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Lead(II) complexes of oligopeptides containing two cysteine residues
Accepted Manuscript Lead(II) complexes of oligopeptides containing two cysteine residues Györgyi Szunyog, Katalin Várnagy PII: DOI: Reference:
S0020-1693(17)30838-1 http://dx.doi.org/10.1016/j.ica.2017.07.067 ICA 17790
To appear in:
Inorganica Chimica Acta
Received Date: Revised Date: Accepted Date:
28 May 2017 20 July 2017 31 July 2017
Please cite this article as: G. Szunyog, K. Várnagy, Lead(II) complexes of oligopeptides containing two cysteine residues, Inorganica Chimica Acta (2017), doi: http://dx.doi.org/10.1016/j.ica.2017.07.067
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Lead(II) complexes of oligopeptides containing two cysteine residues Györgyi Szunyog and Katalin Várnagy*
Department of Inorganic and Analytical Chemistry, University of Debrecen, H-4032 Debrecen, Hungary Abstract The complex formation processes of three oligopeptides (CysSerSerAlaCysSer-NH2, CSSACSNH2; AlaCysSerSerAlaCysSer-NH2, ACSSACS-NH2; SerSerCysSerSerAlaCysSer-NH2, SSCSSACS-NH2) were studied in the presence of toxic lead(II) ions and compared to those of toxic cadmium(II) and essential zinc(II) ions. The stoichiometry and stability constants of the metal complexes were determined by potentiometry, while their structures were supported by means of UV- and NMR-spectroscopy. For hexapeptide containing cysteine in N-terminal position the (NH2,S–) donor groups are the primary metal binding site forming 5-membered chelate ring, and the coordination of C-terminal thiolate group contributes the stability of mono(ligand) complex excluding the formation of bis(ligand) species. The (S–,S–) donor set is, however, the main binding site, if the –CSSACS– sequence is farther from N-terminal amino group in the molecules, and mono- and bis(ligand) complexes are formed. The metal binding affinity of all three ligands are higher for lead(II) ion than zinc(II) ion resulting good selectivity of ligands for lead(II) over zinc(II). This paper is dedicated to Prof. Imre Sóvágó on the occasion of his 70th birthday.
1. Introduction The lead is serious environmental toxin, because it has accumulated in the environment much above the natural level due to the human activity during thousands of years. Based on the soft character of the metal ion, it can bind to the proteins and first of all the side chain thiol groups provide effective binding site for it. As a consequence, this metal ion is able to substitute the essential metal ions (e.g. zinc(II), nickel(II), calcium(II)) in the metalloenzymes and metalloproteins resulting drastic change in their biochemical functions [1-4]. The accumulation *
Corresponding author: Tel.: +36 52 512900/22405 Fax.: +36 52 518660, E-mail: [email protected] (K. Várnagy)
1
of toxic metal ions can cause carcinogenic effect or physiological change in the bones, kidneys or indirectly might take part in the development of neurodegenerative disorders [5-7]. To protect the human body against heavy metal toxicity, cells synthesize cysteine containing and thiol-rich small molecules (glutathione, metallothionein) which can sequester toxic metal ions before they can cause irreversible physiological alteration. On the other hand the chelator therapy could be the most effective method for detoxification of human body. The widely used chelating agents for removal of toxic metal ions are EDTA (ethylenediamine-tetraacetic acid), BAL (2,3mercaptopropanol), DMSA (dimercaptocuccinic acid) and DTPA (diethylenetriaminepentaacetic acid) [8]. An important requirement for ligand to be used in chelation treatment against the metal toxicity is the high selectivity for the metal ion which should be removed with respect to the essential metal ions. According to the experiences, it is relatively easy to find chelator molecules with good selectivity for Pb(II) over Ca(II), but this is not easy in the case over Zn(II), because this latter metal ion usually forms complexes with similar stability to Pb(II) [9]. The systematic studies of complexes formed in Pb(II)-amino acid or small peptides systems reveal that among the amino acids the cysteine and penicillamine [10], while in the case of cysteine containing dipeptides [10] and glutathione [11] show the highest selectivity for Pb(II) over Zn(II). The presence of more sulphur donor atoms in the peptides may increase the metal binding ability and selectivity of the molecules. The previously synthesized and studied hexa- and heptapeptides containing two cysteine residues in separated positions (CSSACS-NH2, ACSSACS-NH2) have outstanding zinc(II) and cadmium(II) binding affinity and the selectivity in the cadmium(II) binding was proved [12]. In the case of the hexapeptide containing N-terminal Cys residue, the amino terminus is the primary metal binding site in the form of a stable (NH2,S–) 5-membered chelate enhanced by coordination of the distant cysteinyl residue. This tridentate coordination mode is especially favored by cadmium(II) and hinders the formation of bis(ligand) complexes. The heptapeptide ACSSACS-NH2 is a slightly less effective metal binder but its coordination chemistry is more versatile. The thiolate groups are the primary binding sites for both metal ions resulting in formation mono- and bis(ligand) complexes. Moreover, the interaction of the terminal amino-N and the thiolate-S of Cys(2) moiety can promote the deprotonation and metal ion coordination of the amide group, [ZnH–1L]– and [CdH–1L]– species exist in basic solution with the (NH2,N–,S–) fused chelates supported by the thiolate of the distant cysteinyl residue. 2
Since lead(II) ion has similar soft character as cadmium(II) ion it seemed to be worth the continuation of these studies on one hand with characterization of lead(II) complexes of both oligopeptides, on the other hand with synthesis and studies of octapeptides containing two cysteine residues. The studied three peptides are CSSACS-NH2 hexapeptide, ACSSACS-NH2 heptapeptide and SSCSSACS-NH2 octapeptide, in which the longer is the peptide the farther is the –CSSAC– sequence from N terminus. It means that in these ligands one cysteine is in the first, second and third N-terminal position, respectively, together with a distant cysteine residue on the C-termini. In this paper we submitted the results obtained from solution equilibrium studies of lead(II) complexes of these oligopeptides and completed these measurements with characterization of Cd(II)- and Zn(II)-complexes of octapeptides in order to compare the metal binding ability and selectivity of all three ligands. 2. Experimental 2.1. Materials N-acetyl-cysteine (N-Ac-Cys) were obtained from Fluka, N-acetyl-penicillamine (N-Ac-Pen) from Bachem and used without further purification. Chemicals and solvents used for synthetic purposes were purchased from commercial sources in the highest available purity and used without further purification. Rink Amide AM resin, 2-(1-H-benzotriazole1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and all Nfluorenylmethoxycarbonyl (Fmoc)-protected amino acids (Fmoc-Cys(Trt)-OH, FmocSer(Trt)-OH and Fmoc-Ala-OH) are Novabiochem (Switzerland) products. 2,2’(ethylenedioxy)diethanethiol (DODT), N-hydroxybenzotriazole (HOBt), N-methylpyrrolidone (NMP), acetic acid (AcOH), acetonitrile (ACN), dichloromethane (DCM), peptide-synthesis grade N,N-dimethylformamide (DMF), diethyl ether (Et2O), piperidine, triisopropylsilane (TIS) and trifluoroacetic acid (TFA) were purchased from SigmaAldrich Co., while N,N-diisopropyl-ethylamine (DIPEA) and 2-methyl-2-butanol was Merck Millipore Co. products. Acetic anhydride (Ac2O) was bought from VWR International. Lead(II)-trifluoracetate ((CF3COO)2Pb) solution was prepared from Pb(OH)2 and TFA. The Pb(OH)2 was get from reaction of lead(II) nitrate with ammonia solution. The exact extra acid content of solution was determined by potentiometric titration using Gran 3
method [13]. Stock solutions of other metal ions were prepared from analytical grade reagents (Pb(NO3)2, Cd(NO3)2, CdSO4 and Zn(NO3)2 (Reanal)) and their concentrations were checked gravimetrically via the precipitation of oxinates or using pH-potentiometric titrations with EDTA. Concentrations of the peptide stock solution were determined by potentiometric titrations.
2.2. Peptide synthesis and purification The peptide sequences (CSSACS-NH2, ACSSACS-NH2, SSCSSACS-NH2, Scheme 1) contain two or more serine amino acids to increase the solubility of the ligands. For these oligopeptides solid phase peptide synthesis was performed using a microwave-assisted Liberty 1 Peptide Synthesizer (CEM, Matthews, NC). Fmoc/tBtu technique and TBTU/HOBt/DIPEA activation strategy were used. The detailed description of the procedure has already been reported in our previous papers [12]. The purity of the synthesized peptides was checked by analytical RPHPLC analyses using a Jasco instrument equipped with a Jasco MD 2010 plus wavelength detector monitoring the absorbance at 222 nm. The chromatographic conditions were the following: Column: Teknokroma Europa Peptide C18 (250 x 4.6 mm, 120 Å pore size, 5 µm particle size); Elution: gradients elution was carried out using solvent A (0.1 % TFA in water) and solvent B (0.1 % TFA in acetonitrile) at a flow rate of 1 mL/min. From 1 min to 15 min 100% to 85% A, from 15 min to 16 min 85% A and from 16 min to 20 min 85% to 100% A was applied. Scheme 1. Scheme 1. The structural formulae of the studied peptides
4
O H2N
CH
C
O H N
CH
C
O H N
CH
CH2
CH2
CH2
SH
OH
OH
C
O H N
CH
C
O H N
CH3
CSSACS-NH2
CH
C
O H N
CH
CH2
CH2
SH
OH
C
NH2
ACSSACS-NH2
SSCSSACS-NH2
2.3. Potentiometric measurements The titrations were accomplished in a 10 or 15 mL samples at 1 mM or 2 mM total ligand concentration with the use of carbonate free stock solution (0.2 M) of potassium hydroxide. The metal to ligand ratios were selected as 1:1 and 1:2. During the titrations, argon was bubbled through the samples to ensure the absence of oxygen and carbon dioxide. All pH-potentiometric measurements were carried out at 298 K and a constant ionic strength of 0.2 M KNO3 was used in all cases to avoid the formation of precipitation (PbCl2) or chlorido complexes. The pH-potentiometric titrations were performed with a computer controlled METTLER TOLEDO titrator equipped with a 6.0255.100 combined glass electrode (Metrohm). The recorded pH readings were converted to hydrogen ion concentration as described by Irving et al. [14]. Protonation constants of the ligands and overall stability (logβpqr) constants of the metal complexes were calculated by means of the general computational programs (PSEQUAD [15] and SUPERQUAD [16]) based on Eqs. (1) and (2). 5
pM + qH + rL = M p H q L r (2)
β pqr =
(1)
[M p H q L r ] [ M ] p ⋅ [ H ]q ⋅ [ L ]r
The hydrolysis models of lead(II) ions were determined in previous works. The following stability constants (log β) were calculated: [Pb(OH)]+ (log β = −7.32), [Pb4(OH)4]4+ (log β = −19.98) and [Pb6(OH)8]4+ (log β = −42.62) (H–1 relates to the metal-induced ionization of the coordination water [17]).
2.4. Spectroscopic studies UV-visible spectra of the cadmium(II) and the lead(II) complexes were recorded from 200 nm to 400 nm on a PerkinElmer Lambda 25 scanning spectrophotometer at 0.15 mM total ligand concentration. To avoid the disturbing effect of UV absorption of nitrate lead(II)-trifluoroacete and cadmium(II)-sulphate solutions were used for the preparation of samples. 400 MHz 1H-NMR, COSY and HSQC spectra were recorded on a Bruker Avance 400 spectrometer at 298 K. In the case of 1H-NMR spectra chemical shifts were referenced to internal sodium 3-(trimethylsilyl)-1-propane sulfonate (TSP, δTSP=0 ppm) and D2O was used as a solvent. DNO3 and NaOD were used to set the pH of the samples.
