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Mixed-ligand complexes of copper(II) ions with AMP and CMP in the systems with polyamines and non-covalent interaction between bioligands
www.elsevier.nl/locate/jinorgbio Journal of Inorganic Biochemistry 78 (2000) 139–147
Mixed-ligand complexes of copper(II) ions with AMP and CMP in the systems with polyamines and non-covalent interaction between bioligands Anna Gasowska, Lechoslaw Lomozik *, Renata Jastrzab Faculty of Chemistry, A. Mickiewicz University, 60-780 Poznan, Poland Received 15 April 1999; received in revised form 26 November 1999; accepted 30 November 1999
Abstract The occurrence of non-covalent interactions and formation of molecular complexes between adenosine 59-monophosphate (AMP) or cytidine 59-monophosphate (CMP) and the polyamines, putrescine, 1,7-diamino-4-azaheptane (3,3-tri), spermidine and 1,11-diamino-4,8diazaundecane (3,3,3-tet), were detected in metal-free systems. The stoichiometric composition of the adducts and their stability constants were determined on the basis of computer analysis of the titration data, taking into account the fact that the acid–base properties of the system change as a result of these interactions. Spectral analysis allowed an identification of the interaction centers in the adducts as protonated amine groups of polyamines, phosphate groups as well as nitrogen atoms of high electron density from nucleotides. Unexpectedly, no participation of the phosphate group from AMP in the formation of molecular complexes with tetramine-3,3,3-tet was detected. The stoichiometric composition and stability constants of mixed-ligand complexes in the systems of Cu(II) with AMP or CMP and polyamines were obtained. Analysis of the results of equilibrium studies and 13C, 31P NMR, UV–Vis, IR and EPR data permitted determination of the mode of coordination. In the systems with metal ions, the formation of molecular complexes Cu(CMP)H4(3,3-tri) was found, apart from heteroligand complexes of the MLL9 and MLL9Hx type. In protonated complexes the occurrence of non-covalent interactions leading to stabilization of the coordination compounds was observed. The differences in the character of coordination biogenic amines and their biologically inactive analogs were identified. q2000 Elsevier Science Inc. All rights reserved. Keywords: Copper(II) complexes; Adenosine 59-monophosphate; Cytidine 59-monophosphate; Polyamines; Bioligands
1. Introduction Aliphatic polyamines (PAs), putrescine (Put; NH2(CH2)4NH2), spermidine (Spd; NH2(CH3)3NH(CH2)4NH2) and spermine (Spm; NH2(CH2)3NH(CH2)4NH(CH2)3NH2), belong to the fundamental components of living cells and play a very important role in many biological processes. [1–7]. These compounds occur practically in all types of cells [8] and their elevated level is observed in young cells, including cancer cells [9]. PAs in physiological fluid occur in the protonated form and mainly in such a form they interact with other bioligands. Of particular importance are the interactions with nucleic acids, which lead to structural changes in the latter on a few levels of organization. These reactions determine the role of PA in the transfer of genetic information [10–12]. The ligand–ligand interactions are * Corresponding author. Fax: q48-61-865-8008; e-mail: lomozik@ chem.amu.edu.pl
observed in the range of pH in which the polyamine is protonated while the nucleoside or nucleotide is deprotonated. The tendency towards formation of molecular complexes depends not only on the number of –NHxq groups but also on the length of the polyamine methyl chain [13,14]. The model of electrostatic interactions sometimes suggested does not explain the high specificity of certain processes. Moreover, the centers of interactions between bioligands are also the potential sites of metal ion bonding, which influence the process of formation of molecular complexes [13]. It has already been established that the presence of putrescine in the systems with Cu(II) and adenosine leads to the disappearance of the coordination dichotomy observed in the binary systems. On the other hand, the presence of spermidine or spermine extends the range of this dichotomy [15,16]. Relative to the reactions taking place in binary systems, the process of complexation in ternary systems (in particular in the systems with nucleotides) occurs at lower pH, and bio-
0162-0134/00/$ - see front matter q2000 Elsevier Science Inc. All rights reserved. PII S 0 1 6 2 - 0 1 3 4 ( 9 9 ) 0 0 2 2 3 - 8
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ligands occur in the complexed form under physiological conditions [13,17–19]. This paper reports results of the study on interactions in the systems of Cu(II) ions with adenosine 59-monophosphate (AMP) or cytidine 59-monophosphate (CMP), as well as the polyamines, 1,4-diaminobutane (putrescine; Put), 1,7diamino-4-azaheptane (3,3-tri), 1,8-diamino-4-azaoctane (Spd), and 1,11-diamino-4,8-diazaundecane (3,3,3-tet), and interactions between bioligands in metal-free systems.
