Solution equilibria and structural characterisation of the transition metal complexes of glycyl-l -cysteine disulfide

www.elsevier.nl/locate/poly Polyhedron 19 (2000) 1849 – 1857

Solution equilibria and structural characterisation of the transition metal complexes of glycyl-L-cysteine disulfide ´ goston a, Katalin Va´rnagy a, Attila Be´nyei b, Daniele Sanna c, Csaba G. A Giovanni Micera d, Imre So´va´go´ a,* a

Department of Inorganic and Analytical Chemistry, Uni6ersity of Debrecen, H-4010 Debrecen, Hungary b Department of Physical Chemistry, Uni6ersity of Debrecen, H-4010 Debrecen, Hungary c Instituto CNR per l’Applicazione delle Tecniche Chimiche A6anzate ai Problemi Agrobiologici, Via Vienna 2, I-07100 Sassari, Italy d Department of Chemistry, Uni6ersity of Sassari, Via Vienna 2, I-07100 Sassari, Italy Received 5 May 2000; accepted 16 June 2000

Abstract Stoichiometry, stability constants and structure of the complexes formed in the reaction of copper(II), nickel(II), zinc(II), cobalt(II) and cadmium(II) with (GlyCys)2 containing disulfide bond were determined by potentiometric, UV – Vis, NMR and EPR spectroscopic methods. The complex [Ni(GlyCys)2·H2O] was prepared in the solid state and its structure determined by single crystal X-ray diffraction method. Disulfide sulfur atoms of (GlyCys)2 were not metal-binding sites in any of the systems studied in solution or in the solid state. It was found that copper(II) is able to induce deprotonation and coordination of the peptide amide nitrogen donor atoms. This resulted in the formation of [CuL], [Cu2H − 2L] and [CuH − 1L]− as the major species in solution. In the case of nickel(II), cobalt(II), zinc(II) and cadmium(II) the complex [ML] predominates, and the outstanding stability of this species was explained by the formation of a macrochelate between the (NH2, CO)-coordinated five-membered chelates in solution. Crystal structure of [Ni(GlyCys)2·H2O] revealed the octahedral geometry of nickel(II) in a polymeric structure. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Disulfide; Peptides; Transition metal ions

1. Introduction Disulfide bonds (SS) have an outstanding significance in the stabilisation of protein structures. As a consequence, both complex formation and redox reactions of this moiety are of much biological importance. In general the disulfide bond easily undergoes reductive cleavage giving rise to sulfur radicals, thiols, thioethers or thiolate anions. The metal ion promoted disproportionation of disulfides has already been well clarified [1], while the insertion of a metal-bond nitrogen atom into a disulfide bond has been described recently [2]. The various redox reactions of disulfide bridges are often involved in the catalytic activity of various metalloenzymes including nickel containing methyl-coenzyme M reductase [3]. * Corresponding author. Tel.: + 36-52-512-900; fax: + 36-52-489667. E-mail address: [email protected] (I. So´va´go´).

There are many examples in the literature of the interactions of metal ions and organic ligands containing disulfide bonds and the most important aspects of the coordination chemistry of organodisulfide ligands have already been reviewed [4]. It is also obvious from these studies that apart from the soft metal ions the stable coordination of disulfide sulfur atoms generally takes place in aprotic solvents or in the absence of other strongly coordinating donor groups. Consequently, in the case of the simplest and biologically important disulfide ligands (cystine, oxidized glutathione and D-penicillamine disulfide) the amino, carboxylate and amide functions are the major metal binding sites for the 3d divalent metal ions, while the disulfide sulfur atoms are uncoordinated or take part in a weak interaction. The structure of the dimeric species obtained in the reaction of copper(II) and D-penicillamine disulfide has been described via the equatorial coordination of the amino acid binding sites with a

