Inorganica Chimica Acta 339 (2002) 373 /382 www.elsevier.com/locate/ica
Copper(II), nickel(II) and zinc(II) complexes of amino acids containing bis(imidazol-2-yl)methyl residues ˝ sz a, Katalin Va´rnagy a, Helga Su¨li-Vargha b, Daniele Sanna c, Katalin O Giovanni Micera d, Imre So´va´go´ a,* a
Department of Inorganic and Analytical Chemistry, University of Debrecen, H-4010 Debrecen, Hungary b Research Group of Peptide Chemistry, Hungarian Academy of Sciences, H-1518 Budapest, Hungary c Istituto C.N.R. per l’Applicazione delle Tecniche Chimiche Avanzate ai Problemi Agrobiologici, Via Vienna 2, I-07100 Sassari, Italy d Department of Chemistry, University of Sassari, Via Vienna 2, I-07100 Sassari, Italy Received 26 November 2001; accepted 8 February 2002 Dedicated in honor of Professor Helmut Sigel
Abstract Copper(II), nickel(II) and zinc(II) complexes of Phe-BIMA and His-BIMA were studied by potentiometric, UV /Vis and EPR spectroscopic methods. The nitrogen donor atoms of the bis(imidazol-2-yl)methyl residues were described as the primary metal binding sites in all systems studied. Deprotonation and coordination of the terminal amino and amide nitrogen atoms took place in the copper(II) complexes of both ligands and resulted in the formation of dinuclear complexes ([Cu2H 2L2]2 ) containing [NH2, N , N(Im)] tridentate ligands and imidazole bridging. The presence of the histidyl side chain provides a great versatility in the complex formation reactions of His-BIMA. The existence of the species [Cu2L2]4 was detected in slightly acidic solution and its structure was described as a mixture of three isomeric forms. Deprotonation of the imidazole-N(1)H donor functions was detected under slightly alkaline conditions with pK values of 8.13 and 8.93. An excess of copper(II) ions shifted this reactions even into the slightly acidic pH range and resulted in the formation of a trinuclear complex ([Cu3H 4L2]2). # 2002 Published by Elsevier Science B.V. Keywords: Transition metal ion; Histidine; Bis(imidazolyl); EPR; Potentiometry; Stability constants
1. Introduction Imidazole nitrogen donor atoms of histidyl residues are among the most common metal binding sites of metalloproteins. As a consequence, a huge number of peptides and model compounds have been synthesised to mimic the binding properties and catalytic activities of imidazole nitrogen donor atoms in different environments. The results obtained for the metal complexes of the most common peptides containing histidine have already been reviewed [1,2] and the data provide an unambiguous proof for the outstanding metal binding ability of histidyl residues. The importance of the X /Y /
* Corresponding author. Tel.: /36-52-316 666; fax: /36-52-489 667. E-mail address:
[email protected] (I. So´va´go´).
His- amino acid sequence at the N-termini of peptides (ATCUN motif) is especially well documented [1,3]. The most recent studies on the metal complexes of Nprotected peptides revealed the anchoring ability of imidazole residues [4,5], while the formation of various macrochelates was reported for the metal complexes of oligopeptides containing histidyl residues at the Ctermini [6]. The great variety in the coordination chemistry of these peptides provides a reliable base to mimic the binding properties of metalloenzymes including superoxide dismutase (SOD) [7]. Another group of enzyme models contains more imidazole residues linked via various carbon chains [8 /12]. Derivatives of bis(imidazol-2-yl)methane (BIM) in which the imidazole rings are linked via a single tetrahedral carbon atom are the most simple representatives of the polyimidazole ligands. BIM was reported to be a very effective complexing agent for a great
0020-1693/02/$ - see front matter # 2002 Published by Elsevier Science B.V. PII: S 0 0 2 0 - 1 6 9 3 ( 0 2 ) 0 0 9 5 4 - 4
374
˝ sz et al. / Inorganica Chimica Acta 339 (2002) 373 /382 K. O
variety of transition elements forming stable six-membered chelates via the coordination of the imidazole nitrogen atoms [13 /16]. The coordination chemistry of the ligands containing two imidazole rings is more versatile when the chelating nitrogen donors are linked to other chelating ligands creating multi- and/or ambidentate ligands [10,17]. In our previous papers, we reported the results on the metal complexes of multidentate ligands containing the bis(imidazol-2-yl)methyl residues (later abbreviated as bis(imidazolyl) residues) at the C- or N-termini of amino acids or peptides [18 /22]. It is obvious from these studies that the imidazole nitrogen donor atoms are the primary metal binding sites in the copper(II), nickel(II) and zinc(II) complexes of these molecules. It was also proved that in the absence of a terminal amino group the peptide backbone could not compete with the chelating side chains [18]. The presence of histidyl residues resulted in further increase in the thermodynamic stability of metal complexes, but without a significant change in the metal binding sites [20]. The results obtained on the metal complexes of Gly-BIMA containing an unprotected amino group revealed that the amino group has a significant impact on the coordination properties of the bis(imidazolyl) ligands [19]. A very stable dinuclear complex was obtained in equimolar solutions of copper(II) and Gly-BIMA, in which all metal ions were coordinated by three N donors including amino, amide and one of the imidazole in two linked chelates and the metal centres were joined via imidazole bridging [19 /21]. Now in this paper, we report the results of combined potentiometric and spectroscopic (EPR and UV /Vis) studies on the copper(II), nickel(II) and zinc(II) complexes of N -phenylalanylbis(imidazol-2-yl)methylamine (Phe-BIMA) and N -histidylbis(imidazol-2-yl)methylamine (His-BIMA) (see Scheme 1). The donor groups of Phe-BIMA are exactly the same as those of GlyBIMA, but the former contains a bulky phenyl residue. His-BIMA is also a bulky ligand, but it is more important that it contains a third imidazole residue in chelating position with the terminal amino group.