3. Results and discussion 3.1. Acid-base properties of the ligands The acid-base processes of CSSACS-NH2 and ACSSACS-NH2 were studied earlier [12] and these studies clarified that the deprotonation order follows the pK(NH3+) < pK(N-terminal Cys) < pK(C-terminal Cys) trend. Similar conslusion can be drawn from formation constants and pK values of SSCSSACS-NH2 ligand (Table 1.) Table 1. Its first pK value around 7 belongs mainly to the deprotonation of ammonium group, while the two cysteinyl side chain thiol groups are characterized by higher pK values. These assumptions are established by 1H NMR spectra of the ligands. Similarly to the other two peptides the deprotonation processes of ammonium and thiol groups are overlapped, but the exact calculation 6
of microconstants was not possible because of the significant overlapping of cysteinyl and seryl methylene and methine groups. On the other hand the change of first pK values of the ligands follow an unexpected trend: pK1(CSSACS-NH2) < pK1(SSCSSACS-NH2) < pK1(ACSSACSNH2). The lowest pK1 value of CSSACS-NH2 correspond to the formation of intramolecular interaction between the thiolate group and the terminal amino group. The increasing of the distance between the terminal amino group and cysteinyl side chain explains the higher pK1 value of ACSSACS-NH2, similar trend was observed for pK values of CysGly (pK2 = 6.89) and AlaCys [18] (pK2 = 7.96). The pK2 values of AlaCys and AlaAlaCys [18] (pK2 = 7.97), however are similar to each other, in contrast with those of ACSSACS-NH2 and SSCSSACS-NH2. The difference in the deprotonation processes of terminal amino group of present studied ligands is due to the presence of N-terminal seryl side chain –OH group in the SSCSSACS-NH2 molecule. The interaction between the terminal amino and N-terminal seryl –OH groups significantly decreases the pK(NH3+) value. Similar effect of serine side chains was observed for triserine compared with tetraalanine (SSS [19]: pK(NH3+) = 7.13, AAAA [20]: pK(NH3+) = 8.13) or in the case of rat amylin fragment/mutants [19] (SSNN-NH2: pK(NH3+) = 6.99, SSAA-NH2: pK(NH3+) = 7.01, AANN-NH2: pK(NH3+) = 7.86). The environment of cysteine residues, however, is similar for all three peptides resulting in similar pK2 and pK3 values. 3.2. Lead(II) complexes of CSSACS-NH2 The stoichiometries and stability constants of lead(II) complexes of CSSACS-NH2 are collected in Table 2 together with data of cadmium(II) and zinc(II) complexes and lead(II) complexes of CysGly for comparison. Table 2. It is clear from these data that stoichiometries of complexes are the same for all three metal ions and their stability follow the Cd(II) > Pb(II) > Zn(II) order in all cases. Only 1:1 complexes are formed and the [ML] complexes are the exclusive species around physiological pH (pH ~ 6-8) (Figure 1.) Figure 1. Similarly to the Zn(II) and Cd(II) complexes the N-terminal part of the molecule is the primary binding site, the amino and N-terminal cysteinyl thiolate groups bind the lead(II) ion in the [MHL]+ complex and the other thiolate group on the C-termini is protonated. The same 7
coordination mode was proved previously for [PbHL]+ complex of CysGly, where the (N,S) donor set binds metal ion and the carboxyl group is protonated. For both cases the logK(M+HL) values can be estimated with calculation of logβ(MHL)–pK(SH(av.) (18.57–8.66 = 9.91) for CSSACS-NH2 and logβ(MHL)–pK(COOH) (13.78–3.17=10.61) for CysGly. Taking into account the significant difference between the sizes of molecules these values are in agreement with each other. The deprotonation of [PbHL]+ complex results in the formation of [PbL] complex and this process is characterized by pK = 5.74. This value is lower than the pK values of free ligand but around one log unit higher than that of Zn(II) and Cd(II) complexes. It means that the ligand coordinates all three metal ions tridentately, but the stabilizing effect of C-terminal thiolate group is the lowest in the case of lead(II). The higher stability of [PbL] complexes of CSSACS-NH2 compared to that of CysGly confirms also the coordination of third donor group of CSSACS-NH2 molecule. Moreover, the (NH2,S–,S–) coordination in the Pb(II) complex hinders the hydrolysis of [PbL] species, mixed hydroxido complex exists only above pH 9 and precipitation was not observed in the measurable pH range. The lower pK(hydrolysis) = pK(ML/MH–1L) in the case of CysGly provides an additional proof for stabilizing effect of C-terminal thiolate group via its coordination in [PbL] complex. The formation of complexes can be followed by UV-spectrophotometry. The deprotonation process of thiolate groups of the ligand is accompanied by appearance of a very intensive band around 230-240 nm (ε ∼ 10000 M–1·cm–1). The binding of thiolate group to the lead(II) ion, however, results in a new absorption band in the 290-330 nm range. The series of spectra detected in Pb(II)-CSSACS-NH2 systems at 1:2 and 1:1 metal-ligand ratios are similar to each other supporting that the same complexes are formed in equimolar solution and at excess of ligand and it is in agreement with the fact that presence of bis(ligand) complexes could not be observed even at excess of ligand. The λmax and ε values of different complexes are collected in Table 3. For comparison we measured the UV spectra of Pb(II)-N-acetyl-cysteine (N-Ac-Cys) and N-acetyl-penicillamine (N-Ac-Pen) at 1:2 and 1:1 metal to ligand ratios and Table 3 are completed with their parameters. Table 3. The λmax value of [PbHL]+ species is 293 nm, which corresponds to binding of one thiolate group. Similarly one thiolate donor atom binds metal ion in the [PbL] complex of N-Ac-Cys [10] 8
and N-Ac-Pen [10], and λmax = 276 nm and 277 nm are characteristic for these complexes, respectively. The coordination of more thiolate group usually results in the shift of absorption maximum to higher wavelengts, as it seems for [PbL2]2– complexes of N-Ac-Cys or N-Ac-Pen (Table 3). In the case of CSSACS-NH2 similar shift can be observed for [PbL] due to the presence of two sulphur atom in the coordination sphere. On the other hand the 1H NMR measurements of Pb(II)-CSSACS-NH2 = 1:1 solution proves the tridentate coordination of ligand as well. The 1H NMR spectra of the free peptide and those of the corresponding Pb(II) containing system at pH 7.9 was detected. The significant line broadening in the lead(II) containing system supports the complex formation and the slight change of CH3(Ala) doublet and drastic change of two CH2(Cys) signs confirm the coordination of both thiolate groups in [PbL]2+ complex.
3.3. Lead(II) complexes of ACSSACS-NH2 The stoichiometries and stability constants of complexes formed in Pb(II)-ACSSACS-NH2 system are collected in Table 4 completed with literature data of Cd(II) and Zn(II) complexes of ACSSACS-NH2 and Pb(II) complexes of AlaCys. Table 4. The stoichiometry of complexes formed in the Pb(II)-ACSSACS-NH2 system are different from those of Pb(II)-CSSACS-NH2. Formation of protonated and non-protonated mono- and bisligand complexes can be detected (Figure 2.) Figure 2. Complexes with same stoichiometry exist in the Cd(II)- and Zn(II)-ACSSACS-NH2 systems too. The coordination mode, however, are different in some complexes. For Zn(II) and Cd(II) the (S–,S–) binding mode was detected in the protonated mono- and bis-ligand complexes and only the weak interaction of amino group exists in the [ML] complex. Similarly, the (S–,S–) coordination mode with formation of 18-membered macrochelate could be suggested in [PbHL]+ and [PbL] complexes too. The stability order follows the same trend (Cd(II) > Pb(II) > Zn(II)) for both complexes like in the case of CSSACS-NH2 These observations are in agreement with data obtained for Pb(II)-AlaCys complexes [10] (Table 4). In this ligand the C-termial thiolate group is the primary binding site and this dipeptide binds lead(II) ion through (S–,COO–) donor set. As a consequence the (NH2,S–) coordination is not 9
preferred if the cysteine is in the second position and the NH2 and S– donors are not in chelatable position. The deprotonation process of [PbHL]+ complex of ACSSACS-NH2 takes place at higher pH than in the case of CSSACS-NH2 and the pK(MHL/ML) value is close to the pK values of free ligand. This data suggests the bidentate coordination of the ligand in the [PbL] species as well, which explains the lower stability of [PbL] complex than that of CSSACS-NH2. The 1H NMR data are in agreement with this assumption. The spectra were detected in the D2O solution of ACSSACS-NH2 and in the sample containing lead(II) ion in 1:1 ratio, at different pH. There is no complex formation process around pD 3.6, while [PbHL]+ and [PbL] species dominate at pD 6.5 and 9.5, respectively. (Table 5.) Table 5. The significant line broadening of cysteinyl methylene groups supports the coordination of both thiolate groups in monocomplexes. The sharp signal of methyl group of the alanyl residues, however, can be assigned in all spectra. NMR peaks of methyl group of the terminal alanyl residue are shifted from 1.55 ppm to 1.26 pp in the pH range 6-10, while the signals of the other Ala side chain remain intact in the whole pH range. In the presence of lead(II) ions there are no any measurable changes in the peaks of the terminal alanyl moiety, although the whole amount of ligand is bound in lead(II) complexes. This observations unambigously proves that the N-terminal amino group is protonated in the protonated complexes and the two thiolate residues are the major metal binding sites. A further increase of pH results in the upfield shift of the methyl protons but this occurs in parallel with the free ligand suggesting that the terminal amino group does not take part in the metal binding. On the other hand the presence of two thiolate sulphur atoms in the coordination sphere gives possibility to bind a second ligand forming bis(ligand) complexes and these complexes predominate in the presence of excess of ligand (Fig. 2(b)). The UV spectroscopic measurements give further evidence for coordination of two thiolate groups both in the [PbHL]+ and [PbL] complex, because both complexes are characterized by similar λmax and ε values (Table 3) and these parameters correspond to the presence of two sulphur atom in the coordination sphere of lead(II) ion (similarly to [PbL2]2– complexes of N-Ac-Cys and N-Ac-Pen). The series of UV spectra detected in Pb(II)-ACSSCS-NH2 system at 1:2 ratio, however, shows a significant shift of absorption maximum to the higher wavelenghts and a new absorption band appears at λmax = 10
333 nm (Figure S1 in Supplement). This absorption maximum corresponds to bis(ligand) complexes (Fig. 2b), which suggests the existence of 3S coordinated species. Similar spectroscopic parameters are characteristic for [PbL3] complex of glutathione (λmax=334 nm, ε = 3500 dm3· mol–1·cm–1) [21]. As a consequence the second ligand is bound only monodentately through one thiolate group. It is agreement with the observations that in the cysteine rich environment Pb(II) usually is coordinated in a trigonal pyramidal geometry with a lone pair occupying the apical position. This trigonal pyramidal PbS3 geometry was determined in the binding of lead(II) to cysteine rich, zinc binding proteins [22-24] for example in solid form of Pb(II)-ALAD metalloenzyme [25-27] or in the lead(II) complexes of „de novo designed” TRI family of peptides [28]. The different coordination of two ligands in bis(ligand) complexes explains the unusual high log(K1/K2)H and log(K1/K2) values (Table 4). At high pH the complex formation processes are totally different in the case of Zn(II) or Cd(II) and Pb(II). In the basic solution (NH2,N–,S–) coordinated [ZnH–1L]– and [CdH–1L]– complexes are formed, while lead(II) ion is not able to induce the deprotonation of peptide nitrogen, the [PbH–1L]– species is a mixed hydroxido complex and it is present above pH 10.