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weak axial interaction of sulfur atoms [5]. In the case of oxidised glutathione a 2:1 = Cu(II):GSSG complex was prepared with (ON3) equatorial binding sites and weak axial interaction of both sulfur atoms [6]. Copper(II) complexes of pyridine- or imidazole-containing disulfide ligands were also characterised by the formation of CuS(disulfide) bonds in solution on the basis of charge transfer transition around 310 – 330 nm [7]. The existence of a relatively short Cu(II)S bond length was reported in the complexes of dithiodipropanoate [8] and synthetic macrocyclic bis(disulfide) tetramine ligands, while other examples of copper(II) complexes of disulfides have been reviewed by Fox et al. [9]. As concerns the other 3d transition metal ions a strong interaction of nickel(II) and sulfur atom was reported in the nickel(II) complex of a ligand containing both pyridine and disulfide donor functions [10]. In a previous study we reported the results of potentiometric and spectroscopic studies in solution on the cobalt(II), nickel(II), copper(II) and zinc(II) complexes of three disulfide ligands: D-penicillamine disulfide, oxidised glutathione and L-cysteinylglycine disulfide [11]. The amino, carboxylate and the amide functions were detected as the exclusive metal binding sites and no indication on the formation of MS bonds was obtained. In the case of the copper(II)-oxidised glutathione system many other studies have been performed in solution, but the Cu(II)S interaction was excluded in all cases [12 – 15]. The peptide hormones oxytocin and vasopressin are other examples of simple peptides containing disulfide bonds. The twenty-membered ring linked by the disulfide bridges (between Cys1 and Cys6 residues) provides a unique conformation of these molecules. This resulted in the increased stability of metal complexes [16 – 18]. The enhancement of thermodynamic stability was, however, not attributed to a significant Cu(II)S interaction, which was only detected in the 2N complexes of oxytocin [16]. The development of a more intense charge transfer band around 380 nm in the 2N complex of the copper(II) – glipressin (GlyGlyGly-Lys8 –vasopressin) system also was explained by the formation of Cu(II)S(disulfide) bond [18]. In contrast with the above-mentioned 3d transition metal ions the soft palladium(II) and platinum(II) interact easily with disulfides and the existence of strong PdS and PtS bonds were reported in the complexes of cystine or oxidized glutathione [19 – 21]. The survey of the literature data obtained for the complexes of disulfide derivatives of peptides with 3d transition element reveals that the formation of MS bonds are generally negligible in solution. On the other hand, the results obtained for the copper(II) complexes of glipressin suggest that the formation of this bond requires a specific arrangement of donor groups in the peptide chain [18]. The role of specific amino acid sequences in the enhanced metal binding ability of

peptide molecules is well known and it is probably best represented by the C-terminal His peptides, e.g. GlyHis and GlyGlyHis [22,23]. Similar increased stability of the copper(II) or nickel(II) complexes of peptides were reported with several other peptide sequences including GlyGlyCys [24], XXAsp [25] and to a smaller extent with the tripeptides of methionine [26]. The oxidised form of the dipeptide glycyl-L-cysteine, (GlyCys)2, contains the disulfide sulfur atoms in a chelating position with the amide nitrogen. Therefore this molecule can be a promising candidate for the metal ion promoted formation of disulfide complexes. In this paper we report the results of combined potentiometric and spectroscopic studies on the copper(II), nickel(II), cobalt(II), zinc(II) and cadmium(II) complexes of (GlyCys)2 in solution. The crystal structure of the nickel complex of (GlyCys)2 also has been determined by single crystal X-ray diffraction method.

2. Experimental

2.1. Materials The peptides glycyl-L-cysteine disulfide, (GlyCys)2, and glycineamide were purchased from Bachem and their purity checked via potentiometric titrations. Stock solutions of the metal ions (CoCl2, NiCl2, CuCl2, ZnCl2 and Cd(NO3)2) were prepared from analytical grade reagents and their concentrations were checked gravimetrically via precipitation of oxinates.