Scheme 1.
2.2. Synthesis Merck Kieselgel-precoated sheets number 5553 were used for thin layer chromatography (TLC) with eluents: ethyl acetate: pyridine: acetic acid: water, 120:20:6:11 (A), ethyl acetate: pyridine: acetic acid: water, 60:20:6:11 (B). For staining the chromatograms beside ninhydrin the chlorine /tolidine reaction [23] has been used, slightly modified for TLC: the plates developed with ninhydrin, then chlorinated for 1 min and aerated, were sprayed with the following reagent: 2 g of tolidine dissolved in 5 cm3 warm acetic acid were diluted with 100 cm3 of water, then 100 cm3 of 0.05 mol dm 3 KI solution and 100 cm3 ethanol were added. Highperformance liquid chromatography (HPLC) was performed on a Knauer instrument using analytical Vydac C18 column for analysis (detection at 220 nm). NMR spectra were recorded on a Bruker DRX equipment. 2.2.1. Synthesis of H-Phe-BIMA ×/3HCl
2. Experimental
2.1. Materials CuCl2, NiCl2, ZnCl2, KCl, KOH (Reanal) were purchased from commercial sources, and used without further purification. Aqueous metal ion stock solutions were prepared from analytical grade reagents and their concentration was checked gravimetrically via oxinates.
2.2.1.1. Z-Phe-BIMA. Z-Phe-OPcp (Z, benzyloxycarbonyl, Pcp, pentachlorophenyl) (1.1 g, 2 mmol) was dissolved in dimethylformamide (DMF) (5 cm3) and BIMA ×/3HCl (545 mg, 2 mmol) and triethylamine (TEA) (0.84 cm3, 6 mmol) were added under stirring at room temperature (r.t.). Stirring was continued overnight, then the reaction mixture was evaporated in vacuo, and the residue was triturated with ether and filtered. Yield: 650 mg (73%). The crude product was dissolved in warm ethanol and precipitated with water. After filtration, homogenous Z-Phe-BIMA, Rf 0.66 (A) was
˝ sz et al. / Inorganica Chimica Acta 339 (2002) 373 /382 K. O
obtained. Z-Phe-BIMA (445 mg, 1 mmol) was dissolved in 1 mol dm 3 HCl (4 cm3) and hydrogenated in the presence of 10% Pd/charcoal catalyst (50 mg) at r.t. for 2 h. The catalyst was filtered off, the filtrate was evaporated in vacuo, dissolved in water and lyophilised resulting in the title compound with quantitative yield; homogenous according TLC, Rf 0.35 (B), and HPLC analysis (water as an eluent). NMR: 13 C (DMSO), d 37.57 (methylene), 45.77 (Phemethyne), 54.46 (methyne), 122.37 (Im-5), 122.61 (Im4), 127.99 (Aryl-4), 129.37 (Aryl-2), 130.28 (Aryl-3), 135.66 (Aryl-1), 144.19, 143.77 (Im-2, prochiral), 169.22 (carbonyl); 1H (DMSO), d 4.14 (1H, t, Phe-methyne), 6.41 (1H, s, methyne), 2.92, 3.20 (2H, m, CH2), 7.20 / 7.40 (9H). 2.2.2. Synthesis of H-His-BIMA ×/4HCl 2.2.2.1. Z-His-BIMA. The solution of Z-His-N2H3 (0.91 g, 3 mmol, in 8 cm3 DMF) was cooled to /5 8C. At this temperature azeotropic HCl solution (1.6 cm3, 9 mmol) and NaNO2 (231 mg, 3.3 mmol) were added, and the reaction mixture was stirred for 20 min. This reaction mixture was poured into the solution of BIMA ×/3HCl (0.82 g, 3 mmol) and of TEA (2.52 cm3, 18 mmol) in DMF (8 cm3) at /5 8C. The pH was adjusted to 8 with TEA, and it was stirred for 1 h at /5 8C and then let it stand overnight in refrigerator. The precipitated TEA ×/HCl was filtered off, the remaining solution was evaporated in vacuo, the residue was triturated with saturated NaHCO3 solution and filtered. The row product (1.216 g) was crystallised from 50% aqueous ethanol to give Z-His-BIMA 0.886 g (68%), Rf 0.27 (B). Z-His-BIMA (0.434 g, 1 mmol) was dissolved in 1 mol dm 3 HCl (4 cm3) and hydrogenated in the presence of 10% Pd/charcoal catalyst (50 mg) at r.t. for 4 h. The catalyst was filtered off, the filtrate was evaporated in vacuo, the residue was dissolved in water and lyophilised to give the title compound, H-His-BIMA ×/4HCl, in quantitative yield showing a single peak in HPLC analysis using water as eluent. NMR: 13C (DMSO), d 26.2 (methylene), 43.5 (His-methyne), 52.4 (methyne), 118.7 (His-Im-5), 121.4 (Im-5), 121.7 (Im-4), 125.9 (His-Im-4), 135,0 (HisIm-2), 139.3 (Im-2), 169.0 (carbonyl); 1H (DMSO), d 3.40 (2H, m), 4.45 (1H, t, His-methyne), 7.00 (1H, s, methyne), 7.23 (1H, s, His-Im-5), 7.49 (1H, s, Im-4), 7.50 (1H, s, Im-5), 8.62 (1H, s, His-Im-2), 14.4 (1H, amideNH). 2.3. Potentiometric studies The pH-potentiometric titrations in the pH range 2.2 /11.0 were performed in aqueous solutions on 3 /10
375
cm3 samples in the concentration range 2 /8 /10 3 mol dm 3 at metal ion to ligand ratios between 3:1 and 1:3. The number of experimental points reached around 50/ 70 data (cm3, pH) for each titration curve. Argon was bubbled through the samples to ensure the absence of oxygen and for stirring the solutions. All pH-potentiometric measurements were carried out at 298 K and at a constant ionic strength of 0.2 mol dm 3 KCl. The measurements were carried out with a Radiometer pHM 84 pH-meter equipped with a 6.0234.100 combination glass electrode (Metrohm) and a Dosimat 715 automatic burette (Metrohm) containing carbonate-free potassium hydroxide in known concentration. The pH-readings were converted to hydrogen ion concentration as described earlier [24]. Protonation constants of the ligands and the overall stability constants (log bpqr ) of the various species ([Mp Hq Lr ]) were calculated by means of a general computational program, PSEQUAD [25], using eqn. (1) and (2). pMqHrL XMp Hq Lr p