3.4. Lead(II), cadmium(II) and zinc(II) complexes of SSCSSACS-NH2 The studies of lead(II) complexes of third ligand were completed by the investigation of its cadmium(II) and zinc(II) complexes. The stability constants are presented in Table 6. Table 6. The stoichiometry of Pb(II) complexes and the complex formation processes are practically the same as those of Pb(II)-ACSSACS-NH2. (Fig. 3) Figure 3. In the [PbHL]+ complex two cysteinyl side chains bind the lead(II) ion, while the terminal amino group is protonated. The coordination of two thiolate groups in the PbL complex is unambigously proved by 1H-13C HSQC spectrum (Figure 4.) too. Figure 4. The spectra of free ligand and Pb(II)-ligand system were registered at pH 5 where the [PbHL]+ complex dominates (Fig 4.). The position of cross peak of N-terminal seryl CH2 and CH group does not affected by addition of lead(II) to the ligand supporting the non-coordinated terminal ammonium group in [PbHL]+ complex. The cross peaks of cysteinyl methylene groups, 11
however, are significantly shifted, which proves the binding of both thiolate groups to lead(II) ion. With the increasing of pH a small difference in the shift of the N-terminal seryl CH sign compared to the free ligand can be observed in the 1H-NMR spectrum, on the basis of which the weak interaction of NH2-group cannot be excluded. The spectral parameters of complexes of SSCSSACS-NH2 and ACSSACS-NH2 are close to each others (Table 3) supporting also the (S–,S–) coordination mode in [PbHL]+ and [PbL] species and (S–,S–)+S– coordination mode in bis(ligand) complexes. The spectra which is characteristic for different complexes of SSCSSACS-NH2 are demonstrated by Figure 5. Figure 5. These coordination modes result in stable [PbL] and [PbL2]2– complexes, which hinder the hydrolysis of metal ion and mixed hidroxido complexes are formed around pH 10. The stoichiometry of complexes formed in the Zn(II)- and Cd(II)-SSCSSACS-NH2 systems seems to be very similar for that of Pb(II) containing system, and the only different is the formation of polynuclear [Cd2H3L3] species in the Cd(II)-SSCSSACS-NH2 system (Figure 6). Figure 6. The detailed analysis, however, shows some other difference between the coordination mode of the complexes with same stoichiometry. In the mono(ligand) complexes two thiolate groups are coordinated both in Cd(II) and Zn(II) complexes. The pK(CdHL/CdL) (Table 6) is lower than pK values of ligand, which suggest the weak interaction of terminal amino group. The 2D homonuclear 1H NMR spectra detected in equimolar Cd(II)-SSCSSACS-NH2 solution at pH 8.5 confirms the supposed binding mode: the presence of the metal ion caused a significant change in the Cys-CH2 resonances (Figure 8). Figure 7. These resonances are the most sensitive for the deprotonation and coordination of thiol groups of the peptide. In addition a small shift of sign of the N-terminal seryl CH proton can be observed at this pH, similarly to [PbL] complexes. The bis(ligand) Cd(II) and Zn(II) complexes are, however, 4S coordinated species similarly to those of ACSSACS-NH2 peptide and unlike from the bis(ligand) complexes formed in Pb(II)-SSCSSACS-NH2 system, which can be explained by different coordination geometry of the metal complexes. The coordination of two or four thiolate group in mono- and bis(ligand) 12
complexes, respectively was supported by UV spectroscopic measurements too. The series of spectra detected in the solution containing free ligand and Cd(II)-SSCSSACS-NH2 in 1:2 ratio show that both the deprotonation of free ligand and formation of Cd(II) complexes are accompanied by appearance of UV band around 230-240 nm. Therefore the single spectra of the deprotonated form of the ligand and different cadmium(II) complexes was calculated by means of PSEQUAD program. The molar absorption coefficient are presented in Table 7 together with some literature data for comparison. Table 7. The molar absorption coefficient of free ligand is close to those of mononuclear complexes, while the values becomed doubled for bis(ligand) complexes. All the UV parameters of free peptide, mono- and bis(ligand) complexes are in good agreement with those of previously studied one cysteine containing hexapeptide [29] (ADAAAC-NH2, Table 7), where the molar absorption coefficient of the 2S coordinated cadmium(II) complexes falls in the 9000-11300 M–1·cm–1 range. On the other hand the formation of polynuclear species can be observed in the Cd(II)SSCSSACS-NH2 system. In this species thiolate groups behave as bridging ligand. The existence of polynuclear structures was proved in numerous cysteine or penicillamine derivatives, e.g. Cys-OMe, N-Ac-Cys, N-Ac-Pen etc.[18] In the case of this octapeptide the formation of polynuclear complexes could be supported on one hand by UV spectroscopic measurements. As it seen from Table 7 an extremly high molar absorbtion coefficient belongs to the [Cd2H3L3]+ species reinforced the polynuclear structure. On the other hand similar conlusion can be drawn from the 1H NMR spectra registered in Cd(II)-SSCSSACS-NH2 solution at 1:2 metal-ligand ratio. In the spectra detected at pH 5 two sign can be assigned to the methylene protons of cysteinyl residues: one sharper peak at 2.96 ppm corresponds to the non-coordinated, while a wide peak at 3.12 ppm to the bounded cysteinyl side chain protons, respectively. This means the formation of those complexes in which the Cd:L ratio is less than 1:2, indirectly confiming the existence of polynuclear species, in which this ratio is 1:1.5. The increasing of the pH (pH 8.5) results in the disappear of the sign of free ligand, which supports the coordination of all thiolate group in CdL2 complex.
13
Conclusion A hexa-, hepta- and octa-peptides containing two cysteines were synthesised, in which one cysteine is the first, second or third N-terminal position, respectively, while the second cysteine is on the C-termini. The lead(II) complexes of these ligands were studied and compared the complex formation processes to those of zinc(II) and cadmium(II) containing systems. It can be concluded that similarly to other two metal ions lead(II) forms very stable complexes with all three oligopeptides. The stoichiometry of complexes are usually similar in all three cases, but there are some differences in the coordination modes. These data reveal that for all three ligands the stability of complexes follows the Cd(II) > Pb(II) > Zn(II) order. This is well demonstrated by Figure 8, where the stability constants of [ML] complex are depicted. Figure 8. This diagram reflects that the stability of [ML] complex of ligand containing N-terminal cysteine group is different from that of other two peptides due to the stable tridentate coordination of the ligand. If the –CSSAC– sequence is farther from the N-terminal part of the molecule the (S–,S–) sequence is the main binding site and the contribution of N-terminal amino group in the metal binding significantly decreases or it is negligible. The formation of bis(ligand) complexes can be observed only for the ACSSACS-NH2 and SSCSSACS-NH2. The main difference is between these bis(ligand) complexes is that Pb(II) is bounded through 3S– donor atoms, while the 2×(S– ,S–) coordination mode is characteristic for CdHxL2 and ZnHxL2 (x=0-2) complexes, which can be explained the different coordination geometry of metal ions. As a summary we can state that these ligands have high affinity to bind zinc(II), cadmium(II) and lead(II) ion. The higher thermodynamic stability of lead(II) and cadmium(II) complexes, however, results in a good selectivity for Pb(II) and/or Cd(II) over Zn(II) and this selectivity increases with the increase of distance between the terminal amino group and –CSSAC– sequences. This selectivity is demonstrated by theoretical distribution curves of the complexes formed in Pb(II):Cd(II):Zn(II):ligand = 1:1:1:1 systems (Figure 9, S3, S4) and by diagram (Figure S5), where the mole ratio of different metal complexes at pH 7.4 is depicted. Figure 9.
14
Acknowledgements The research was supported by the EU and co-financed by the European Regional Development Fund under the project GINOP-2.3.2-15-2016-00008 and the Hungarian Scientific Research Fund (K115480).
References [1] R.M. Harrison, D.R.H. Laxen, Lead Pollution, Chapman and Hall, London, 1981. [2] H.G. Sailer (ed.) Handbook on Toxicity of Inorganic Compounds, Chap. 31, Marcel Dekker, New York, 1988. [3] A. Sigel, H. Sigel and K.O.R. Sigel (eds.) Lead: Its Effect on Environment and Health, Metal Ions in Life Sciences, Vol. 17, Walter De Gruyter Inc., Hawthorne, New York, 2017. [4] N.H. Zawia, T. Crupmton, M. Brydie, G.R. Reddy, M. Razmiafshari, Neurotoxicology, 21 (2000) 1069-1080. [5] M.A. Variety, Neurotoxicology 20 (1999) 489-497. [6] P.S. Barry, D.B. Mossman, Br. J. Ind. Med. 27 (1970) 339-351. [7] D.H. Hare, N.G. Faux, B.R. Roberts, I. Volitakis, R.N. Martins, A.I. Busch, Metallomics 8 (2016) 628-632. [8] O. Andersen, Chem. Rev. 99 (1999)2683-2710. [9] R. Ferreirós-Martínez, D. Esteban-Gómez, C. Platas-Iglesias, A. de Blas, T. Rodíguer-Blas, Inorg. Chem. 48 (2009) 10976-10987. [10] E. Farkas, B. Bóka, B. Szőcs, A.J. Godó, I. Sóvágó, Inorg. Chim. Acta 423 (2014) 242-249. [11] K.P. Neupane, V.L. Pecoraro, J. Inorg. Biochem. 105 (2011) 1030-1034. [12] N. Lihi, Á. Grenács, S. Timári, I. Turi, I. Bányai, I. Sóvágó, K. Várnagy, New J. Chem. 83 (2015) 8364-8372. [13] G. Gran, Acta Chem. Scand. 4 (1950) 559-577. [14] H. Irving, G. Miles, L. D. Pettit, Anal. Chim. Acta, 38 (1967) 475-488. [15] P. Gans, A. Sabatini, A. Vacca, J. Chem. Soc., Dalton Trans., (1985) 1195-1200. [16] L. Zékány, I. Nagypál, in: D.J. Leggett (Ed.), Computational Methods for the Determination of Formation Constants, Plenum Press, New York (1985) 291-299. [17] E. Farkas, D. Bátka, Z. Pataki, P. Buglyó, M.A. Santos, Dalton Trans. (2004) 1248-1253. [18] H. Kozlowski, J. Urbanska, I. Sóvágó, K. Várnagy, A. Kiss, J. Spychala, K. Cherifi, Polyhedron 9 (1990) 831837. [19] Á. Dávid, C. Kállay, D. Sanna, N. Lihi, I. Sóvágó, K. Várnagy, Dalton Trans. 44 (2015) 17091–17099. [20] J.-F. Galey, B. Decock-Le Reverend, A. Lebkiri, L. D. Pettit, S. I. Pyburn, H. Kozlowski, J. Chem. Soc., Dalton Trans. (1991) 2281-2287. [21] K.P. Neupane, V.L. Pecoraro, J. Inorg. Biochem. 105 (2011) 1030-1034. [22] J.C. Payne, M.A. Horst, H.A. Godwin, J. Am. Chem. Soc. 121 (1990) 6850-6855. [23] A.B. Ghering, L.M.M. Jenkins, B.L. Schenk, S. Deo, R.A. Mayer, M.J. Pikaart, J.G. Omichinski, H.A. Godwin, J. Am. Chem. Soc. 127 (2005) 3751-3759. [24] J.S. Magyar, T.C. Weng, M.S. Charlotte, D.F. Dye, B.W. Rous, J.C. Payne, B.M. Bridgewater, A. Mijovilovich, G. Parkin, J.M. Zaleski, J.E. Penner-Hahn, H.A. Godwin, J. Am. Chem. Soc. 127 (2005) 9495[25] P.T. Erskine, E.M.H. Duke, I.J. Tickle, N.M. Senior, M.J. Warren, J.B. Cooper, Acta Crystallogr. Spect. D 56 (2000) 421-430. [26] C.W. Nogueira, F.A. Soares, P.C. Nascimento, D. Muller, J.B.T. Rocha, Toxicology 184 (2003) 85-95. [27] V.M. Morsch, M.R.C. Schetinger, A.F. Martins, J.B. T. Rocha, Biol. Plant 45 (2001) 85-89. [28] M. Matzapetakis, D. Ghosh, T.-C. Weng, J.E. Penner-Hahn, V.L. Pecoraro, J. Biol. Inorg. Chem. 11 (2006) 876-890. [29] N. Lihi, M. Lukács, D. Szűcs, K. Várnagy, I. Sóvágó, Polyhedron, 133 (2017) 364-373.