2.2. Potentiometric studies The pH-potentiometric titrations were performed in 5 cm3 samples in the metal ion concentration range 2– 4× 10 − 3 mol dm − 3 and with metal ion to ligand ratios between 2:1 and 1:2. The measurements were made with a Radiometer pHM64 pH-meter equipped with a Russel CWR/320/757 combined electrode and an ABU13 automatic burette containing a carbonate free stock solution of potassium hydroxide. 40–60 titration points were recorded for all different ratios. During the titration argon was bubbled through the samples to ensure the absence of oxygen and carbon dioxide, and stirring of the solutions. All pH-potentiometric measurements were carried out at a constant ionic strength of 0.2 mol dm − 3 KCl (KNO3 for cadmium(II)) and at constant temperature (298 K). The pH-readings were converted to hydrogen ion concentration and the overall stability constants (log bpqr ) were calculated by means of a general computational program (PSEQUAD) [27]. pM+qH+ rL X MpHqLr [MpHqLr] bpqr = [M]p[H]q[L]r

´ goston et al. / Polyhedron 19 (2000) 1849–1857 C.G. A

2.3. Spectroscopic studies UV–Vis absorption spectra of copper(II) and nickel(II) complexes were recorded on an HP 8453 diode array spectrophotometer in the concentration range of 4× 10 − 3 mol dm − 3 for copper(II) and 10 − 2 mol dm − 3 for nickel(II). Anisotropic X-band EPR spectra of copper(II) complexes were recorded at 120 K using a Varian E-9 spectrometer in frozen solution after addition of ethylene glycol to ensure good glass formation. Proton NMR spectra of the free ligand and zinc(II) complexes (cL = cZn =10 − 2 mol dm − 3) were measured in D2O as a function of pD. The spectra were recorded on a Bruker MA 360 MHz instrument using TSP (sodium 3-trimethylsilylpropionate) as internal standard.

2.4. Crystallisation of [Ni(GlyCys)2 ·H2O] (C10H18N4NiO7S2) One equivalent of (GlyCys)2 was added to a solution of NiCl2 in water (c =0.1 mol dm − 3) and the pH was adjusted to 8.50. The solution was transferred into a glass tube and methanol was layered on it. Green crystals were formed at the interface which were filtered off, washed with a mixture of cold water and methanol and dried in air.

2.5. Structure analysis and refinement Green prism crystals (0.3 × 0.25 × 0.2 mm) of C10H18N4NiO7S2, M =429.11, trigonal, a = 12.988(4), b=12.988(6), c= 20.665(7) A, , V = 3019 A, 3, Z= 6, space group: P3121, rcalc =1.416 g cm − 3. Data were collected at 293(1) K, Enraf Nonius MACH3 diffractometer, Mo Ka radiation l = 0.71073 A, , v-2u motion, umax =25.5°, 2017 measured, 2017 unique reflections of which 1519 were with I\ 2s(I), decay: 1%. The struc-

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ture was solved using the SIR-92 software [28] and refined on F 2 using SHELX-97 [29] program, publication material was prepared with the WINGX-97 suite [30]. R(F)=0,0853 and wR(F 2)= 0.246 for 2017 reflections, 220 parameters. The maximum and minimum residual electron densities are 1.057 and − 0.607 e A, − 3, respectively.

3. Results and discussion Stability constants of the proton and metal ion complexes of (GlyCys)2 were determined by pH-potentiometric titrations of the samples containing the metal ion and ligands at different ratios. The stability constants of the complexes and the corresponding pK values are included in Table 1. The lowest pK values (around 2–3) correspond to the deprotonation of the carboxylic groups, while the other two deprotonation processes in the pH range 7–9 results from the ammonium groups. The potentiometric titration curves provide an unambiguous proof that extra deprotonation processes occur only in the copper(II) containing systems. In the copper(II)–(GlyCys)2 = 2:1 system two equivalents of base are titrated in addition to the carboxylic and ammonium groups suggesting that copper(II) is able to induce deprotonation and coordination of amide nitrogen donor atoms. In the case of cobalt(II), nickel(II), zinc(II) and cadmium(II) the parent mono and bis complexes can form and the coordination of the amide groups can be ruled out below pH 10. On the other hand, stable pH values cannot be determined in more alkaline solutions, because all metal ions are able to induce disproportionation of disulfide bonds in alkaline solutions as was reported for oxidised glutathione [1]. As a consequence, only the experimental points below pH 10 were used for calculation of stability constants of metal complexes.

