redox potential of copper complexes is one of the important factors for the SOD activities. Cyclic

K. Jitsukawa et al. / Inorganica Chimica Acta 324 (2001) 108–116

114

Table 3 Summary of the structural data for the copper complexes (1–7) Complexes

UV–Vis spectra (nm) (m)

ESR spectra

~ value

Structure a

1

mixture of several species more than 2

0.89

TBP

g = 2.00, A =92 G gÞ =2.20, AÞ =76G mixture of several species more than 2

0.99

TBP

4

(158) 870 (239) (sh, 113) 995 (281) 663 (sh, 135) 798 (178) 734 (155)

5

650 (200)

6 7b

780 (378) 870 (213)

2 3

a b

(TBP)

g = 2.22, A =182 G gÞ =2.06 g = 2.41, A =120 G gÞ =2.08 mixture of several species more than 2 g = 2.00, A =63 G gÞ =2.20, AÞ =97G

SQ 0.53 0.46 0.49 0.97

TBP+SQP TBP+SQP TBP

TBP, trigonal bipyramidal structure; SQ, square planar structure; SQP, square pyramidal structure. Refs. [33,36].

voltammograms of the complexes 1 – 6 were examined in aqueous acetonitrile media under argon atmosphere. The mononuclear Cu(II) complexes 1 – 4, as listed in Table 4, showed quasi-reversible one-electron redox potentials with a pair of cathodic and anodic waves of the Cu(II)/Cu(I) couple, which are in the range between − 330 mV (vs. NHE at pH 7; O2/O2’−) and + 890 mV (vs. NHE at pH 7; O2’−/H2O2), indicating that they are in good range for superoxide dismutation. In the cases of the dinuclear complexes 5 and 6 those were also within the range suitable for superoxide dismutation, although they did not give anodic peak potentials. The redox potentials of 2 and 3 were quite different from each other, in spite of the similarity of their structures in both crystal and solution states. The same behavior was also observed between 5 and 6. The chloride species might exhibit higher potential in comparison with the other corresponding complexes and the hydroxide ones might show lower potential.

3.3. Correlation between the structures of copper complexes and their SOD acti6ities

In the view of the SOD activity, it is also important to examine the electrochemical properties of the copper complexes, because the native Cu,Zn– SOD exhibits the higher redox potential (+ 420 mV [31] or + 350 mV [32] vs. NHE at pH 7), which leads higher stability of Cu(I) state in the first step of the catalysis, as is understandable from Eq. (1). However, the redox potential (E1/2) of 2 (+ 267 mV), as listed in Table 4, is quite different from those of 1 (−28 mV) and 3(−68 mV), in spite of the higher structural similarity between them. Similar correlation in cathodic peaks appeared Table 4 Electrochemical data of the copper complexes (1–6) E1/2 a,b

−238 +192 −133 +57 +152

+182 +342 −3 +122 not identified

−28 +267 −68 +90 not identified

+302

not identified

not identified

Epc

[Cu(tnpa)(OH)]ClO4 (1) [Cu(tapa)Cl]ClO4 (2) [Cu(tapa)(OH)]ClO4 (3) [Cu(bppa)]ClO2 (4) [Cu2(tppen)(H2O)2](ClO4)4 (5) [Cu2(tppen)Cl4] (6) a

The role of the positively charged Arg 141 residue in the bovine Cu,Zn –SOD has been considered to assist the attraction of a negatively charged superoxide anion to the copper center in the catalytic cycle [29,30]. In the crystal structure of the resting state of the enzyme reaction, a water molecule coordinates to copper [11], which probably indicates that guanidinium group of the arginine residue acts as a hydrogen bonding moiety to stabilize dioxygen species bound to the copper center. Therefore, we tried to examine the disproportionation activities for superoxide anion using the mono and dinuclear copper complexes (1 – 6) with hydrogen bonding moiety.

Epa a

Complexes

b

a

vs. NHE. E1/2 =(Epa+Epc)/2.