15
Scheme 1. The structural formulae of the studied peptides O H2N
CH
C
O H N
CH
C
O H N
CH
CH2
CH2
CH2
SH
OH
OH
C
O H N
CH
C
O H N
CH3
CSSACS-NH2
CH
H N
CH
CH2
CH2
SH
OH
ACSSACS-NH2
SSCSSACS-NH2
16
C
O C
NH2
Figure 1 Concentration distribution curves of the complexes formed in the lead(II)–CSSACS-NH2 system at 1 : 2 metal-ligand ratio (solid lines) and the absorption values at 308 nm (squares) in function of pH (cL = 0.2 mM).
1
Pb(II)
[PbL]
0.4
0.8 0.3
0.6
0.2
0.4
[PbH-1L]-
0.1
0.2
0
0
4.00
5.00
6.00
7.00
8.00
17
9.00
pH
10.00
A (308 nm)
Fraction of Pb(II)
[PbHL]+
Figure 2. Concentration distribution curves of the complexes formed in the lead(II)–ACSSACS-NH2 system at 1 : 1 (a) and 1 : 2 ratio (b) in the function of pH (cL = 0.3 mM). 1
[PbHL]+
Pb(II)
[PbHL]+
1
Pb(II)
[PbL]
0.8
0.8
Fraction of Pb(II)
[PbL2]-2 Fraction of Pb(II)
0.6
0.4
[PbH-1L]-
0.6
0.4
[PbH2L2]
0.2
[PbHL2]-
[PbL]
0.2
0
[PbH-1L]-
0
4.00
5.00
6.00
7.00
8.00
9.00
10.00
4.00
5.00
6.00
7.00
pH
(a)
(b)
18
8.00
9.00
pH
10.00
Figure 3 Concentration distribution curves of the complexes formed in the lead(II)–SSCSSACS-NH2 system at 1 : 1 ratio (cL = 5 mM). 1
[PbHL]+ [PbL]
Fraction of Pb(II)
0.8
[PbH-1L]-
0.6
0.4
Pb(II) 0.2
0 4.00
5.00
6.00
7.00
8.00
19
9.00
pH
10.00
Figure 4. Selected region of 1H – 13C HSQC spectra of the free ligand(red) and the lead(II) (blue) MHL complex.
-CH2 (Cys) [PbHL]+
-CH2 (Cys) ligand
-CH (Ser, N-term.) -CH2 (Ser, N-term.)
20
Figure 5. The single UV-spectra of complexes formed in lead(II)-SSCSSACS-NH2 system; a: [PbHL]+ (pH = 5.6, 1:1 ratio), b: [PbL] (pH = 7.7, 1:1 ratio), c: [PbH–1L]– (pH 11.0, 1:1 ratio), d: [PbL2]2– (pH = 9.1, 1:2 metal ion ligand ratio) 1
ε 0.9 0.8 0.7
c
0.6
b
a
0.5 0.4
d
0.3 0.2 0.1 0 260
280
300
320
340
360
λ (nm)
21
380
400
Figure 6. Concentration distribution curves of the complexes formed in the cadmium(II)–SSCSSACSNH2 system at 1 : 2 ratio (cL = 5 mM) 1.0
[Cd2H3L3]+ [CdHL2]-
[CdL2]2-
Fraction of Cd(II)
0.8
0.6
0.4
[CdHL]+
0.2
Cd(II) [CdL]
0.0 4.00
5.00
6.00
7.00
8.00
22
9.00
pH
10.00
Figure 7. 1H NMR COSY spectra of [CdL] (pH 8.5) (dark purple) compared with SSCSSACSNH2 ligand (red)
-CH (Ser, N-term.)
-CH2 (Cys)
23
Figure 8. The stability constants of [ML] complexes of three studied oligopeptides log β 16 14 12 PbL CdL ZnL
10 8 6 4 2 0
CSSACS-NH2
ACSSACS-NH2
SSCSSACS-NH2
24
Figure 9 Theoretical distribution curves of complexes formed in the system containing Pb(II):Cd(II):Zn(II)- SSCSSACS-NH2 in 1:1:1:1 ratio (cM = cL = 0.1 mM) 1 0.9
Fraction of SSCSSACS-NH2
0.8
Cd(II) complexes
free ligand
0.7 0.6 0.5 0.4
Pb(II) complexes
0.3 0.2 0.1
Zn(II) complexes 0 4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
pH
25
8.00
8.50
9.00
Table 1 Protonation (logβ) and deprotonation (pK) constants of the ligands. I = 0.2 M (KNO3), T = 298 K logβ(HL–) logβ(H2L) logβ(H3L+) pK1 pK2 pK3
CSSACS-NH2 9.08(1) 17.32(1) 23.83(1) 6.51(1) 8.24(1) 9.08(1)
ACSSACS-NH2 8.96(1) 17.19(1) 24.74(1) 7.55(1) 8.23(1) 8.96(1)
26
SSCSSACS-NH2 8.83(1) 16.89(1) 23.73(1) 6.84(1) 8.06(1) 8.83(1)
Table 2. Stability constants of the lead(II), cadmium(II) and zinc(II) complexes of CSSACS-NH2 and lead(II) complexes of CysGly. I = 0.2 M, KNO3 for Pb(II) and KCl for Cd(II) and Zn(II), T = 298 K
logβ [MH2L]2+ [MHL]+ [ML] [MH–1L]– [MH2L2] [MHL2]– [ML2]2– [ML3]4– pK(MHL/ML) pK(ML/MH–1L)
CSSACS-NH2 Pb(II)
Cd(II) [12]
Zn(II) [12]
—
—
—
18.57(4) 12.83(7) 2.8(1)
18.87 14.38 3.20
16.79 12.02 2.99
— — — —
— — — —
— — — —
5.74 10.0
4.49 11.18
4.77 9.03
27
CysGly [10] Pb(II) 17.96 13.78 10.40 2.32 28.46 23.26 15.96 19.0 3.38 8.08
Table 3 Spectroscopic data of lead(II) complexes of ligands containing thiol functional group
[MHL]+ [ML] [MHxL2]2–x (x=0-2)
N-Ac-Cys λ ε — — 276 2400 309 2600
N-Ac-Pen λ ε — — 277 2800 313 2200
CSSACS-NH2 λ ε 293 3000 306 4000 — —
28
ACSSACS-NH2 λ ε 312 3600 303 5000 333 4500
SSCSSACS-NH2 λ ε 307 2800 307 3000 331 6000
Table 4. Stability constants of the lead(II), cadmium(II) and zinc(II) complexes of ACSSACSNH2 and lead(II) complexes of AlaCys. I = 0.2 M, KNO3 for Pb(II) and KCl for Cd(II) and Zn(II), T = 298 K ACSSACS-NH2 logβ Pb(II) [MH2L]2+ — [MHL]+ 18.39(3) [ML] 10.15 (7) – [MH–1L] 0.0(5) [MH2L2] 31.9(1) – [MHL2] 23.6(2) [ML2]2– 15.0(1) pK(MHL/ML) 8.24 pK(ML/MH–1L) 10.1 pK(MH2L2/MHL2) 8.3 pK(MHL2/ML2) 8.6 log(K1/K2)H 4.9 log(K1/K2) 5.3
Cd(II) [12] — 19.23 11.98 3.41 34.35 26.40 17.29 7.25 8.57 7.95 9.11 4.11 6.67
29
Zn(II) [12] — 17.00 9.88 1.92 32.68 24.94 16.80 7.12 7.96 7.74 8.14 1.32 2.96
AlaCys [10] Pb(II) 20.49 15.99 8.3 0.80 30.1 22.0 13.7 7.7 7.5 8.1 8.3 1.88 2.9
Table 5. The NMR chemical shifts (ppm) measured in the solution of free ligand and in equimolar solution of Pb(II)–ACSSACS-NH2 system at different pH values
pH 2xCys-CH2 +Pb(II) 2.95, 2.97 3.0 (wide) ∼3.5 2.95, 2.96 a ∼6.5 2.86, 2.88 b ∼9.5 a wide, not well resolved signal b
Ala(1)-CH3 1.54, 1.55 1.51, 1.53 1.26, 1.27
no evaluable signals
30
+Pb(II) 1.52, 1.54 1.51, 1.53 1.27, 1.29
Ala(5)-CH3 1.40, 1.42 1.41, 1.43 1.41, 1.42
+Pb(II) 1.38, 1.40 1.38, 1.40 1.38, 1.40
Table 6. Stability constants of the lead(II), cadmium(II) and zinc(II) complexes of SSCSSACSNH2. I = 0.2 M (KNO3), T = 298 K
[MHL]+ [ML] [MH–1L]– [MH–2L]– [M2H3L3]+ [MH2L2] [MHL2]– [ML2]2– pK(MHL/ML) pK(ML/MH–1L) pK(MH2L2/MHL2) pK(MHL2/ML2) log(K1/K2)H log(K1/K2)
Pb(II) 17.76(1) 10.66(3) 1.41(4)
Cd(II) 18.15(2) 11.77(5) 1.69(5)
— —
—
Zn(II) 15.56(2) 8.54(6) 0.61(3) –9.53(4)
56.6(2)
—
—
30.69(3)
26.61(7) 17.6(1) 6.38 10.08
16.60(6) 7.02 7.93
31.61(6) 24.04(7) 15.89(6) 7.10 9.25 7.57 8.15 3.91 5.43
—
8.97 —
5.94
31
7.04 0.43 0.48
Table 7. The UV spectroscopic parameters of Cd(II) complexes of SSCSSACS-NH2 and ADAAAC-NH2 ligands
species 2–
L [CdHL]+ [CdL] [CdH–1L] [Cd2H3L3]+ [CdH2L2] [CdHL2]– [CdL2]2–
SSCSSACS-NH2 λmax (nm) 232 238 238 238 238 — 238 238
–1
–1
ε (M ·cm ) 12000 9300 8800 11600 43000 — 24700 22200
32
ADAAAC-NH2 [29] λmax (nm) ε (M–1·cm–1) 232 6400 232 5000 232 5200 232 5300 — — 232 9000 232 11300 232 10700
[PbL2]2– L = SSCSSACS-NH2
The lead(II) complexes of three oligopeptides containing two cysteinyl residues were characterized and compared to those of cadmium(II) and zinc(II) ions. The metal binding affinity of all three ligands are higher for lead(II)
ion than zinc(II) ion resulting good selectivity of ligands for Pb(II) over Zn(II).