Table 1 pK values and stability constants (log bpqr ) of the metal complexes of (GlyCys)2a Cu(II) pK1 pK2 pK3 pK4 [MHL]+ [ML] [MH−1L]− [MH−2L]2− [ML2]2− [M2H−1L]+ [M2H−2L] [M2H−3L]− [M2H−4L]2− a

Ni(II)

Co(II)

Zn(II)

Cd(II)

12.85(3) 7.41(2) −2.83(7)

12.01(5) 5.51(1)

12.55(9) 6.69(2)

11.70(20) 5.40(3)

2.06(4) 3.21(2) 7.75(2) 8.77(2) 14.55(4) 10.35(3) 3.55(4) −7.03(9)

8.87(8) 8.13(8) 3.55(3) −4.27(6) −14.63(7)

I= 0.2 mol dm−3, T = 298 K (standard deviations are in parenthesis).

8.40(10)

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Fig. 1. Concentration distribution of the complexes formed in the copper(II) – (GlyCys)2 system. (a) cCu =8 × 10 − 3 mol dm − 3, cL =4 × 10 − 3 mol dm − 3; and (b) cCu =cL = 4×10 − 3 mol dm − 3.

In agreement with the conclusions drawn from the titration curves, Table 1 shows a significant difference in the complex formation processes of copper(II) and the other metal ions. Namely, in the case of copper both mono- and dinuclear species can form, while the other systems are characterised by the formation of [ML] as the major species. Concentration distributions of the various complexes formed in the copper(II)– (GlyCys)2 system at 1:1 and 2:1 ratios are shown in Fig. 1. It can be seen from Fig. 1 that in the copper(II)– (GlyCys)2 system the complex formation starts above pH 3 and the stoichiometry of the first species is

[CuHL]+, which can correspond to the coordination of the terminal amino and neighbouring carbonyl oxygen donor atoms in a five-membered chelate, while the amino group at the other side of the disulfide bridge remains uncoordinated and protonated. EPR spectra of equimolar solutions support this conclusion (see Table 2), because the parallel region of EPR spectra provides the parameters reminescent to those of (NH2, CO) species of GlyGly [31]. The species [CuL] is present in the pH range 4.5–6.5 and its spectral parameters show a close similarity to those of [CuH − 1L] of GlyGly or other common dipeptides. It supports that one dipeptide unit of the

´ goston et al. / Polyhedron 19 (2000) 1849–1857 C.G. A

molecule is coordinated as the common dipeptides, (NH2, N−, COO−), while the other side is still free with protonated amino group. The visible spectra of equimolar solutions do support this conclusion, because lmax =635 and 640 nm and o =74 and 84 dm3 mol − 1 cm − 1 can be obtained for the [CuL] of (GlyCys)2 and [CuH − 1L] of GlyGly, respectively [31]. Further increase of pH results in a blue shift of absorption spectra (lmax =612 nm and o = 80 dm3 mol − 1 cm − 1) and it is accompanied with a characteristic change of EPR parameters. The most reasonable explanation for the binding sites of the species [CuH − 1L]− that one more nitrogen donor from the other dipeptide unit of the molecule coordinates to copper(II) as it is represented by Scheme 1. Table 2 Parameters of the parallel region of EPR spectra of the complexes formed in copper(II)–(GlyCys)2 and copper(II)–GlyGly systemsa Species

Coordination

Cu(II)–(GlyCys)2 [CuHL]+ [CuL] [CuH−1L]− Cu(II)–GlyGly [CuL]+ [CuH−1L] [CuH−1L2]−

g

10−4A /cm−1

(NH2, CO) 2.330 (NH2, N−, COO−) 2.236 (NH2, N−, COO−) 2.215 + (NH2, CO)

153 193 171

(NH2, CO) 2.332 (NH2, N−, COO−) 2.248 (NH2, N−, COO−) 2.232 + (NH2, CO)

161 185 168

b

a

For the [CuH−1LB]− complex of GlyGly and N-acetyllysine g = 2.226 and A =168×10−4 cm−1 were measured. b Data are taken from Ref. [31]

Scheme 1.