Table 5 IC50 values of various copper complexes (1–7) Run

Complexes

IC50

1 2 3 4 5 6 7 8

[Cu(tnpa)OH]ClO4 [Cu(tapa)Cl]ClO4 [Cu(tapa)OH]ClO4 [Cu(bppa)](ClO4)2 [Cu2(tppen)(H2O)2](ClO4)4 [Cu2(tppen)Cl4] [Cu(tpa)(H2O)](ClO4)2 Cu, Zn–SOD

11.03 91.72 5.02 9 0.87 7.46 9 0.97 140.0 915.0 0.76 90.29 0.549 0.10 12.50 90.22 2.819 0.04

mM mM mM mM mM mM mM nM

K. Jitsukawa et al. / Inorganica Chimica Acta 324 (2001) 108–116

also in the cases of the complexes 5 ( + 152 mV) and 6 ( + 302 mV). As described below (Table 5), the SOD activities of complexes 1 and 7, complexes 2 and 3, and complexes 5 and 6, demonstrated almost similar values, respectively [33]. However, the respective redox potentials are rather different. These facts indicate that the redox potentials in the Cu(II) complexes employed here are not essentially associated with their SOD activities. The assay data for the SOD activities by the complexes 1–7, which were examined by following the reduction rate of cytochrome c by superoxide generated from xanthine oxidase [4], are given in Table 5. Complexes 1–3 demonstrated moderate activities (IC50 values; 5–11 mM), which are slightly high in comparison with the other mononuclear copper complexes reported previously [2,14,34], although all these values are considerably lower than that in native SOD (IC50 values; 2.81 nM). Interestingly, the IC50 values of these complexes are significantly high in comparison with that of 7 without hydrogen bonding site, although it also forms a trigonal bipyramidal structure in both solution and solid states [25,33], as well as complexes 1, 2 and 3. This finding indicates that the Cu(II) complexes with the hydrogen bonding moiety such as 6-neopentylamino and 6-amino groups may accelerate the SOD activity. The hydrogen bonding interaction site may assist the induction of the superoxide ion to copper ion, as the Arg141 residue near the copper site in the native bovine SOD. On the other hand, the [Cu(bppa)](ClO4)2 complex 4, having hydrogen bonding site, showed quite low dismutation activity (IC50 values; 140 mM). The mononuclear complexes, 1, 2 and 3, that indicate considerably higher activity are all five-coordinate trigonal bipyramidal, whereas only the complex 4 is four-coordinate square planar [21]. Similar behavior has been observed in the four-coordinate square planar Cu(II) complex [14,15]. The geometry around the copper site in native Cu,Zn– SOD [9] is rather distorted tetrahedron with a vacant site for binding of superoxide, in which the vacant site has been transiently occupied by labile H2O in the crystal structure [35]. Therefore, the distorted structure around the copper center is quite important for the SOD activity. This may be explained in terms of the tight square planar geometry, having been forced by the stronger coordination, would prevent the attack of superoxide anion to the metal ion and the reduction process from Cu(II) to Cu(I). Interestingly, as shown in Table 5, the SOD activities of the dinuclear Cu(II) complexes, 5 and 6, are tenfold higher than those of the mononuclear complexes, 1, 2 and 3. Such a behavior is consistent with previous reports of the investigation of the use of homo or hetero dinuclear metal complexes [9,10]. Although the metal–metal interaction, as is clear from the results of the above ESR studies, is not observed in 5 and 6, the

115

geometries around the Cu(II) ions were a mixture of five-coordinate trigonal bipyramidal and square pyramidal. The above findings indicate that the flexibility in the copper coordination geometries and dinuclear center could accelerate the catalytic cycle, and the dual metal center formed with transition metal ions and multidentate ligand can function both of the dioxygen binding and electron transfer sites in this cycle. In conclusion, we designed and prepared some copper complexes with tetra-coordinate tris(2-pyridylmethyl)amine derivatives containing pivalamido, neopentylamino, or amino substitutents at pyridine sixposition in order to examine the effect of hydrogen bonding interaction, and they have been characterized by the use of electronic and ESR spectroscopic and electrochemical methods and X-ray structural analysis in both solution and solid states. It has become apparent that the SOD activity depends strongly on the geometry around the copper ion rather than the redox potentials; the activity decreases in the order of structurally flexible five-coordinate geometry (a mixture of trigonal bipyramidal and square pyramidal)\ five-coordinate trigonal bipyramidal four-coordinate square planar. The Cu(II) complexes with a trigonal bipyramidal geometry and hydrogen bonding interaction site, 1, 2 and 3, exhibited significantly higher activity in comparison with the corresponding Cu(II) complex without hydrogen bonding group, indicating that the hydrogen bond may promote the SOD activity.

Acknowledgements This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (11228203), to which our thanks are due.