33
Highlights • • • •
oligopeptides containing two cysteinyl residues form stable lead(II) complexes the two thiolate group are the main binding site for lead(II) ion binding affinity of the studied ligands follows the Cd(II) > Pb(II) > Zn(II) order
the peptides have a good selectivity for Pb(II) and/or Cd(II) over Zn(II)
34
S0020-1693(17)30838-1 http://dx.doi.org/10.1016/j.ica.2017.07.067 ICA 17790
To appear in:
Inorganica Chimica Acta
Received Date: Revised Date: Accepted Date:
28 May 2017 20 July 2017 31 July 2017
Please cite this article as: G. Szunyog, K. Várnagy, Lead(II) complexes of oligopeptides containing two cysteine residues, Inorganica Chimica Acta (2017), doi: http://dx.doi.org/10.1016/j.ica.2017.07.067
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Lead(II) complexes of oligopeptides containing two cysteine residues Györgyi Szunyog and Katalin Várnagy*
Department of Inorganic and Analytical Chemistry, University of Debrecen, H-4032 Debrecen, Hungary Abstract The complex formation processes of three oligopeptides (CysSerSerAlaCysSer-NH2, CSSACSNH2; AlaCysSerSerAlaCysSer-NH2, ACSSACS-NH2; SerSerCysSerSerAlaCysSer-NH2, SSCSSACS-NH2) were studied in the presence of toxic lead(II) ions and compared to those of toxic cadmium(II) and essential zinc(II) ions. The stoichiometry and stability constants of the metal complexes were determined by potentiometry, while their structures were supported by means of UV- and NMR-spectroscopy. For hexapeptide containing cysteine in N-terminal position the (NH2,S–) donor groups are the primary metal binding site forming 5-membered chelate ring, and the coordination of C-terminal thiolate group contributes the stability of mono(ligand) complex excluding the formation of bis(ligand) species. The (S–,S–) donor set is, however, the main binding site, if the –CSSACS– sequence is farther from N-terminal amino group in the molecules, and mono- and bis(ligand) complexes are formed. The metal binding affinity of all three ligands are higher for lead(II) ion than zinc(II) ion resulting good selectivity of ligands for lead(II) over zinc(II). This paper is dedicated to Prof. Imre Sóvágó on the occasion of his 70th birthday.
1. Introduction The lead is serious environmental toxin, because it has accumulated in the environment much above the natural level due to the human activity during thousands of years. Based on the soft character of the metal ion, it can bind to the proteins and first of all the side chain thiol groups provide effective binding site for it. As a consequence, this metal ion is able to substitute the essential metal ions (e.g. zinc(II), nickel(II), calcium(II)) in the metalloenzymes and metalloproteins resulting drastic change in their biochemical functions [1-4]. The accumulation *
Corresponding author: Tel.: +36 52 512900/22405 Fax.: +36 52 518660, E-mail: [email protected] (K. Várnagy)
1
of toxic metal ions can cause carcinogenic effect or physiological change in the bones, kidneys or indirectly might take part in the development of neurodegenerative disorders [5-7]. To protect the human body against heavy metal toxicity, cells synthesize cysteine containing and thiol-rich small molecules (glutathione, metallothionein) which can sequester toxic metal ions before they can cause irreversible physiological alteration. On the other hand the chelator therapy could be the most effective method for detoxification of human body. The widely used chelating agents for removal of toxic metal ions are EDTA (ethylenediamine-tetraacetic acid), BAL (2,3mercaptopropanol), DMSA (dimercaptocuccinic acid) and DTPA (diethylenetriaminepentaacetic acid) [8]. An important requirement for ligand to be used in chelation treatment against the metal toxicity is the high selectivity for the metal ion which should be removed with respect to the essential metal ions. According to the experiences, it is relatively easy to find chelator molecules with good selectivity for Pb(II) over Ca(II), but this is not easy in the case over Zn(II), because this latter metal ion usually forms complexes with similar stability to Pb(II) [9]. The systematic studies of complexes formed in Pb(II)-amino acid or small peptides systems reveal that among the amino acids the cysteine and penicillamine [10], while in the case of cysteine containing dipeptides [10] and glutathione [11] show the highest selectivity for Pb(II) over Zn(II). The presence of more sulphur donor atoms in the peptides may increase the metal binding ability and selectivity of the molecules. The previously synthesized and studied hexa- and heptapeptides containing two cysteine residues in separated positions (CSSACS-NH2, ACSSACS-NH2) have outstanding zinc(II) and cadmium(II) binding affinity and the selectivity in the cadmium(II) binding was proved [12]. In the case of the hexapeptide containing N-terminal Cys residue, the amino terminus is the primary metal binding site in the form of a stable (NH2,S–) 5-membered chelate enhanced by coordination of the distant cysteinyl residue. This tridentate coordination mode is especially favored by cadmium(II) and hinders the formation of bis(ligand) complexes. The heptapeptide ACSSACS-NH2 is a slightly less effective metal binder but its coordination chemistry is more versatile. The thiolate groups are the primary binding sites for both metal ions resulting in formation mono- and bis(ligand) complexes. Moreover, the interaction of the terminal amino-N and the thiolate-S of Cys(2) moiety can promote the deprotonation and metal ion coordination of the amide group, [ZnH–1L]– and [CdH–1L]– species exist in basic solution with the (NH2,N–,S–) fused chelates supported by the thiolate of the distant cysteinyl residue. 2
Since lead(II) ion has similar soft character as cadmium(II) ion it seemed to be worth the continuation of these studies on one hand with characterization of lead(II) complexes of both oligopeptides, on the other hand with synthesis and studies of octapeptides containing two cysteine residues. The studied three peptides are CSSACS-NH2 hexapeptide, ACSSACS-NH2 heptapeptide and SSCSSACS-NH2 octapeptide, in which the longer is the peptide the farther is the –CSSAC– sequence from N terminus. It means that in these ligands one cysteine is in the first, second and third N-terminal position, respectively, together with a distant cysteine residue on the C-termini. In this paper we submitted the results obtained from solution equilibrium studies of lead(II) complexes of these oligopeptides and completed these measurements with characterization of Cd(II)- and Zn(II)-complexes of octapeptides in order to compare the metal binding ability and selectivity of all three ligands. 2. Experimental 2.1. Materials N-acetyl-cysteine (N-Ac-Cys) were obtained from Fluka, N-acetyl-penicillamine (N-Ac-Pen) from Bachem and used without further purification. Chemicals and solvents used for synthetic purposes were purchased from commercial sources in the highest available purity and used without further purification. Rink Amide AM resin, 2-(1-H-benzotriazole1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and all Nfluorenylmethoxycarbonyl (Fmoc)-protected amino acids (Fmoc-Cys(Trt)-OH, FmocSer(Trt)-OH and Fmoc-Ala-OH) are Novabiochem (Switzerland) products. 2,2’(ethylenedioxy)diethanethiol (DODT), N-hydroxybenzotriazole (HOBt), N-methylpyrrolidone (NMP), acetic acid (AcOH), acetonitrile (ACN), dichloromethane (DCM), peptide-synthesis grade N,N-dimethylformamide (DMF), diethyl ether (Et2O), piperidine, triisopropylsilane (TIS) and trifluoroacetic acid (TFA) were purchased from SigmaAldrich Co., while N,N-diisopropyl-ethylamine (DIPEA) and 2-methyl-2-butanol was Merck Millipore Co. products. Acetic anhydride (Ac2O) was bought from VWR International. Lead(II)-trifluoracetate ((CF3COO)2Pb) solution was prepared from Pb(OH)2 and TFA. The Pb(OH)2 was get from reaction of lead(II) nitrate with ammonia solution. The exact extra acid content of solution was determined by potentiometric titration using Gran 3
method [13]. Stock solutions of other metal ions were prepared from analytical grade reagents (Pb(NO3)2, Cd(NO3)2, CdSO4 and Zn(NO3)2 (Reanal)) and their concentrations were checked gravimetrically via the precipitation of oxinates or using pH-potentiometric titrations with EDTA. Concentrations of the peptide stock solution were determined by potentiometric titrations.
2.2. Peptide synthesis and purification The peptide sequences (CSSACS-NH2, ACSSACS-NH2, SSCSSACS-NH2, Scheme 1) contain two or more serine amino acids to increase the solubility of the ligands. For these oligopeptides solid phase peptide synthesis was performed using a microwave-assisted Liberty 1 Peptide Synthesizer (CEM, Matthews, NC). Fmoc/tBtu technique and TBTU/HOBt/DIPEA activation strategy were used. The detailed description of the procedure has already been reported in our previous papers [12]. The purity of the synthesized peptides was checked by analytical RPHPLC analyses using a Jasco instrument equipped with a Jasco MD 2010 plus wavelength detector monitoring the absorbance at 222 nm. The chromatographic conditions were the following: Column: Teknokroma Europa Peptide C18 (250 x 4.6 mm, 120 Å pore size, 5 µm particle size); Elution: gradients elution was carried out using solvent A (0.1 % TFA in water) and solvent B (0.1 % TFA in acetonitrile) at a flow rate of 1 mL/min. From 1 min to 15 min 100% to 85% A, from 15 min to 16 min 85% A and from 16 min to 20 min 85% to 100% A was applied. Scheme 1. Scheme 1. The structural formulae of the studied peptides
4
O H2N
CH
C
O H N
CH
C
O H N
CH
CH2
CH2
CH2
SH
OH
OH
C
O H N
CH
C
O H N
CH3
CSSACS-NH2
CH
C
O H N
CH
CH2
CH2
SH
OH
C
NH2
ACSSACS-NH2
SSCSSACS-NH2
2.3. Potentiometric measurements The titrations were accomplished in a 10 or 15 mL samples at 1 mM or 2 mM total ligand concentration with the use of carbonate free stock solution (0.2 M) of potassium hydroxide. The metal to ligand ratios were selected as 1:1 and 1:2. During the titrations, argon was bubbled through the samples to ensure the absence of oxygen and carbon dioxide. All pH-potentiometric measurements were carried out at 298 K and a constant ionic strength of 0.2 M KNO3 was used in all cases to avoid the formation of precipitation (PbCl2) or chlorido complexes. The pH-potentiometric titrations were performed with a computer controlled METTLER TOLEDO titrator equipped with a 6.0255.100 combined glass electrode (Metrohm). The recorded pH readings were converted to hydrogen ion concentration as described by Irving et al. [14]. Protonation constants of the ligands and overall stability (logβpqr) constants of the metal complexes were calculated by means of the general computational programs (PSEQUAD [15] and SUPERQUAD [16]) based on Eqs. (1) and (2). 5
pM + qH + rL = M p H q L r (2)
β pqr =
(1)
[M p H q L r ] [ M ] p ⋅ [ H ]q ⋅ [ L ]r
The hydrolysis models of lead(II) ions were determined in previous works. The following stability constants (log β) were calculated: [Pb(OH)]+ (log β = −7.32), [Pb4(OH)4]4+ (log β = −19.98) and [Pb6(OH)8]4+ (log β = −42.62) (H–1 relates to the metal-induced ionization of the coordination water [17]).
2.4. Spectroscopic studies UV-visible spectra of the cadmium(II) and the lead(II) complexes were recorded from 200 nm to 400 nm on a PerkinElmer Lambda 25 scanning spectrophotometer at 0.15 mM total ligand concentration. To avoid the disturbing effect of UV absorption of nitrate lead(II)-trifluoroacete and cadmium(II)-sulphate solutions were used for the preparation of samples. 400 MHz 1H-NMR, COSY and HSQC spectra were recorded on a Bruker Avance 400 spectrometer at 298 K. In the case of 1H-NMR spectra chemical shifts were referenced to internal sodium 3-(trimethylsilyl)-1-propane sulfonate (TSP, δTSP=0 ppm) and D2O was used as a solvent. DNO3 and NaOD were used to set the pH of the samples.