Scheme 2.

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The binding mode in Scheme 1 is similar to that of GlyGly in [CuH − 1L2]−, but in the case of GlyGly the second ligand coordinates via the amino-N and carbonyl-O donor atoms in an equatorial-axial equilibrium. The mixed complex [CuH − 1LB]− provides a good model for the structure of [CuH − 1L]− of (GlyCys)2. In the ternary species B represents N-acetyllysine, which is a monodentate amino donor via the o-amino group and the spectral parameters A =168× 10 − 4 cm − 1, g = 2.226 and lmax = 612 nm, o= 96 dm3 mol − 1 cm − 1 were reported for the mixed ligand complex [32]. The spectral data for the species [CuH − 1L]− of (GlyCys)2 are slightly different from those of the complexes described above. However, the spectral trend observed on passing from (NH2, N−, COO−) to (NH2, N−, COO−)+ (NH2, CO) coordination, namely the simultaneous decrease of both A and g , is the same in all the three systems. This trend is unusual for a regular geometry and indicates the achievement of a distorted structure even if further data are needed for a complete understanding of the details of structural changes. In any case, it means that in the species [CuH − 1L]− the two dipeptide residues linked by the disulfide bridge are not identical, being tridentately coordinated from one end and monodentately from the other one. This binding mode, of course, should result in some enhancement of the thermodynamic stability of the complexes and suppresses the hydroxo complex formation. It is reflected in the increase of pK for the formation of the hydroxo complexes [CuH − 2L]2 − , because the values 9.37 and 10.58 can be obtained for GlyGly and (GlyCys)2, respectively. The potentiometric titration curves indicate that in the copper(II)–(GlyCys)2 system two additional equivalents of base can be titrated in the presence of excess of metal ion. In principle, the extra base consumption can correspond to both deprotonation of the second amide nitrogen or only hydrolytic processes. The spectral changes during titration definitely support the amide coordination above pH 5 and it results in dinuclear complex formation as it is shown by Fig. 1. A comparison of Fig. 1(a, b) reveals that the dimeric species are also present in equimolar solution but their concentration will predominate only in the presence of metal ion excess. The absorption maximum of the complex [Cu2H − 2L] appears at lmax = 639 nm, which corresponds to the assumption that both copper(II) is coordinated in the (NH2, N−, COO−) environment as it is depicted by Scheme 2. The dimeric complex [Cu2H − 2L] has two free coordination sites, which are occupied by water molecules or hydroxide ions in slightly basic media (see Fig. 1). EPR spectra of the samples containing metal ion excess are poorly resolved, but the signals of free copper(II) ions disappear by pH 6 and the significant line

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Fig. 2. Concentration distribution of the complexes formed in the nickel(II) – (GlyCys)2 system. cNi =cL =4 × 10 − 3 mol dm − 3.