q
(1) r
b [Mp Hq Lr ]=[M] [H] [L]
(2)
2.4. Spectroscopic studies UV /Vis spectra of the copper(II) and nickel(II) complexes were recorded on Hewlett Packard HP 8453 or JASCO UVIDEC-610 spectrophotometers in the same concentration range as used for the potentiometry. Anisotropic X-band EPR spectra (9.15 GHz) of frozen solutions were recorded at 120 K, using a Varian E-9 spectrometer after addition of 10% ethylene glycol to ensure good glass formation in frozen solutions. Copper(II) stock solutions for EPR measurements were prepared from CuSO4 ×/5H2O enriched for 63Cu to get better resolution of EPR spectra. For this purposes metallic copper (99.3% 63Cu and 0.7% 65Cu) was purchased from JV Isoflex, Moscow, Russia and converted to sulphate. Table 1 Protonation constants (log bpqr ) and pK values of Phe-BIMA, HisBIMA and Gly-BIMA [T 298 K; I 0.2 mol dm 3 (KCl)] Species
[HL] [H2L]2 [H3L]3 [H4L]4 pK1 pK2 pK3 pK4 a
Gly-BIMA
a
7.95 13.46 16.68 7.95 5.51 3.22
Data from ref. [19].
Phe-BIMA
His-BIMA
Protonation site
7.17(1) 12.45(1) 15.54(1)
7.28(1) 13.09(2) 17.62(2) 20.23(4) 7.28 5.81 4.53 2.61
NH2 N(Im) N(Im) N(Im)
7.17 5.28 3.09
˝ sz et al. / Inorganica Chimica Acta 339 (2002) 373 /382 K. O
376
3. Results and discussion 3.1. Protonation equilibria of the ligands Protonation constants and pK values of the ligands Phe-BIMA and His-BIMA are collected in Table 1 and the parameters of Gly-BIMA are also shown for comparison. It is clear from Table 1 that the presence of the bulky phenyl or imidazolyl side chains results in some decrease in the basicity of the amino groups, similarly to those of simple amino acids [26]. On the other hand, the pK values of the bis(imidazolyl) nitrogen donors of PheBIMA and Gly-BIMA are very similar to each other and to those of simple tripeptides containing the bis(imidazolyl) residues at the C-termini [18,19]. HisBIMA has four nitrogen donor atoms, of which the protonation equilibria significantly overlap. Comparison of the data in Table 1, however, supports that amino and histidyl imidazole nitrogen donors have the highest basicities. 3.2. Metal complexes of Phe-BIMA Stability constants of the metal complexes of PheBIMA are included in Table 2, and the data reveal that the complex formation processes of Phe-BIMA are rather similar to those of Gly-BIMA [19] containing the same set of donor groups. Namely, the species [MHL]3 and [MH2L2]4 are present in acidic solutions containing six-membered chelate rings via the coordination of the bis(imidazolyl) residues, while the amino groups remain protonated at low pH values. The EPR parameters of these species are as follows: gjj /2.303 and 2.235, Ajj /166 and 192 (10 4 cm 1), for [CuHL]3 and [CuH2L2]4, respectively. The gjj values are almost the same as those reported for the corresponding species of Gly-BIMA
[19], but the coupling constants (Ajj) are slightly smaller. The small differences in the Ajj values, however, can be explained by the use of different stock solution in the present (63Cu) and in the previous EPR measurements (mixture of isotopes [19]). The comparison of the stepwise stability constants reveals some differences in the thermodynamic stability of copper(II) complexes of Gly-BIMA and Phe-BIMA. In the case of the copper(II) complexes the ratio of the stepwise stability constants log(K1/K2)H are smaller for Phe-BIMA, which reflects the increased stability of bis(ligand) complex formation with this ligand. Similar observations have already been reported for phenylalanine itself and it can be explained by a weak hydrophobic or stacking interaction between the aromatic side chains [26,27]. A new base consuming process starts above pH 5, which corresponds to the titration of 2 equiv. of base resulting the dinuclear species [Cu2H 2L2]2. The EPR spectrum of the species formed at pH 7 is almost exactly the same as reported for Gly-BIMA and the metal binding sites of this species can be described by the tridentate [NH2,N ,N(Im)]-coordination of each ligand with imidazole bridging as shown by Scheme 2. The EPR spectrum of the [Cu2H 2L2]2 species of Phe-BIMA makes it possible to calculate the approximate Cu /Cu distances in the dinuclear complexes [28]. On the low field side of the EPR spectra there is a partially resolved seven-line structure assigned to the parallel transitions. In the DM /1 region there are also two very strong peaks at both sides of g /2, which should be assigned to the perpendicular features. The parameters g /2.057 and D /0.044 cm 1 can be calculated from the perpendicular signals, which provide a value of 393 pm for the Cu /Cu distance in the dinuclear complex of Phe-BIMA. This value is very close to that reported for the Cu /Cu distance in the
Table 2 Stability constants (log bpqr ) of the metal complexes of Phe-BIMA [T 298 K; I 0.2 mol dm 3 (KCl)] Species [MH2L2]4 [MHL2]3 [ML2]2 [MH 1L2] [MHL]3 [MH 1L] [M2H 2L2]2 [MH 2L] log(K1/K2)H a pK1complex b pK2complex b
Cu(II) 28.89(8)
15.44(8) 14.94(16) 2.41(8) 1.99
Ni(II)
Zn(II)
26.04(1) 20.33(4) 13.70(4) 5.90(7) 13.48(1) 0.01(4)
22.60(4)
0.89 5.79 6.44
11.78(1)
0.96
a
log(K1/K2)H 2 log b111 log b122. pKi complex values correspond to the formation of the complex CuH(2 i )L2 where i 1, 2. b
Scheme 2.