3. Results and discussion 3.1. Acid-base properties of the ligands The acid-base processes of CSSACS-NH2 and ACSSACS-NH2 were studied earlier [12] and these studies clarified that the deprotonation order follows the pK(NH3+) < pK(N-terminal Cys) < pK(C-terminal Cys) trend. Similar conslusion can be drawn from formation constants and pK values of SSCSSACS-NH2 ligand (Table 1.) Table 1. Its first pK value around 7 belongs mainly to the deprotonation of ammonium group, while the two cysteinyl side chain thiol groups are characterized by higher pK values. These assumptions are established by 1H NMR spectra of the ligands. Similarly to the other two peptides the deprotonation processes of ammonium and thiol groups are overlapped, but the exact calculation 6
of microconstants was not possible because of the significant overlapping of cysteinyl and seryl methylene and methine groups. On the other hand the change of first pK values of the ligands follow an unexpected trend: pK1(CSSACS-NH2) < pK1(SSCSSACS-NH2) < pK1(ACSSACSNH2). The lowest pK1 value of CSSACS-NH2 correspond to the formation of intramolecular interaction between the thiolate group and the terminal amino group. The increasing of the distance between the terminal amino group and cysteinyl side chain explains the higher pK1 value of ACSSACS-NH2, similar trend was observed for pK values of CysGly (pK2 = 6.89) and AlaCys [18] (pK2 = 7.96). The pK2 values of AlaCys and AlaAlaCys [18] (pK2 = 7.97), however are similar to each other, in contrast with those of ACSSACS-NH2 and SSCSSACS-NH2. The difference in the deprotonation processes of terminal amino group of present studied ligands is due to the presence of N-terminal seryl side chain –OH group in the SSCSSACS-NH2 molecule. The interaction between the terminal amino and N-terminal seryl –OH groups significantly decreases the pK(NH3+) value. Similar effect of serine side chains was observed for triserine compared with tetraalanine (SSS [19]: pK(NH3+) = 7.13, AAAA [20]: pK(NH3+) = 8.13) or in the case of rat amylin fragment/mutants [19] (SSNN-NH2: pK(NH3+) = 6.99, SSAA-NH2: pK(NH3+) = 7.01, AANN-NH2: pK(NH3+) = 7.86). The environment of cysteine residues, however, is similar for all three peptides resulting in similar pK2 and pK3 values. 3.2. Lead(II) complexes of CSSACS-NH2 The stoichiometries and stability constants of lead(II) complexes of CSSACS-NH2 are collected in Table 2 together with data of cadmium(II) and zinc(II) complexes and lead(II) complexes of CysGly for comparison. Table 2. It is clear from these data that stoichiometries of complexes are the same for all three metal ions and their stability follow the Cd(II) > Pb(II) > Zn(II) order in all cases. Only 1:1 complexes are formed and the [ML] complexes are the exclusive species around physiological pH (pH ~ 6-8) (Figure 1.) Figure 1. Similarly to the Zn(II) and Cd(II) complexes the N-terminal part of the molecule is the primary binding site, the amino and N-terminal cysteinyl thiolate groups bind the lead(II) ion in the [MHL]+ complex and the other thiolate group on the C-termini is protonated. The same 7
coordination mode was proved previously for [PbHL]+ complex of CysGly, where the (N,S) donor set binds metal ion and the carboxyl group is protonated. For both cases the logK(M+HL) values can be estimated with calculation of logβ(MHL)–pK(SH(av.) (18.57–8.66 = 9.91) for CSSACS-NH2 and logβ(MHL)–pK(COOH) (13.78–3.17=10.61) for CysGly. Taking into account the significant difference between the sizes of molecules these values are in agreement with each other. The deprotonation of [PbHL]+ complex results in the formation of [PbL] complex and this process is characterized by pK = 5.74. This value is lower than the pK values of free ligand but around one log unit higher than that of Zn(II) and Cd(II) complexes. It means that the ligand coordinates all three metal ions tridentately, but the stabilizing effect of C-terminal thiolate group is the lowest in the case of lead(II). The higher stability of [PbL] complexes of CSSACS-NH2 compared to that of CysGly confirms also the coordination of third donor group of CSSACS-NH2 molecule. Moreover, the (NH2,S–,S–) coordination in the Pb(II) complex hinders the hydrolysis of [PbL] species, mixed hydroxido complex exists only above pH 9 and precipitation was not observed in the measurable pH range. The lower pK(hydrolysis) = pK(ML/MH–1L) in the case of CysGly provides an additional proof for stabilizing effect of C-terminal thiolate group via its coordination in [PbL] complex. The formation of complexes can be followed by UV-spectrophotometry. The deprotonation process of thiolate groups of the ligand is accompanied by appearance of a very intensive band around 230-240 nm (ε ∼ 10000 M–1·cm–1). The binding of thiolate group to the lead(II) ion, however, results in a new absorption band in the 290-330 nm range. The series of spectra detected in Pb(II)-CSSACS-NH2 systems at 1:2 and 1:1 metal-ligand ratios are similar to each other supporting that the same complexes are formed in equimolar solution and at excess of ligand and it is in agreement with the fact that presence of bis(ligand) complexes could not be observed even at excess of ligand. The λmax and ε values of different complexes are collected in Table 3. For comparison we measured the UV spectra of Pb(II)-N-acetyl-cysteine (N-Ac-Cys) and N-acetyl-penicillamine (N-Ac-Pen) at 1:2 and 1:1 metal to ligand ratios and Table 3 are completed with their parameters. Table 3. The λmax value of [PbHL]+ species is 293 nm, which corresponds to binding of one thiolate group. Similarly one thiolate donor atom binds metal ion in the [PbL] complex of N-Ac-Cys [10] 8
and N-Ac-Pen [10], and λmax = 276 nm and 277 nm are characteristic for these complexes, respectively. The coordination of more thiolate group usually results in the shift of absorption maximum to higher wavelengts, as it seems for [PbL2]2– complexes of N-Ac-Cys or N-Ac-Pen (Table 3). In the case of CSSACS-NH2 similar shift can be observed for [PbL] due to the presence of two sulphur atom in the coordination sphere. On the other hand the 1H NMR measurements of Pb(II)-CSSACS-NH2 = 1:1 solution proves the tridentate coordination of ligand as well. The 1H NMR spectra of the free peptide and those of the corresponding Pb(II) containing system at pH 7.9 was detected. The significant line broadening in the lead(II) containing system supports the complex formation and the slight change of CH3(Ala) doublet and drastic change of two CH2(Cys) signs confirm the coordination of both thiolate groups in [PbL]2+ complex.
3.3. Lead(II) complexes of ACSSACS-NH2 The stoichiometries and stability constants of complexes formed in Pb(II)-ACSSACS-NH2 system are collected in Table 4 completed with literature data of Cd(II) and Zn(II) complexes of ACSSACS-NH2 and Pb(II) complexes of AlaCys. Table 4. The stoichiometry of complexes formed in the Pb(II)-ACSSACS-NH2 system are different from those of Pb(II)-CSSACS-NH2. Formation of protonated and non-protonated mono- and bisligand complexes can be detected (Figure 2.) Figure 2. Complexes with same stoichiometry exist in the Cd(II)- and Zn(II)-ACSSACS-NH2 systems too. The coordination mode, however, are different in some complexes. For Zn(II) and Cd(II) the (S–,S–) binding mode was detected in the protonated mono- and bis-ligand complexes and only the weak interaction of amino group exists in the [ML] complex. Similarly, the (S–,S–) coordination mode with formation of 18-membered macrochelate could be suggested in [PbHL]+ and [PbL] complexes too. The stability order follows the same trend (Cd(II) > Pb(II) > Zn(II)) for both complexes like in the case of CSSACS-NH2 These observations are in agreement with data obtained for Pb(II)-AlaCys complexes [10] (Table 4). In this ligand the C-termial thiolate group is the primary binding site and this dipeptide binds lead(II) ion through (S–,COO–) donor set. As a consequence the (NH2,S–) coordination is not 9
preferred if the cysteine is in the second position and the NH2 and S– donors are not in chelatable position. The deprotonation process of [PbHL]+ complex of ACSSACS-NH2 takes place at higher pH than in the case of CSSACS-NH2 and the pK(MHL/ML) value is close to the pK values of free ligand. This data suggests the bidentate coordination of the ligand in the [PbL] species as well, which explains the lower stability of [PbL] complex than that of CSSACS-NH2. The 1H NMR data are in agreement with this assumption. The spectra were detected in the D2O solution of ACSSACS-NH2 and in the sample containing lead(II) ion in 1:1 ratio, at different pH. There is no complex formation process around pD 3.6, while [PbHL]+ and [PbL] species dominate at pD 6.5 and 9.5, respectively. (Table 5.) Table 5. The significant line broadening of cysteinyl methylene groups supports the coordination of both thiolate groups in monocomplexes. The sharp signal of methyl group of the alanyl residues, however, can be assigned in all spectra. NMR peaks of methyl group of the terminal alanyl residue are shifted from 1.55 ppm to 1.26 pp in the pH range 6-10, while the signals of the other Ala side chain remain intact in the whole pH range. In the presence of lead(II) ions there are no any measurable changes in the peaks of the terminal alanyl moiety, although the whole amount of ligand is bound in lead(II) complexes. This observations unambigously proves that the N-terminal amino group is protonated in the protonated complexes and the two thiolate residues are the major metal binding sites. A further increase of pH results in the upfield shift of the methyl protons but this occurs in parallel with the free ligand suggesting that the terminal amino group does not take part in the metal binding. On the other hand the presence of two thiolate sulphur atoms in the coordination sphere gives possibility to bind a second ligand forming bis(ligand) complexes and these complexes predominate in the presence of excess of ligand (Fig. 2(b)). The UV spectroscopic measurements give further evidence for coordination of two thiolate groups both in the [PbHL]+ and [PbL] complex, because both complexes are characterized by similar λmax and ε values (Table 3) and these parameters correspond to the presence of two sulphur atom in the coordination sphere of lead(II) ion (similarly to [PbL2]2– complexes of N-Ac-Cys and N-Ac-Pen). The series of UV spectra detected in Pb(II)-ACSSCS-NH2 system at 1:2 ratio, however, shows a significant shift of absorption maximum to the higher wavelenghts and a new absorption band appears at λmax = 10
333 nm (Figure S1 in Supplement). This absorption maximum corresponds to bis(ligand) complexes (Fig. 2b), which suggests the existence of 3S coordinated species. Similar spectroscopic parameters are characteristic for [PbL3] complex of glutathione (λmax=334 nm, ε = 3500 dm3· mol–1·cm–1) [21]. As a consequence the second ligand is bound only monodentately through one thiolate group. It is agreement with the observations that in the cysteine rich environment Pb(II) usually is coordinated in a trigonal pyramidal geometry with a lone pair occupying the apical position. This trigonal pyramidal PbS3 geometry was determined in the binding of lead(II) to cysteine rich, zinc binding proteins [22-24] for example in solid form of Pb(II)-ALAD metalloenzyme [25-27] or in the lead(II) complexes of „de novo designed” TRI family of peptides [28]. The different coordination of two ligands in bis(ligand) complexes explains the unusual high log(K1/K2)H and log(K1/K2) values (Table 4). At high pH the complex formation processes are totally different in the case of Zn(II) or Cd(II) and Pb(II). In the basic solution (NH2,N–,S–) coordinated [ZnH–1L]– and [CdH–1L]– complexes are formed, while lead(II) ion is not able to induce the deprotonation of peptide nitrogen, the [PbH–1L]– species is a mixed hydroxido complex and it is present above pH 10.