broadening can be explained by the dipolar coupling between the copper(II) units of the species [Cu2H − 2L]. Another question which arises in connection with the structural characterisation of the copper(II) complexes of (GlyCys)2 is whether the disulfide moiety took part in any interaction with the metal ion. The absence of any characteristic charge transfer band below 400 nm and the clear evidence for the existence of the (NH2, N−, COO−) binding site seem to rule out the involvement of sulfur atom in metal binding in solution. In part this is in contradiction with our expectations, but can be explained by the more effective coordination of the charged carboxylate residue. On the other hand, it should be emphasized that this result was obtained in solution and a weak metal ion interaction with the disulfide bond is still a possibility in the solid state as it was found for the copper(II) complex of D-penicillamine disulfide [5,11]. The pH-potentiometric titration curves of nickel(II)– (GlyCys)2 system at different metal ion to ligand ratios show that there is no any extra base consuming process below pH 9. This is clear evidence that the amide nitrogen is not a metal binding site and the 1:1 complex should have an outstanding thermodynamic stability as compared to that of the bis complex. Equilibrium data in Table 1 reveal that the formation of only three species [NiHL]+, [NiL] and [NiH − 1L]− can be detected in this system. The corresponding species distribution curves are shown by Fig. 2 and it can be seen that [NiHL]+ and [NiH − 1L]− are only minor species and the interaction of nickel(II) and (GlyCys)2 can be described almost exclusively by the formation of the species [NiL]. A reasonable explanation for the outstanding thermodynamic stability of the species [NiL] and for the lack of bis(ligand) complex formation can be given by the assumption of the binding mode shown

by Scheme 3. The enhanced thermodynamic stability of the complex [NiL] probably stems from the formation of 16-membered loop around the central metal ion. This loop is a consequence of the presence of the disulfide bond in the molecule supporting that this residue affects the thermodynamic stability of the complexes via the change of the conformation of the molecules without the formation of a direct NiS bond. The increased nickel binding ability of (GlyCys)2 as compared to that amino acid amides is reflected in the corresponding stability constants, too. Namely, log b1 = 7.41 was obtained for [NiL] of (GlyCys)2, while log b2 = 4.93 was reported for [NiL2]2 + of valineamide [33]. The formation of a similar loop or macrochelate and extra stabilization of the complex was reported in the copper(II)-oxidised glutathione system [11]. According to Scheme 3 the species [NiL] has the same binding sites as that of [NiL2]2 + of glycineamide [33,34]. The absorption spectra of the complexes show the presence of octahedral central metal ions in both species and in the visible range the spectral parameters l1 = 373 and 373 nm and l2 = 620 and 621 nm were measured for [NiL] of (GlyCys)2 and [NiL2]2 + of

Scheme 3.

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Fig. 3. ORTEP view and numbering scheme of [Ni(GlyCys)2·H2O] (50% probability displacement ellipsoids).

glycineamide, respectively. These parameters show a very close similarity in the binding sites, but on the other hand there is a big difference in the complex formation processes of (GlyCys)2 and glycineamide. Namely, in the nickel(II) – glycineamide system the species [NiL2]2 + is present only in a narrow pH range (around pH 8) and by pH 9 it is followed by the complete formation of the square planar and diamagnetic [NiH − 2L − 2] complex with the cooperative deprotonation of two amide nitrogens. This reaction and the formation of square planar complexes, however, can not be detected at any pH or metal ion to ligand ratios in the nickel(II)–(GlyCys)2 system. It supports that the binding mode presented by Scheme 3 prevents deprotonation and coordination of the amide groups and also prevents the interaction with the disulfide bond. The exclusive formation of the species [NiL] in the pH range of 6–9 and its structural peculiarity stimulated us to prepare the complex in the solid state. The compound was obtained with the stoichiometric composition of C10H18N4NiO7S2 ([Ni(GlyCys)2·H2O]) and X-ray quality crystals were grown from a mixture of methanol and water. The crystal structure of the complex [Ni(GlyCys)2·H2O] shows octahedral nickel(II) ions (Fig. 3), but in contrast with Scheme 3 in a coordinatively saturated environment. Four coordination sites of the octahedron are occupied by two aminoN and two carbonyl-O donor atoms, while the remaining two co-ordination sites are occupied by the carboxylate residues. Thus all coordination sites of the metal ions and all possible donor functions of the ligands take part in binding in the solid state. The other difference between the structures obtained in solution and in the solid state is that the (NH2, CO)2 coordination environment exists in a polymeric network in the solid state and that the two five-membered chelate rings