˝ sz et al. / Inorganica Chimica Acta 339 (2002) 373 /382 K. O
dinuclear complex of GlyBIMA (390 pm) suggesting that the presence of the bulky phenyl side chains does not have any influence on the dinuclear complex formation. In the presence of excess ligand, however, quite significant differences can be observed in the complex formation processes of Gly-BIMA and Phe-BIMA. The formation of the species [CuL2]2 and [CuH 1L2] was detected in the copper(II) /Gly-BIMA system and their binding sites were described by tridentate [NH2,N ,N(Im)]-coordination of one ligand and monodentate [NH2 or N(Im)]-coordination of the other one. Neither potentiometric nor spectroscopic data, however, provide sufficient proof for the existence of bis(ligand) complexes in the copper(II) /Phe-BIMA system at high pH values. The formation of the dinuclear complex is exclusive at any metal ion to ligand ratios and it probably can be explained by steric requirements caused by the bulky phenyl residues. The base consuming process above pH 8 can be attributed to hydroxo complex formation both in copper(II) /Gly-BIMA and Phe-BIMA systems. In the case of Gly-BIMA polynuclear complexes are formed under mildly alkaline conditions [19]. The EPR spectra of the copper(II) /Phe-BIMA system at pH /8, however, definitely indicate the existence of a mononuclear complex for which the parameters gjj /2.221 and Ajj / 171 /10 4 cm 1 can be calculated. The relatively low value for the coupling constant suggests a tetrahedral distortion in the coordination sphere of the central metal ion, which can be attributed a hydrophobic or stacking interaction between the phenyl and imidazole side chains. It can be seen from Table 2 that complex formation reactions of nickel(II) and zinc(II) with Phe-BIMA are not so complicated. The nitrogen atoms from the bis(imidazolyl) residues are the primary metal binding sites with these metal ions too, and the values of stability constants obey the Irving /Williams order: Cu(II) / Ni(II) /Zn(II). The major difference between copper(II) and the other two metal complexes comes from the involvement of amide nitrogen in metal binding. Namely, the deprotonation and coordination of the amide functions take place only in the copper(II) complexes. In the case of nickel(II) and especially the zinc(II) complexes precipitation of bis(ligand) or mixed hydroxo complexes rules out solution studies above pH 8. As a consequence, the amide nitrogen of Phe-BIMA cannot be considered as a potential binding site of nickel(II) and zinc(II) in aqueous solution and in the physiological pH range. 3.3. Metal complexes of His-BIMA His-BIMA is an ambidentate ligand which is suitable for several different types of coordination: (i) six-
377
membered chelate via the N-termini (histamine-like coordination), (ii) six-membered chelate via the nitrogen donors of bis(imidazolyl) residue at the C-termini and (iii) tridentate coordination via the terminal amino, deprotonated amide and one of the imidazole nitrogen donors in the form of two, linked five-membered chelates. Moreover, the last type of coordination makes the dinuclear complex formation possible in which either the histidyl or imidazolyl nitrogen atoms can be the bridging residues. Another set of dinuclear complexes can be obtained by the simultaneous coordination of the N- and C-termini to two different metal ions. As a consequence, the metal ion speciation in the His-BIMA system is rather complicated as it is represented by Table 3 containing the stability constants of the metal complexes of His-BIMA and by Fig. 1 illustrating the concentration distribution in the copper(II) /HisBIMA system at three different metal ion to ligand ratios. Complex formation processes of His-BIMA start in very acidic solutions (below pH 2), which strongly supports that the bis(imidazolyl) residue is the primary metal binding site. The N-terminus of the molecule corresponds to the binding sites of histidine (or histamine) which are very effective metal binding sites, but complex formation of copper(II) with these ligands generally starts above pH 3 [29]. UV /Vis and EPR Table 3 Stability constants (log bpqr ) of the metal complexes of His-BIMA [T 298 K; I 0.2 mol dm 3 (KCl)] Species [MH4L2]6 [MH3L2]5 [MH2L2]4 [MHL2]3 [ML2]2 [MH 1L2] [MH2L]4 [MHL]3 [M2L2]4 [M2H 1L2]3 [M2H 2L2]2 [M2H 3L2] [M2H 4L2] [ML]2 [MH 1L] [MH 2L] [M2L]4 [M3H 4L2]2 [M2H 2L]2 pK1 complex a pK2complex a pK3complex a pK4complex a pK4complex a
Cu(II)
Ni(II)
37.20(5) 33.12(9) 28.51(9) 22.66(13) 16.15(14) 8.81(15) 19.29(9)
35.62(6) 31.63(7) 26.79(7) 20.83(11) 13.90(10) 5.20(12) 18.42(1)
25.68(4) 19.75(13) 13.58(8) 5.45(20) 3.48(13)
21.77(4)
15.36(4) 8.53(8) 4.08 4.61 5.85 6.51 7.34
1.49(3) 7.70(4) 12.62(3)
4.00 4.84 5.96 6.93 8.70
Zn(II)
28.08(7) 22.79(7) 16.85(7) 9.97(7) 16.84(1) 12.16(2)
6.82(1) 0.52(1)
5.29 5.94 6.88
a pKi complex values correspond to the formation of the complex CuH(4 i )L2 where i 1, 2, 3, 4, 5.