3.4. Lead(II), cadmium(II) and zinc(II) complexes of SSCSSACS-NH2 The studies of lead(II) complexes of third ligand were completed by the investigation of its cadmium(II) and zinc(II) complexes. The stability constants are presented in Table 6. Table 6. The stoichiometry of Pb(II) complexes and the complex formation processes are practically the same as those of Pb(II)-ACSSACS-NH2. (Fig. 3) Figure 3. In the [PbHL]+ complex two cysteinyl side chains bind the lead(II) ion, while the terminal amino group is protonated. The coordination of two thiolate groups in the PbL complex is unambigously proved by 1H-13C HSQC spectrum (Figure 4.) too. Figure 4. The spectra of free ligand and Pb(II)-ligand system were registered at pH 5 where the [PbHL]+ complex dominates (Fig 4.). The position of cross peak of N-terminal seryl CH2 and CH group does not affected by addition of lead(II) to the ligand supporting the non-coordinated terminal ammonium group in [PbHL]+ complex. The cross peaks of cysteinyl methylene groups, 11
however, are significantly shifted, which proves the binding of both thiolate groups to lead(II) ion. With the increasing of pH a small difference in the shift of the N-terminal seryl CH sign compared to the free ligand can be observed in the 1H-NMR spectrum, on the basis of which the weak interaction of NH2-group cannot be excluded. The spectral parameters of complexes of SSCSSACS-NH2 and ACSSACS-NH2 are close to each others (Table 3) supporting also the (S–,S–) coordination mode in [PbHL]+ and [PbL] species and (S–,S–)+S– coordination mode in bis(ligand) complexes. The spectra which is characteristic for different complexes of SSCSSACS-NH2 are demonstrated by Figure 5. Figure 5. These coordination modes result in stable [PbL] and [PbL2]2– complexes, which hinder the hydrolysis of metal ion and mixed hidroxido complexes are formed around pH 10. The stoichiometry of complexes formed in the Zn(II)- and Cd(II)-SSCSSACS-NH2 systems seems to be very similar for that of Pb(II) containing system, and the only different is the formation of polynuclear [Cd2H3L3] species in the Cd(II)-SSCSSACS-NH2 system (Figure 6). Figure 6. The detailed analysis, however, shows some other difference between the coordination mode of the complexes with same stoichiometry. In the mono(ligand) complexes two thiolate groups are coordinated both in Cd(II) and Zn(II) complexes. The pK(CdHL/CdL) (Table 6) is lower than pK values of ligand, which suggest the weak interaction of terminal amino group. The 2D homonuclear 1H NMR spectra detected in equimolar Cd(II)-SSCSSACS-NH2 solution at pH 8.5 confirms the supposed binding mode: the presence of the metal ion caused a significant change in the Cys-CH2 resonances (Figure 8). Figure 7. These resonances are the most sensitive for the deprotonation and coordination of thiol groups of the peptide. In addition a small shift of sign of the N-terminal seryl CH proton can be observed at this pH, similarly to [PbL] complexes. The bis(ligand) Cd(II) and Zn(II) complexes are, however, 4S coordinated species similarly to those of ACSSACS-NH2 peptide and unlike from the bis(ligand) complexes formed in Pb(II)-SSCSSACS-NH2 system, which can be explained by different coordination geometry of the metal complexes. The coordination of two or four thiolate group in mono- and bis(ligand) 12
complexes, respectively was supported by UV spectroscopic measurements too. The series of spectra detected in the solution containing free ligand and Cd(II)-SSCSSACS-NH2 in 1:2 ratio show that both the deprotonation of free ligand and formation of Cd(II) complexes are accompanied by appearance of UV band around 230-240 nm. Therefore the single spectra of the deprotonated form of the ligand and different cadmium(II) complexes was calculated by means of PSEQUAD program. The molar absorption coefficient are presented in Table 7 together with some literature data for comparison. Table 7. The molar absorption coefficient of free ligand is close to those of mononuclear complexes, while the values becomed doubled for bis(ligand) complexes. All the UV parameters of free peptide, mono- and bis(ligand) complexes are in good agreement with those of previously studied one cysteine containing hexapeptide [29] (ADAAAC-NH2, Table 7), where the molar absorption coefficient of the 2S coordinated cadmium(II) complexes falls in the 9000-11300 M–1·cm–1 range. On the other hand the formation of polynuclear species can be observed in the Cd(II)SSCSSACS-NH2 system. In this species thiolate groups behave as bridging ligand. The existence of polynuclear structures was proved in numerous cysteine or penicillamine derivatives, e.g. Cys-OMe, N-Ac-Cys, N-Ac-Pen etc.[18] In the case of this octapeptide the formation of polynuclear complexes could be supported on one hand by UV spectroscopic measurements. As it seen from Table 7 an extremly high molar absorbtion coefficient belongs to the [Cd2H3L3]+ species reinforced the polynuclear structure. On the other hand similar conlusion can be drawn from the 1H NMR spectra registered in Cd(II)-SSCSSACS-NH2 solution at 1:2 metal-ligand ratio. In the spectra detected at pH 5 two sign can be assigned to the methylene protons of cysteinyl residues: one sharper peak at 2.96 ppm corresponds to the non-coordinated, while a wide peak at 3.12 ppm to the bounded cysteinyl side chain protons, respectively. This means the formation of those complexes in which the Cd:L ratio is less than 1:2, indirectly confiming the existence of polynuclear species, in which this ratio is 1:1.5. The increasing of the pH (pH 8.5) results in the disappear of the sign of free ligand, which supports the coordination of all thiolate group in CdL2 complex.
13
Conclusion A hexa-, hepta- and octa-peptides containing two cysteines were synthesised, in which one cysteine is the first, second or third N-terminal position, respectively, while the second cysteine is on the C-termini. The lead(II) complexes of these ligands were studied and compared the complex formation processes to those of zinc(II) and cadmium(II) containing systems. It can be concluded that similarly to other two metal ions lead(II) forms very stable complexes with all three oligopeptides. The stoichiometry of complexes are usually similar in all three cases, but there are some differences in the coordination modes. These data reveal that for all three ligands the stability of complexes follows the Cd(II) > Pb(II) > Zn(II) order. This is well demonstrated by Figure 8, where the stability constants of [ML] complex are depicted. Figure 8. This diagram reflects that the stability of [ML] complex of ligand containing N-terminal cysteine group is different from that of other two peptides due to the stable tridentate coordination of the ligand. If the –CSSAC– sequence is farther from the N-terminal part of the molecule the (S–,S–) sequence is the main binding site and the contribution of N-terminal amino group in the metal binding significantly decreases or it is negligible. The formation of bis(ligand) complexes can be observed only for the ACSSACS-NH2 and SSCSSACS-NH2. The main difference is between these bis(ligand) complexes is that Pb(II) is bounded through 3S– donor atoms, while the 2×(S– ,S–) coordination mode is characteristic for CdHxL2 and ZnHxL2 (x=0-2) complexes, which can be explained the different coordination geometry of metal ions. As a summary we can state that these ligands have high affinity to bind zinc(II), cadmium(II) and lead(II) ion. The higher thermodynamic stability of lead(II) and cadmium(II) complexes, however, results in a good selectivity for Pb(II) and/or Cd(II) over Zn(II) and this selectivity increases with the increase of distance between the terminal amino group and –CSSAC– sequences. This selectivity is demonstrated by theoretical distribution curves of the complexes formed in Pb(II):Cd(II):Zn(II):ligand = 1:1:1:1 systems (Figure 9, S3, S4) and by diagram (Figure S5), where the mole ratio of different metal complexes at pH 7.4 is depicted. Figure 9.
14
Acknowledgements The research was supported by the EU and co-financed by the European Regional Development Fund under the project GINOP-2.3.2-15-2016-00008 and the Hungarian Scientific Research Fund (K115480).
References [1] R.M. Harrison, D.R.H. Laxen, Lead Pollution, Chapman and Hall, London, 1981. [2] H.G. Sailer (ed.) Handbook on Toxicity of Inorganic Compounds, Chap. 31, Marcel Dekker, New York, 1988. [3] A. Sigel, H. Sigel and K.O.R. Sigel (eds.) Lead: Its Effect on Environment and Health, Metal Ions in Life Sciences, Vol. 17, Walter De Gruyter Inc., Hawthorne, New York, 2017. [4] N.H. Zawia, T. Crupmton, M. Brydie, G.R. Reddy, M. Razmiafshari, Neurotoxicology, 21 (2000) 1069-1080. [5] M.A. Variety, Neurotoxicology 20 (1999) 489-497. [6] P.S. Barry, D.B. Mossman, Br. J. Ind. Med. 27 (1970) 339-351. [7] D.H. Hare, N.G. Faux, B.R. Roberts, I. Volitakis, R.N. Martins, A.I. Busch, Metallomics 8 (2016) 628-632. [8] O. Andersen, Chem. Rev. 99 (1999)2683-2710. [9] R. Ferreirós-Martínez, D. Esteban-Gómez, C. Platas-Iglesias, A. de Blas, T. Rodíguer-Blas, Inorg. Chem. 48 (2009) 10976-10987. [10] E. Farkas, B. Bóka, B. Szőcs, A.J. Godó, I. Sóvágó, Inorg. Chim. Acta 423 (2014) 242-249. [11] K.P. Neupane, V.L. Pecoraro, J. Inorg. Biochem. 105 (2011) 1030-1034. [12] N. Lihi, Á. Grenács, S. Timári, I. Turi, I. Bányai, I. Sóvágó, K. Várnagy, New J. Chem. 83 (2015) 8364-8372. [13] G. Gran, Acta Chem. Scand. 4 (1950) 559-577. [14] H. Irving, G. Miles, L. D. Pettit, Anal. Chim. Acta, 38 (1967) 475-488. [15] P. Gans, A. Sabatini, A. Vacca, J. Chem. Soc., Dalton Trans., (1985) 1195-1200. [16] L. Zékány, I. Nagypál, in: D.J. Leggett (Ed.), Computational Methods for the Determination of Formation Constants, Plenum Press, New York (1985) 291-299. [17] E. Farkas, D. Bátka, Z. Pataki, P. Buglyó, M.A. Santos, Dalton Trans. (2004) 1248-1253. [18] H. Kozlowski, J. Urbanska, I. Sóvágó, K. Várnagy, A. Kiss, J. Spychala, K. Cherifi, Polyhedron 9 (1990) 831837. [19] Á. Dávid, C. Kállay, D. Sanna, N. Lihi, I. Sóvágó, K. Várnagy, Dalton Trans. 44 (2015) 17091–17099. [20] J.-F. Galey, B. Decock-Le Reverend, A. Lebkiri, L. D. Pettit, S. I. Pyburn, H. Kozlowski, J. Chem. Soc., Dalton Trans. (1991) 2281-2287. [21] K.P. Neupane, V.L. Pecoraro, J. Inorg. Biochem. 105 (2011) 1030-1034. [22] J.C. Payne, M.A. Horst, H.A. Godwin, J. Am. Chem. Soc. 121 (1990) 6850-6855. [23] A.B. Ghering, L.M.M. Jenkins, B.L. Schenk, S. Deo, R.A. Mayer, M.J. Pikaart, J.G. Omichinski, H.A. Godwin, J. Am. Chem. Soc. 127 (2005) 3751-3759. [24] J.S. Magyar, T.C. Weng, M.S. Charlotte, D.F. Dye, B.W. Rous, J.C. Payne, B.M. Bridgewater, A. Mijovilovich, G. Parkin, J.M. Zaleski, J.E. Penner-Hahn, H.A. Godwin, J. Am. Chem. Soc. 127 (2005) 9495[25] P.T. Erskine, E.M.H. Duke, I.J. Tickle, N.M. Senior, M.J. Warren, J.B. Cooper, Acta Crystallogr. Spect. D 56 (2000) 421-430. [26] C.W. Nogueira, F.A. Soares, P.C. Nascimento, D. Muller, J.B.T. Rocha, Toxicology 184 (2003) 85-95. [27] V.M. Morsch, M.R.C. Schetinger, A.F. Martins, J.B. T. Rocha, Biol. Plant 45 (2001) 85-89. [28] M. Matzapetakis, D. Ghosh, T.-C. Weng, J.E. Penner-Hahn, V.L. Pecoraro, J. Biol. Inorg. Chem. 11 (2006) 876-890. [29] N. Lihi, M. Lukács, D. Szűcs, K. Várnagy, I. Sóvágó, Polyhedron, 133 (2017) 364-373.