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come from two different ligands. Selected examples of bond lengths and angles are collected in Table 3. It can be seen from Fig. 3 and also from Table 3 that coordination geometry of the central Ni(II) ion corresponds to a slightly distorted octahedron. The bond lengths of the three different types of metal–ligand bond (NiN(amino), NiO(carbonyl) and NiO(carboxylate)) are quite close to each other being in the range 2.070–2.111 A, . There are more significant differences in the bond angles. The smallest values are obtained for the chelating sites N(22)Ni(1)O(20) and N(1)Ni(1)O(4) are 78.2 and 80.7°, respectively. The monodentate carboxylate donor functions are more separated, but they are in cis arrangement, while the two nitrogen donors occupy trans positions. The Ni(II)S distance ranges from 5.864 to 6.603 A, , supporting that the disulfide donors are not involved in metal binding in the solid state either. The complex crystallizes in space group of P3121 with significant solvent accessible void volumes. However, only one solvent water molecule with high isotropic atomic displacement parameter for oxygen (O1w) could be located on the electron density map. The void space can be explained by the polymeric nature of the structure. N1, N5, N18, N22, O18 and O1w donors participate in Table 3 Selected bond lengths (A, ) and angles (°) for the compound [Ni(II)(GlyCys)2·H2O] Ni1N22 Ni1O4 Ni1N1 Ni1O16 Ni1O9 Ni1O20 S12C13 S12S12 a S11C10 S11S11 a O9C7 O20C19 O4C3 O17C15 O16C15

2.070(10) 2.074(10) 2.081(12) 2.092(9) 2.104(9) 2.111(9) 1.817(15) 2.017(9) 1.803(17) 2.043(9) 1.267(17) 1.222(15) 1.292(16) 1.220(18) 1.282(17)

N22Ni1O4 N22Ni1N1 O4Ni1N1 N22Ni1O16 O4Ni1O16 N1Ni1O16 N22Ni1O9 O4Ni1O9 N1Ni1O9 O16Ni1O9 N22Ni1O20 O4Ni1O20 N1Ni1O20 O16Ni1O20 O9Ni1O20

86.1(5) 163.5(5) 80.7(4) 94.9(4) 90.3(4) 95.0(4) 98.4(4) 174.1(4) 94.2(4) 93.0(4) 78.2(4) 88.8(4) 91.6(4) 173.2(4) 88.5(4)

a

−x, −x+y, 4/3−z.

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Fig. 4. Coordination modes of nickel on one (GlyCys)2 ligand. Symmetry code: – 5: − x, − x+ y, 4/3− z.

an infinite H-bond network while N1 and N22 form intramolecular H-bonds, too. For the numbering scheme see Fig. 3. There are 14 and 26 membered macrocycles in the structure formed by the sigma bond network of ligands coordinated to Ni(II). Fig. 4 is another view of the complex showing that one ligand is coordinated to four independent metal ions providing a polymeric network, in which all coordination sites of the metal ions and all donor groups of the ligands (except disulfide sulfur atoms) are involved in coordinative bonds. The results obtained for the structure of [NiL] in the solid state seem to be in contradiction with those suggested in solution. However, it should be considered that the structure presented in Scheme 3 was suggested for rather diluted solutions, where the existence of coordinatively unsaturated metal ions is quite common. Steric requirements rule out the binding of free carboxylate residues to the same metal ion in the monomeric complex, but they can act as additional binding sites for another [NiL] unit in more concentrated solutions. As a consequence, the coordinatively unsaturated loop structure will be destroyed during crystallisation, but the major metal binding sites (NH2, CO)2 will remain the same. The complex formation processes of cobalt(II), zinc(II) and cadmium(II) with (GlyCys)2 are rather similar to those of nickel(II). Namely, the species [ML] predominates in all cases and it should have the same binding sites as reported for the nickel(II) complexes. The corresponding stability constants are, however, smaller than those of nickel(II). This results in two major differences between nickel(II) and the other metal ions. On the one hand, the lower thermodynamic stability of the complexes is not able to prevent metal ion hydrolysis and results in precipitation in slightly basic media. On the other hand, there is an increased tendency for the formation of bis(ligand) complexes. This is especially true for cobalt(II) and cadmium(II), but the ratio of stepwise stability constants is rather high even with these metal ions (log (K1/K2) = 2.15 and