378
˝ sz et al. / Inorganica Chimica Acta 339 (2002) 373 /382 K. O
Fig. 1. Species distribution of the complexes formed in the copper(II) / His-BIMA system as a function of pH, at different metal to ligand ratios. ((a): cCu(II) /2 /10 3 mol dm 3, cL /4 /10 3 mol dm 3; (b): cCu(II) /cL /4 /10 3 mol dm 3; (c): cCu(II) /6 /10 3 mol dm 3, cL /4 /10 3 mol dm 3).
parameters of the major species (Table 4) provide further support for the exclusive binding of the bis(imidazolyl) residues in strongly acidic solutions. The spectral parameters of the species [CuH2L]4 corresponds well to those of 2N-coordination of the bis(imidazolyl) residue [18 /22], while the other two nitrogen donors (amino and histidyl imidazole) remain un-coordinated and protonated. Bis(ligand) complexes are formed at 1:2 metal ion to ligand ratio with the
general formulae of [CuH(4 i )L2](6 i ) (where i /0, 1, 2, 3, 4). Spectral parameters of these complexes are almost the same supporting the existence of the same 4N bis(imidazolyl) binding sites in all bis(ligand) complexes. Even the nine-line superhyperfine splitting of EPR spectra remains intact in the pH range of 3.5 /6.5 in agreement with the metal ion coordination of 4 equiv. nitrogen donors in the equatorial plane. The spectral changes of the various samples are, however, completely different in the equimolar solutions of copper(II) and His-BIMA. The complex [CuH2L]4 can be detected as the major species below pH 3, but the broad unresolved spectra obtained in the pH range 4/7 suggest the coexistence of several different species with the same stoichiometry. The EPR spectra of equimolar solutions can be explained if we assume that several isomeric forms of [Cu2L2]4 exist in the slightly acidic pH range. One set of these isomers can be described via the chelation of the N- and C-termini to different metal ions as it is shown by Scheme 3 Two different forms of [Cu2L2]4 can be obtained by this type of coordination. Scheme 3(a) corresponds to the symmetrical arrangement of the donor sites, while Scheme 3(b) corresponds to the assymetrical one as in a mixed ligand complex. Earlier studies on the copper(II) complexes of different nitrogen donors came to the conclusion that the mixed ligand complex formation is unfavoured if both aromatic and aliphatic nitrogen donors are present [30]. As a consequence, the symmetrical arrangement of the donor sites is more probable and it is supported by the appearance of the superhyperfine splitting in the parallel region of EPR spectra. The other set of the isomers of [Cu2L2]4 can be described in the various protonated forms of imidazole bridged dinuclear complexes as it is depicted in Scheme 2. The dinuclear complex [Cu2H 2L2]2 is the major species at pH 7 in equimolar solutions and its EPR spectrum is almost the same as those of the corresponding species of Gly-BIMA or Phe-BIMA. As a consequence, the terminal amino, deprotonated amide and one of the imidazole nitrogen donor atoms are the binding sites of [Cu2H 2L2]2 with imidazole bridging from the other nitrogen atom of the bis(imidazolyl) residue. Taking into account that the deprotonation of the amide function starts around pH 5, the third imidazole residue of His-BIMA can be partially protonated in the dinuclear complexes resulting the existence of the species [Cu2H 1L2]3 and [Cu2L2]4. Thus, we can conclude that the EPR spectra are the mixture of at least three different types of metal ion coordination including those described by Schemes 2 and 3(a) and (b). The pK values for the deprotonation of the [Cu2L2]4 dinuclear complex are 5.93 and 6.17. The difference of these values is much smaller than statistically expected supporting that the deprotonation of the amide function and the non-coordinated histidyl residues takes place in
˝ sz et al. / Inorganica Chimica Acta 339 (2002) 373 /382 K. O
379
Table 4 EPR and UV /Vis spectral parameters of the complexes formed in the Cu(II) /His-BIMA system Species
[CuH(4 i )L2](6 i ) [CuH 1L2] [CuH2L]4 [Cu2H 2L2]2 [Cu2H 4L2] [Cu3H 4L2]2 a
Conditions
a
UV /Vis
EPR
Cu:L ratio
pH
lmax [nm](o [dm3 mol 1 cm 1])
gjj
Ajj (10 4 cm 1)
1:2 1:2 1:1 1:1 1:1 3:2
5.10 8.12 2.53 7.88 10.44 7.00
605(50) 585(95) 683(32) 592(111) 565(117) 580(84)
2.234 2.221 2.298 dinuclear dinuclear polynuclear
197 175 176
Where i 0, 1, 2, 3, 4.