References [1] (a) J.H. Dawson, Science 240 (1988) 433; (b) E.I. Solomon, M.D. Lowery, Science 259 (1993) 1575; (c) M. Momenteau, C.A. Reed, Chem. Rev. 94 (1994) 659; (d) N. Kitajima, Y. Moro-oka, Chem. Rev. 94 (1994) 737; (e) A.L. Feig, S.J. Lippard, Chem. Rev. 94 (1994) 759; (f) E.I. Solomon, F. Tuczek, D.E. Root, C.A. Brown, Chem. Rev. 94 (1994) 827; (g) J.S. Valentine, C.S. Foote, A. Greenberg, J.F. Liebman (Eds.), Active Oxygen in Biochemistry, Blackie Academic & Professional, Chapman & Hall, Glasgow, 1995; (h) H.H. Thorp, V.L. Pecoraro (Eds.), Mechanistic Bioinorganic Chemistry, American Chemical Society, Washington, DC, 1995; (i) J.P. Klinman, Chem. Rev. 96 (1996) 2541. [2] J.R.J. Sorenson, Prog. Med. Chem. 26 (1989) 437. [3] (a) I. Fridovich, J. Biol. Chem. 264 (1989) 7761; (b) I. Fridovich, Annu. Rev. Biochem. 64 (1995) 97. [4] (a) J.M. McCord, I. Fridovich, J. Biol. Chem. 244 (1969) 6049; (b) C. Beauchamp, I. Fridovich, Anal. Biochem. 44 (1971) 276.

116

K. Jitsukawa et al. / Inorganica Chimica Acta 324 (2001) 108–116

[5] (a) D.P. Riley, R.H. Weiss, Cattech 1 (1997) 42; (b) D.P. Riley, Chem. Rev. 99 (1999) 2573; (c) D. Salvemini, Z.-Q. Wang, J.L. Zweier, A. Samouilov, H. Macarthur, T.P. Misko, M.G. Currie, S. Cuzzocrea, J.A. Sikorski, D.P. Riley, Science 286 (1999) 304. [6] (a) D.P. Riley, R.H. Weiss, J. Am. Chem. Soc. 116 (1994) 387; (b) D.P. Riley, P.L. Lennon, W.L. Neumann, R.H. Weiss, J. Am. Chem. Soc. 119 (1997) 6522. [7] D.-L. Zhang, D.H. Busch, P.L. Lennon, R.G. Weiss, W.L. Neumann, D.P. Riley, Inorg. Chem. 37 (1998) 956. [8] (a) Y. Mori, M. Sato, T. Iida, Chem. Lett. (1992) 469; (b) M. Nakagawa, K. Hanaoka, A. Terasaki, Y. Mori, H. Hokazono, M. Sato, T. Iida, Chem. Lett. (1994) 1721. [9] (a) For hetero dinuclear (Cu –Zn) complexes: J.-L. Pierre, P. Chautemps, S.M. Rafaif, C.G. Beguin, A. El-Marzouki, G. Serratrice, P. Rey, J. Laugier, J. Chem. Soc., Chem. Commun. (1994) 1117; (b) J.-L. Pierre, P. Chautemps, S.M. Rafaif, C.G. Beguin, A. El-Marzouki, G. Serratrice, E. Saint-Aman, P. Rey, J. Am. Chem. Soc. 117 (1995) 1965; (c) Z.-W. Mao, M.-Q. Chen, X.-S. Tan, J. Liu, W.-X. Tang, Inorg. Chem. 34 (1995) 2889; (d) H. Ohtsu, Y. Shimazaki, A. Odani, O. Yamauchi, Chem. Commun. (1999) 2393; (e) H. Ohtsu, Y. Shimazaki, A. Odani, O. Yamauchi, W. Mori, S. Itoh, S. Fukuzumi, J. Am. Chem. Soc. 122 (2000) 5733. [10] (a) For homo dinuclear (Cu –Cu) complexes G. Tabbi, W.L. Driessen, J. Reedijk, R.P. Bonomo, N. Veldman, A.L. Spek, Inorg. Chem. 36 (1997) 1168; (b) J.-J. Zhang, Q.-H. Luo, D.-L. Lomg, J.-T. Chen, F.-M. Li, A.-D. Liu, J. Chem. Soc., Dalton. Trans. (2000) 1893. [11] J.A. Tainer, E.D. Getzoff, J.S. Richardson, D.C. Richardson, Nature 306 (1983) 284. [12] C. Beauchamp, I. Fridovich, Anal. Biochem. 44 (1971) 276. [13] K. Wada, Y. Fujibayashi, N. Tajima, A. Yokoyama, Biol. Pharm. Bull. 17 (1994) 701. [14] (a) E. Kimura, A. Sakonaka, M. Nakamoto, Biochem. Biophys. Acta 678 (1981) 172; (b) E. Kimura, A. Yatsunami, A. Watanabe, R. Machida, T. Koike, H. Fujioka, Y. Kuramoto, M. Sumomogi, H. Kunimitsu, A. Yamashita, Biochem. Biophys. Acta 745 (1983) 37; (c) E. Kimura, A. Yatsunami, Tanpakushitsu Kakusan Kouso Bessatsu 26 (1983) 177.