2.40 for cobalt(II) and cadmium(II), respectively) supporting the fact that [ML] is the major species with all metal ions. Proton NMR spectra of the ligand and zinc(II) containing systems provided further evidence for the existence of [ZnL] as major species in zinc(II)– (GlyCys)2 system. Protons of CH2(Gly) moieties are detected as a singlet both in the free ligand and in the zinc(II) complex, but the chemical shift of their protons is a function of pD and the extent of complexation. The NMR peaks were obtained as follows: 3.892 and 3.893 ppm (pD 3.90), 3.871 and 3.674 ppm (pD 6.50), 3.783 and 3.659 ppm (pD 7.40), 3.639 and 3.643 ppm (pD 8.05) for the free ligand and Zn(II): (GlyCys)2 =1:1 solutions, respectively. These data clearly indicate that there is no complex formation below pH 4 while the very small changes of CH2 peaks in the zinc(II) containing systems between pD 6.50 and 8.05 suggest the existence of one major species in agreement with the complexation with nickel(II) shown by Fig. 2. None of the above mentioned results provided evidence for the existence of MS bond in complexes. In some sense it contradicts with our previous expectations based upon the location of disulfide bond in the amino acid sequence of the peptide. After the coordination of the first two nitrogen donors (amino and amide) both carboxylate-O and disulfide-S atoms are in favourable position for metal ion coordination. The preference of (NH2, N−, COO−)-binding to that of (NH2, N−, S) probably comes from the charge neutralization and from the higher flexibility of the small carboxylate function. On the other hand, these results indicate that sulfur atoms of disulfide bonds are even weaker donors towards 3d transition metal ions than the thioether sulfur atoms [26]. To summarize the results obtained for the metal complexes of the disulfide bridged peptide molecule (GlyCys)2 it can be concluded that in the case of 3d transition elements including copper(II), nickel(II), cobalt(II) and zinc(II) the stable coordination of nitrogen and oxygen donors in chelates rules out the formation of MS bonds both in solution and, at least in the case of nickel(II), in the solid state. The complex formation processes of (GlyCys)2 are, however, significantly different from those of simple di- or tetrapeptides (e.g. from diglycine or tetraglycine), which is caused by the presence of the disulfide bonds. In the case of copper(II) it results in the enhanced stability of the species with (N3O) donor sites, [CuH − 1L]−, in equimolar solution and in the formation of dinuclear complexes in the presence of excess of copper(II). As concerns the other 3d transition metal ions the stable coordination of the amino and carbonyl functions prevent amide and disulfide coordination and this results in the formation of a sixteen-membered loop around the central metal ion (see Scheme 3 for nickel(II)). The complexes of cadmium(II) are very similar to those of

´ goston et al. / Polyhedron 19 (2000) 1849–1857 C.G. A

zinc(II) or especially cobalt(II) and the sulfur coordination was ruled out with this metal ion, too. Cadmium(II) generally prefers the coordination of sulfur donor atoms [35], but in this case the binding in a stable chelate should be accompanied by the formation of Cd(II)N(amide) bond, which has not yet been reported in peptides.

4. Supplementary data Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre, CCDC No. 143861. Copies of this may be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (Fax: +44-1223-336033; e-mail: [email protected] or www: http://www.ccdc.cam. ac.uk).

Acknowledgements This work was supported by the Hungarian Scientific Research Fund (OTKA T19337) and by the Ministry of Education (Hungary, FKP0507). The postdoctoral fellowship of A.B. is sponsored by OTKA D25136.

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