overlapping processes. The absorption maxima of the equimolar solution in the pH range of 4.79 /7.88 is shifted to 592 from 620 nm suggesting that the ligand bridged dimers (Scheme 3) predominate in the lower pH ranges (pH 4/6), while imidazole bridged complexes are the major species at higher pH. The EPR spectrum obtained for the species [Cu2H 2L2]2 makes it possible to calculate a Cu /Cu distance. The values g /2.080 and D /0.0449 cm 1 can be obtained from the perpendicular signal, which corresponds to a value of 397 pm for the Cu /Cu distance in the dinuclear complex. This value is very close to those obtained for Gly-BIMA and Phe-BIMA suggesting the same coordination environments for all three ligands. At the same time, it should be considered that in the copper(II) /His-BIMA system either bis(imidazolyl) or histidyl nitrogen donor atoms could act as bridging ligands. Molecular models, however, reveal that the Cu /Cu distance should be much higher in the case of histidyl bridging. The complex formation processes of His-BIMA are significantly different from those of Phe-BIMA under alkaline conditions. It is clear from Fig. 1(a) that in the presence of excess of His-BIMA the bis(ligand) complex [CuH 1L2] predominates in the pH range 8 /9. The EPR spectra indicate that it is a monomeric species with the parameters of gjj /2.221 and Ajj /175 /10 4 cm 1 (see Table 4). Its formation is accompanied with a small
blue shift of the absorption spectra suggesting that [CuH 1L2] is not a mixed hydroxo complex. Its metal binding sites can be described by the tridentate equatorial [NH2,N ,N(Im)]-coordination of one ligand and monodentate equatorial or bidentate axial /equatorial coordination of nitrogen donors of the second ligand. The major differences in the copper(II) complexes of His-BIMA and Phe-BIMA (or Gly-BIMA) can be observed in equimolar solutions at high pH values. In the case of the latter two ligands the [Cu2H 2L2]2 dinuclear complexes were transformed into mixed hydroxo species above pH 8 in the form of mononuclear and polynuclear complexes for Phe-BIMA and GlyBIMA, respectively. The same base consuming process is observed with His-BIMA, but it is accompanied with a significant blue shift of the absorption spectra (see Table 4) and characteristic changes of the EPR signals. The EPR spectra provide an unambiguous proof that the species [Cu2H 4L2] detected at pH 10.44 is still a dinuclear complex, but the blue shift of the d/d bands suggests that the deprotonation cannot be a simple mixed hydroxo complex formation. The EPR parameters from the perpendicular signal of the spectrum obtained at pH 10.44 are g /2.055 and D /0.048 cm 1 from which a value of 384 pm can be calculated for the Cu /Cu distance in [Cu2H 4L2]. These data can be explained assuming that the base consuming process comes from the deprotonation of the pyrrole-type
Scheme 3.
˝ sz et al. / Inorganica Chimica Acta 339 (2002) 373 /382 K. O
380
N(1)H groups of coordinated imidazole residues. The coordination of charged nitrogen donors generally results in a blue shift of absorption spectra (e.g. in peptide complexes) and shortness of the chemical bond distances. Metal ion induced deprotonation of the coordinated imidazolyl-N(1)H groups have already been reported for many peptide complexes containing histidyl residues and for several model compounds [30]. Palladium(II) and gold(III) were reported to be especially effective in the promotion of ionisation [31 /34], while pK values around or above 10 were reported for copper(II) complexes [29,35,36]. The pK values for the deprotonation of [Cu2H 2L2]2 of His-BIMA are 8.13 and 8.93. These values are lower than those reported in the literature for the process when deprotonation is induced via the imidazole ring without direct metal ion coordination [36]. However, in this case the deprotonation comes from the coordinated bis(imidazolyl) residues having different (positive) net charge and the charge neutralisation probably has a significant contribution to the outstanding stability of [Cu2H 4L2]. As a consequence, the fully deprotonated dinuclear complex is the major species in alkaline solution even in the presence of excess of ligand as it is demonstrated by Fig. 1(a). Another important consequence of the deprotonation of the N(1)H-groups is that they can be considered as additional metal binding sites in the presence of excess of metal ions. In agreement with this expectation there is no precipitation in the solutions containing copper(II) and His-BIMA in the ratio 3:2. Fig. 1(c) shows that the species [Cu3H 4L2]2 is formed almost exclusively at pH 7 under these conditions. The formation of dinuclear complexes starts under acidic conditions (above pH 3), but [Cu2L]4 is the first species, in which the separated N- and C-termini are the metal binding sites. The extra base consuming process appears between pH 5 and 6 resulting the trinuclear [Cu3H 4L2]2 complex. The d / d absorption band of this species is rather wide with a maximum around 580 nm and it shows a broad unresolved EPR spectra suggesting a significant dipolar interaction between the copper ions. These spectral
Scheme 4.
parameters suggest that the trinuclear complex contains copper(II) ions at least in two different environments as it is shown by Scheme 4. Two of the copper(II) ions are coordinated tridentately, while the third copper(II) ion occupies the central position with 4N-coordination of two tridentately coordinated residues. This type of binding requires the deprotonation of one imidazole-N(1)H groups from each tridentately coordinated units in the pH range 5/ 6. It is a surprisingly low value for the metal ion induced deprotonation of pyrrolic type N(1)H-groups, but it should be considered that deprotonation is accompanied with metal ion coordination. Imidazolato bridged di- or polynuclear complexes have already been prepared by several authors and they generally were formed around the physiological pH range with a potential SOD activity [37,38]. The stability constants of the nickel(II) and zinc(II) complexes of His-BIMA are included in Table 3. The species [NiH2L]4 and [NiH4L2]6 are the major complexes under acidic conditions and their binding sites can be explained by the coordination of the bis(imidazolyl) residues, similarly to the corresponding copper(II) complexes. pK values for the deprotonation of the bis(ligand) complex follow the statistical trends suggesting that the binding sites are not changing upon the formation of [NiL2]2 from [NiH4L2]6. In agreement with this conclusion the values of the stability constants of the [ML2]2 