15
Scheme 1. The structural formulae of the studied peptides O H2N
CH
C
O H N
CH
C
O H N
CH
CH2
CH2
CH2
SH
OH
OH
C
O H N
CH
C
O H N
CH3
CSSACS-NH2
CH
H N
CH
CH2
CH2
SH
OH
ACSSACS-NH2
SSCSSACS-NH2
16
C
O C
NH2
Figure 1 Concentration distribution curves of the complexes formed in the lead(II)–CSSACS-NH2 system at 1 : 2 metal-ligand ratio (solid lines) and the absorption values at 308 nm (squares) in function of pH (cL = 0.2 mM).
1
Pb(II)
[PbL]
0.4
0.8 0.3
0.6
0.2
0.4
[PbH-1L]-
0.1
0.2
0
0
4.00
5.00
6.00
7.00
8.00
17
9.00
pH
10.00
A (308 nm)
Fraction of Pb(II)
[PbHL]+
Figure 2. Concentration distribution curves of the complexes formed in the lead(II)–ACSSACS-NH2 system at 1 : 1 (a) and 1 : 2 ratio (b) in the function of pH (cL = 0.3 mM). 1
[PbHL]+
Pb(II)
[PbHL]+
1
Pb(II)
[PbL]
0.8
0.8
Fraction of Pb(II)
[PbL2]-2 Fraction of Pb(II)
0.6
0.4
[PbH-1L]-
0.6
0.4
[PbH2L2]
0.2
[PbHL2]-
[PbL]
0.2
0
[PbH-1L]-
0
4.00
5.00
6.00
7.00
8.00
9.00
10.00
4.00
5.00
6.00
7.00
pH
(a)
(b)
18
8.00
9.00
pH
10.00
Figure 3 Concentration distribution curves of the complexes formed in the lead(II)–SSCSSACS-NH2 system at 1 : 1 ratio (cL = 5 mM). 1
[PbHL]+ [PbL]
Fraction of Pb(II)
0.8
[PbH-1L]-
0.6
0.4
Pb(II) 0.2
0 4.00
5.00
6.00
7.00
8.00
19
9.00
pH
10.00
Figure 4. Selected region of 1H – 13C HSQC spectra of the free ligand(red) and the lead(II) (blue) MHL complex.
-CH2 (Cys) [PbHL]+
-CH2 (Cys) ligand
-CH (Ser, N-term.) -CH2 (Ser, N-term.)
20
Figure 5. The single UV-spectra of complexes formed in lead(II)-SSCSSACS-NH2 system; a: [PbHL]+ (pH = 5.6, 1:1 ratio), b: [PbL] (pH = 7.7, 1:1 ratio), c: [PbH–1L]– (pH 11.0, 1:1 ratio), d: [PbL2]2– (pH = 9.1, 1:2 metal ion ligand ratio) 1
ε 0.9 0.8 0.7
c
0.6
b
a
0.5 0.4
d
0.3 0.2 0.1 0 260
280
300
320
340
360
λ (nm)
21
380
400
Figure 6. Concentration distribution curves of the complexes formed in the cadmium(II)–SSCSSACSNH2 system at 1 : 2 ratio (cL = 5 mM) 1.0
[Cd2H3L3]+ [CdHL2]-
[CdL2]2-
Fraction of Cd(II)
0.8
0.6
0.4
[CdHL]+
0.2
Cd(II) [CdL]
0.0 4.00
5.00
6.00
7.00
8.00
22
9.00
pH
10.00
Figure 7. 1H NMR COSY spectra of [CdL] (pH 8.5) (dark purple) compared with SSCSSACSNH2 ligand (red)
-CH (Ser, N-term.)
-CH2 (Cys)
23
Figure 8. The stability constants of [ML] complexes of three studied oligopeptides log β 16 14 12 PbL CdL ZnL
10 8 6 4 2 0
CSSACS-NH2
ACSSACS-NH2
SSCSSACS-NH2
24
Figure 9 Theoretical distribution curves of complexes formed in the system containing Pb(II):Cd(II):Zn(II)- SSCSSACS-NH2 in 1:1:1:1 ratio (cM = cL = 0.1 mM) 1 0.9
Fraction of SSCSSACS-NH2
0.8
Cd(II) complexes
free ligand
0.7 0.6 0.5 0.4
Pb(II) complexes
0.3 0.2 0.1
Zn(II) complexes 0 4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
pH
25
8.00
8.50
9.00
Table 1 Protonation (logβ) and deprotonation (pK) constants of the ligands. I = 0.2 M (KNO3), T = 298 K logβ(HL–) logβ(H2L) logβ(H3L+) pK1 pK2 pK3
CSSACS-NH2 9.08(1) 17.32(1) 23.83(1) 6.51(1) 8.24(1) 9.08(1)
ACSSACS-NH2 8.96(1) 17.19(1) 24.74(1) 7.55(1) 8.23(1) 8.96(1)
26
SSCSSACS-NH2 8.83(1) 16.89(1) 23.73(1) 6.84(1) 8.06(1) 8.83(1)
Table 2. Stability constants of the lead(II), cadmium(II) and zinc(II) complexes of CSSACS-NH2 and lead(II) complexes of CysGly. I = 0.2 M, KNO3 for Pb(II) and KCl for Cd(II) and Zn(II), T = 298 K
logβ [MH2L]2+ [MHL]+ [ML] [MH–1L]– [MH2L2] [MHL2]– [ML2]2– [ML3]4– pK(MHL/ML) pK(ML/MH–1L)
CSSACS-NH2 Pb(II)
Cd(II) [12]
Zn(II) [12]
—
—
—
18.57(4) 12.83(7) 2.8(1)
18.87 14.38 3.20
16.79 12.02 2.99
— — — —
— — — —
— — — —
5.74 10.0
4.49 11.18
4.77 9.03
27
CysGly [10] Pb(II) 17.96 13.78 10.40 2.32 28.46 23.26 15.96 19.0 3.38 8.08
Table 3 Spectroscopic data of lead(II) complexes of ligands containing thiol functional group
[MHL]+ [ML] [MHxL2]2–x (x=0-2)
N-Ac-Cys λ ε — — 276 2400 309 2600
N-Ac-Pen λ ε — — 277 2800 313 2200
CSSACS-NH2 λ ε 293 3000 306 4000 — —
28
ACSSACS-NH2 λ ε 312 3600 303 5000 333 4500
SSCSSACS-NH2 λ ε 307 2800 307 3000 331 6000
Table 4. Stability constants of the lead(II), cadmium(II) and zinc(II) complexes of ACSSACSNH2 and lead(II) complexes of AlaCys. I = 0.2 M, KNO3 for Pb(II) and KCl for Cd(II) and Zn(II), T = 298 K ACSSACS-NH2 logβ Pb(II) [MH2L]2+ — [MHL]+ 18.39(3) [ML] 10.15 (7) – [MH–1L] 0.0(5) [MH2L2] 31.9(1) – [MHL2] 23.6(2) [ML2]2– 15.0(1) pK(MHL/ML) 8.24 pK(ML/MH–1L) 10.1 pK(MH2L2/MHL2) 8.3 pK(MHL2/ML2) 8.6 log(K1/K2)H 4.9 log(K1/K2) 5.3
Cd(II) [12] — 19.23 11.98 3.41 34.35 26.40 17.29 7.25 8.57 7.95 9.11 4.11 6.67
29
Zn(II) [12] — 17.00 9.88 1.92 32.68 24.94 16.80 7.12 7.96 7.74 8.14 1.32 2.96
AlaCys [10] Pb(II) 20.49 15.99 8.3 0.80 30.1 22.0 13.7 7.7 7.5 8.1 8.3 1.88 2.9
Table 5. The NMR chemical shifts (ppm) measured in the solution of free ligand and in equimolar solution of Pb(II)–ACSSACS-NH2 system at different pH values
pH 2xCys-CH2 +Pb(II) 2.95, 2.97 3.0 (wide) ∼3.5 2.95, 2.96 a ∼6.5 2.86, 2.88 b ∼9.5 a wide, not well resolved signal b
Ala(1)-CH3 1.54, 1.55 1.51, 1.53 1.26, 1.27
no evaluable signals
30
+Pb(II) 1.52, 1.54 1.51, 1.53 1.27, 1.29
Ala(5)-CH3 1.40, 1.42 1.41, 1.43 1.41, 1.42
+Pb(II) 1.38, 1.40 1.38, 1.40 1.38, 1.40
Table 6. Stability constants of the lead(II), cadmium(II) and zinc(II) complexes of SSCSSACSNH2. I = 0.2 M (KNO3), T = 298 K
[MHL]+ [ML] [MH–1L]– [MH–2L]– [M2H3L3]+ [MH2L2] [MHL2]– [ML2]2– pK(MHL/ML) pK(ML/MH–1L) pK(MH2L2/MHL2) pK(MHL2/ML2) log(K1/K2)H log(K1/K2)
Pb(II) 17.76(1) 10.66(3) 1.41(4)
Cd(II) 18.15(2) 11.77(5) 1.69(5)
— —
—
Zn(II) 15.56(2) 8.54(6) 0.61(3) –9.53(4)
56.6(2)
—
—
30.69(3)
26.61(7) 17.6(1) 6.38 10.08
16.60(6) 7.02 7.93
31.61(6) 24.04(7) 15.89(6) 7.10 9.25 7.57 8.15 3.91 5.43
—
8.97 —
5.94
31
7.04 0.43 0.48
Table 7. The UV spectroscopic parameters of Cd(II) complexes of SSCSSACS-NH2 and ADAAAC-NH2 ligands
species 2–
L [CdHL]+ [CdL] [CdH–1L] [Cd2H3L3]+ [CdH2L2] [CdHL2]– [CdL2]2–
SSCSSACS-NH2 λmax (nm) 232 238 238 238 238 — 238 238
–1
–1
ε (M ·cm ) 12000 9300 8800 11600 43000 — 24700 22200
32
ADAAAC-NH2 [29] λmax (nm) ε (M–1·cm–1) 232 6400 232 5000 232 5200 232 5300 — — 232 9000 232 11300 232 10700
[PbL2]2– L = SSCSSACS-NH2
The lead(II) complexes of three oligopeptides containing two cysteinyl residues were characterized and compared to those of cadmium(II) and zinc(II) ions. The metal binding affinity of all three ligands are higher for lead(II)
ion than zinc(II) ion resulting good selectivity of ligands for Pb(II) over Zn(II).
33
Highlights • • • •
oligopeptides containing two cysteinyl residues form stable lead(II) complexes the two thiolate group are the main binding site for lead(II) ion binding affinity of the studied ligands follows the Cd(II) > Pb(II) > Zn(II) order
the peptides have a good selectivity for Pb(II) and/or Cd(II) over Zn(II)
34