[15] E. Kimura, T. Koike, Y. Shimizu, M. Kodama, Inorg. Chem. 25 (1986) 2242. [16] M. Harata, K. Jitsukawa, H. Masuda, H. Einaga, J. Am. Chem. Soc. 116 (1994) 10817. [17] M. Harata, K. Jitsukawa, H. Masuda, H. Einaga, Chem. Lett. (1995) 61. [18] M. Harata, K. Jitsukawa, H. Masuda, H. Einaga, Chem. Lett. (1996) 813. [19] M. Harata, K. Jitsukawa, H. Masuda, H. Einaga, Bull. Chem. Soc. Jpn. 71 (1998) 637. [20] A. Wada, M. Harata, K. Hasegawa, K. Jitsukawa, H. Masuda, M. Mukai, T. Kitagawa, H. Einaga, Angew. Chem., Int. Ed. Engl. 37 (1998) 798. [21] M. Harata, K. Hasegawa, K. Jitsukawa, H. Masuda, H. Einaga, Bull. Chem. Soc. Jpn. 71 (1998) 1031. [22] S. Ogo, S. Wada, Y. Watanabe, M. Iwase, A. Wada, M. Harata, K. Jitsukawa, H. Masuda, H. Einaga, Angew. Chem., Int. Ed. Engl. 37 (1998) 2101. [23] A. Wada, S. Ogo, Y. Watanabe, M. Mukai, T. Kitagawa, K. Jitsukawa, H. Masuda, H. Einaga, Inorg. Chem. 38 (1999) 3592. [24] J. O’M. Bckris, A.K.N. Reddy, Modern Electrochemistry, Plenum Press, New York, 1970. [25] K.D. Karlin, J.C. Hayes, S. Juen, J.P. Hutchinson, J. Zubieta, Inorg. Chem. 21 (1982) 4106. [26] A.W. Addison, T.N. Rao, J. Reedijik, J. Rijn, G.C. Verschoor, J. Chem. Soc., Dalton Trans. (1984) 1349. [27] B.S. Hammes, V.G. Young Jr, A.S. Borovik, Angew. Chem., Int. Ed. Engl. 38 (1999) 666. [28] B.J. Hathaway, in: G. Wilkinson (Ed.), Comprehensive Coordination Chemistry, vol. 5, Pergamon Press, Oxford, 1987, p. 533. [29] E.D. Getzoff, J.A. Tainer, P.K. Weiner, P.A. Kollman, J.S. Richardson, D.C. Richardson, Nature 306 (1983) 287. [30] W.E. Baeyer, I. Fridovich, G.T. Mullenbach, R. Hallewell, J. Biol. Chem. 262 (1987) 11182. [31] H.A. Free, P.E. DiCorlete, Biochemistry 12 (1973) 4893. [32] G.D. Lawrence, D.T. Sawyer, Biochemistry 18 (1979) 3045. [33] H. Nagao, N. Komeda, M. Mukaida, M. Suzuki, K. Tanaka, Inorg. Chem. 35 (1996) 6809. [34] M. Tamura, T. Nagano, Kikan Kagaku Sosetsu 27 (1995) 183. [35] W. Kaim, B. Schwederski (Eds.), Bioinorganic Chemistry: Inorganic Elements in the Chemistry of Life, Wiley, Chichester, 1994, p. 209. [36] R.R. Jacobson, Z. Tyeklar, K.D. Karlin, J. Zubieta, Inorg. Chem. 30 (1991) 2035.