complexes of Phe-BIMA and His-BIMA are very similar to each other. The deprotonation of the [NiH2L]4 complex in equimolar solution, however, takes place in a different way. The species [NiHL]3 cannot be detected suggesting the cooperative deprotonation of the amino and imidazole nitrogen donors of histidyl residues. Stoichiometry of the resulting species can be either [NiL]2 or [Ni2L2]4, but the cooperative deprotonation supports the dinuclear complex formation. Steric requirements rule out the quadridentate binding of His-BIMA in a mononuclear complex, but it is possible in a dinuclear species as it was suggested for copper(II) in Scheme 3(a) and (b). Unfortunately, none of the spectroscopic techniques is sensitive enough to distinguish monomeric and dimeric nickel(II) complexes and to provide a sufficient proof for dinuclear complex formation in solution. The UV / Vis absorption spectra unambiguously indicate the formation of octahedral species at any pH values and according to the potentiometric data the dinuclear complexes are favoured over the mononuclear ones. A further increase of pH results in an extra base consuming process above pH 7 or 8, which can be attributed to the formation of [NiH 1L] and [NiH 1L2]. From the potentiometric studies we cannot distinguish hydroxo complex formation from amide deprotonation, but a comparison with literature data and characteristic changes of visible spectra provide
˝ sz et al. / Inorganica Chimica Acta 339 (2002) 373 /382 K. O
sufficient proof for the latter process. Deprotonation and coordination of the amide functions were reported to occur in the nickel(II) and zinc(II) complexes of GlyBIMA [22], while in the case of Phe-BIMA this process was suppressed by hydrolytic reactions. The nickel(II) induced deprotonation of the amide function, however, also was reported in the nickel(II) complexes of HisBPMA (a bis(pyridyl) analogue of His-BIMA) [21]. The coordination geometry of nickel(II) ions remains octahedral in the species [NiH 1L] and [NiH 1L2], but the formation of [NiH 1L] from [Ni2L2]4 results a significant change of the absorption spectra. For example 10Dq /10 650 cm 1 for [Ni2L2]4 at pH 6 and it is shifted to 11 000 cm 1 by pH 8.6. These spectral changes are accompanied with some decrease of the corresponding Racah parameters. Namely, the B values are 1010 cm 1 for [Ni2L2]4 and 700 cm 1 for [NiH 1L] suggesting the increase of covalency in the latter species in agreement with amide binding instead of hydrolysis. The complex formation processes of zinc(II) with HisBIMA are similar to those of nickel(II). The bis(imidazolyl) nitrogen donors are the primary metal binding sites in acidic media, but in agreement with the reduced thermodynamic stability of zinc(II) complexes the complex formation is shifted to higher pH values and the fully protonated [ZnH4L2]6 cannot be identified. [ZnL]2 (or [Zn2L2]4) are the major species in neutral solution, containing tridentately (or quadridentately) coordinated ligands. Extra deprotonation starts around pH 8, but is followed by precipitation of hydroxo complexes. Thus, in contrast with nickel(II) complexes amide deprotonation and coordination do not occur in the slightly alkaline aqueous solutions of zinc(II) complexes. The mononuclear [ZnL]2 complex is coordinatively unsaturated which makes hydroxo complex formation possible under slightly alkaline conditions.
4. Conclusions The results presented in this paper provide further support on the outstanding metal binding ability of the bis(imidazolyl) derivatives of amino acids. It is also obvious from the data that the amino acid side chains have significant effects on the complex formation processes, especially if additional donor groups are present. Complex formation reactions of Phe-BIMA are very similar to those of Gly-BIMA and the presence of the bulky, non-coordinating phenyl residues influences only the formation of bis(ligand) and mixed hydroxo complexes. Nitrogen donors of the bis(imidazolyl) residues are the major metal binding sites in acidic solutions with all metal ions studied and only copper(II) is able to
381
induce deprotonation and coordination of the amide function of Phe-BIMA. The coordination chemistry of His-BIMA is more versatile and seems to be a well-suited model for the active centre of various metalloenzymes. Similarly to any other bis(imidazolyl) ligands the imidazole-N donor atoms are the primary metal binding sites. The histidyl side chains, however, also have significant metal binding ability, which results in the formation of a dinuclear complex [Cu2L2]4. The EPR spectra of equimolar solutions indicate that the stoichiometry [Cu2L2]4 corresponds to a mixture of several isomeric species including those represented by Schemes 2 and 3(a) and (b). Species with the same stoichiometry were formed with nickel(II) and zinc(II), but the spectral parameters support the favoured formation of dinuclear complexes with nickel(II) and mononuclear complexes with zinc(II). Deprotonation and coordination of the amide function of His-BIMA take place in the copper(II) and nickel(II) complexes. In the case of nickel(II) the outstanding thermodynamic stability of the dinuclear complex shifts this process into the slightly alkaline pH range, thus it overlaps with hydroxo complex formation. However, for copper(II) the species [Cu2H 2L2]2 predominates in the physiological pH range (Scheme 2). The formation of another dinuclear complex [Cu2H 4L2] can be detected as the final species by pH 10. The same binding sites were suggested for this dinuclear complex as for [Cu2H 2L2]2, but containing negatively charged imidazole residues formed by the deprotonation of the pyrrolic type N(1)H-groups. This deprotonation is supported by the blue shift of the absorption spectra and by the decrease in the value of Cu /Cu distance. The N(1)H deprotonation creates a new chelating site which results in the formation of the species [Cu3H 4L2]2 in the presence of excess of metal ions (Scheme 4). Deprotonation of imidazole-N(1)H groups occurs in a surprisingly low pH range (pH 5 /6) in these complexes, but it should be considered that deprotonation is accompanied by metal ion coordination. On the other hand, it is important to note that the formation of the trinuclear complexes with negatively charged imidazole bridges renders the ligand His-BIMA a much promising model for the SOD enzymes.
Acknowledgements This work was supported by the Hungarian Scientific Research Fund (Hungary, OTKA-T029141).
References [1] H. Kozlowski, W. Bal, M. Dyba, T. Kowalik-Jankowska, Coord. Chem. Rev. 184 (1999) 319.
382
˝ sz et al. / Inorganica Chimica Acta 339 (2002) 373 /382 K. O
[2] P. Tsiveriotis, G. Malandrinos, N. Hadjiliadis, Rev. Inorg. Chem. 20 (2000) 305. [3] S.J. Lau, T.P.A. Kruck, B. Sarkar, J. Biol. Chem. 249 (1974) 5878. [4] M.A. Zoroddu, T. Kowalik-Jankowska, H. Kozlowski, H. Molinari, K. Salnikow, L. Broday, M. Costa, Biochim. Biophys. Acta Gen. 1475 (2000) 163. ´ goston, I. So´va´go´, G. Micera, Polyhedron 20 [5] D. Sanna, C.G. .A (2001) 937. [6] K. Va´rnagy, J. Szabo´, I. So´va´go´, G. Malandrinos, N. Hadjiliadis, D. Sanna, G. Micera, J. Chem. Soc., Dalton Trans. (2000) 467. [7] A. Myari, G. Malandrinos, Y. Deligiannakis, J.C. Plakatouras, N. Hadjiliadis, Z. Nagy, I. So´va´go´, J. Inorg. Biochem. 85 (2001) 253. [8] R. Breslow, J.T. Hunt, R. Smiley, T. Tarnowski, J. Am. Chem. Soc. 105 (1983) 5337. [9] G.J.A.A. Koolhaas, W.L. Driessen, J. Reedijk, J.L. van der Plas, R.A.D. de Graaf, D. Gatteschi, H. Kooijman, A.L. Spek, Inorg. Chem. 35 (1996) 1509. [10] I. To¨ro¨k, P. Surdy, A. Rockenbauer, L. Korecz, Jr., G.J.A.A. Koolhaas, T. Gajda, J. Inorg. Biochem. 71 (1998) 7. [11] T. Gajda, R. Kra¨mer, A. Jancso´, Eur. J. Inorg. Chem. (2000) 1635. [12] H. Ohtsu, Y. Shimazaki, A. Odani, O. Yamauchi, W. Mori, S. Itoh, S. Fukuzumi, J. Am. Chem. Soc. 122 (2000) 5733. [13] C.N.C. Drey, J.S. Fruton, Biochemistry 4 (1965) 1. [14] C.N.C. Drey, J.S. Fruton, Biochemistry 4 (1965) 1258. [15] M.S. Mohan, Ind. J. Chem. 20A (1981) 252. [16] H. Sigel, A. Saha, N. Saha, P. Carloni, L.E. Kapinos, R. Griesser, J. Inorg. Biochem. 78 (2000) 129. [17] C.J. Campbell, W.L. Driessen, J. Reedijk, W. Smeets, A.L. Spek, J. Chem. Soc., Dalton Trans. (1998) 2703. ´ goston, Z. Liko´, H. Su¨li-Vargha, D. [18] K. Va´rnagy, I. So´va´go´, K. A Sanna, G. Micera, J. Chem. Soc., Dalton Trans. (1994) 2939. [19] K. Va´rnagy, I. So´va´go´, W. Goll, H. Su¨li-Vargha, G. Micera, D. Sanna, Inorg. Chim. Acta 283 (1998) 233.
[20] K. Va´rnagy, I. So´va´go´, H. Su¨li-Vargha, D. Sanna, G. Micera, J. Inorg. Biochem. 81 (2000) 35. ˝ sz, K. Va´rnagy, I. So´va´go´, L. Lennert, H. Su¨li-Vargha, D. [21] K. .O Sanna, G. Micera, New J. Chem. 25 (2001) 700. ˝ sz, Comments Inorg. Chem. 23 [22] I. So´va´go´, K. Va´rnagy, K. .O (2002) 149. [23] F. Reindel, W. Hoppe, Chem. Ber. 87 (1954) 1103. [24] H. Irving, G. Miles, L.D. Pettit, Anal. Chim. Acta 38 (1967) 475. [25] L. Ze´ka´ny, I. Nagypa´l, in: D.J. Leggett (Ed.), Computation Methods for the Determination of Stability Constants, Plenum Press, New York, 1985, p. 291. [26] T. Kiss, in: K. Burger (Ed.), Biocoordination Chemistry, Ellis Horwood, Chichester, 1990, p. 56. [27] A. Gergely, I. So´va´go´, I. Nagypa´l, R. Kira´ly, Inorg. Chim. Acta 6 (1972) 435. [28] R.L. Belford, N.D. Chasteen, H. So, R.E. Tapscott, J. Am. Chem. Soc. 91 (1969) 4675. [29] I. So´va´go´, T. Kiss, A. Gergely, J. Chem. Soc., Dalton Trans. (1978) 964. [30] I. So´va´go´, A. Gergely, Inorg. Chim. Acta 37 (1979) 233. [31] R.J. Sundberg, R.B. Martin, Chem. Rev. 74 (1974) 471. [32] S.L. Best, T.K. Chattopadhyay, M.I. Djuran, R.A. Palmer, P.J. Sadler, I. So´va´go´, K. Va´rnagy, J. Chem. Soc., Dalton Trans. (1997) 2587. [33] M. Wienken, B. Lippert, E. Zangrando, L. Randaccio, Inorg. Chem. 31 (1991) 1983. [34] M. Wienken, E. Zangrando, L. Randaccio, S. Menzer, B. Lippert, J. Chem. Soc., Dalton Trans. (1993) 3349. [35] H. Aiba, A. Yokoyama, H. Tanaka, Bull. Chem. Soc. Jpn. 47 (1974) 1437. [36] P.J. Morris, R.B. Martin, J. Inorg. Nucl. Chem. 33 (1971) 2913. [37] G. Kolks, C.R. Frihart, H.N. Rabinowitz, S.J. Lippard, J. Am. Chem. Soc. 98 (1976) 5720. [38] G. Tabbi, W.L. Driessen, J. Reedijk, R.P. Bonomo, N. Veldman, A.L. Spek, Inorg. Chem. 36 (1997) 1168.