2. Experimental Adenosine 59-monophosphate (AMP) and cytidine 59monophosphate (CMP) were purchased from Sigma. 1,4Diaminobutane (Put), 1,7-diamino-4-azaheptane (3,3-tri), 1,8-diamino-4-azaoctane (Spd), 1,11-diamino-4,8-diazaundecane (3,3,3-tet) and their hydrochlorides, PutP2HCl, 3,3-triP3HCl, SpdP3HCl, were purchased from Sigma; 3,3,3-tetP4HCl was obtained in the reaction of HCl solution with 3,3,3-tet in methanol. Results of elemental analyses (%C, %N, %H) were consistent with the calculated ones ("0.5%). The source of Cu(II) ions was Cu(NO3)2 prepared in the following way. About 10 g of CuO was wetted with H2O, then concentrated HNO3 was added at about 10% excess of CuO. At the moment when the pH reached a value of 5, the unreacted oxide was removed. After evaporation of the filtrate to about 50% of the initial volume, the crystallized salt was filtered off, washed with ether and dried over P4O10. The method of determination of the Cu(II) concentration was described earlier [20]. The potentiometric measurements were carried out using a Multi-Titrator DTS 800 Radiometer with a GK-2401c electrode calibrated according to the procedure from [21]. The concentrations of AMP, CMP, PutP2HCl, 3,3-triP3HCl, SpdP3HCl and 3,3,3tetP4HCl were in the range from 1.5=10y3 to 3=10y3 M, and the metal-to-ligand ratio was from 1:2 to 1:6.5. Titrations of the metal-free systems were carried out at the ligand-toligand molar ratio of 1:1 (ligand concentration was 1=10y2 M). All measurements were performed in helium atmosphere at an ionic strength ms0.1 (KNO3), Ts(20"1) 8C, using as a titrant CO2-free NaOH at a concentration of 0.2280 or 0.5418 M. In the Cu/AMP/Put system a precipitate appeared at pH;6 and in the Cu/CMP/Put system at pH;9.5. The precipitate dissolved at pH;10.7 in both systems. In the systems, Cu/AMP(CMP)/Spd, Cu/AMP(CMP)/(3,3-tri) and Cu/AMP/(3,3,3-tet), the precipitate appeared at pH;5.5, 8 and 7.5, respectively. The selection of the models and the determination of the stability constants of the complexes were made using the SUPERQUAD program [22], whereas the distribution of particular forms was determined by the HALTFALL program [23]. The criteria used for verification of results are given in [24]. The calculations were performed using 100–350 points for each job taking into account only this part of the titration curve, when there was no precipitate in the solution.
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Samples to be studied by NMR and IR were obtained by dissolving AMP, CMP, Cu(II) and the polyamine hydrochlorides in D2O. The pH values of the samples were corrected according to the formula pDspH meter readings q0.4 [25]. The concentration of ligands in the samples was 0.05 M, and the metal-to-ligand ratio was 1:2 (IR), and 1:100 or 1:200 (NMR). The 13C NMR spectra were recorded on a NMR Gemini 300VT Varian spectrometer using dioxane as an internal standard. The positions of 13C NMR signals were given in the TMS scale. Measurements of 31P NMR were performed on a NMR Unity-300 Varian spectrometer using H3PO4 as a standard. IR spectra were recorded on a Bruker IFS-113v instrument using a KRS5 cell. The UV–Vis spectra were taken on a UV 160 Shimadzu spectrometer at the ligand concentration of 0.1 M and at the metal:ligand ratio of 1:3.5. EPR spectra were recorded on a Radiopan SE/X 2547 instrument (CCus0.001 M or 0.005 M).
3. Results and discussion 3.1. Potentiometric studies of metal-free systems Non-covalent interactions between polyamines and fragments of nucleic acids change the acid–base properties of the system components. The formation of molecular complexes described by the formula: H x PAqH y NMP|(NMP)H(xqyyn)(PA)qn Hq
(1)
was analyzed on the basis of results of the potentiometric studies (for the sake of simplicity for most of the species, charges have been omitted). The procedure of measurements has been described earlier in [13,14,24]. The formulae of the bioligands studied are given in Scheme 1. Table 1 presents the overall stability constants b and the equilibrium constants Ke of the reaction of molecular complex formation. In the system with the diamine-putrescine and AMP or CMP, only molecular complexes of the type (NMP)H2(Put) are formed, while the tetramine 3,3,3-tet forms three adducts with each of the nucleotides studied.
Scheme 1.
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Fig. 1. Distribution diagrams for the systems: (a) AMP/3,3-tri: 1, (AMP)H4(3,3-tri); 2, (AMP)H3(3,3-tri); 3, (AMP)H2(3,3-tri); 4, H2AMP; 5, HAMP; 6, AMP; 7, H3(3,3-tri); 8, H2(3,3-tri); 9, H(3,3-tri); 10, 3,3-tri; CAMPs0.015 M; C3,3-tris0.015 M; (b) CMP/3,3,3-tet: 1, H2CMP; 2, HCMP; 3, CMP; 4, H4(3,3,3-tet); 5, H3(3,3,3-tet); 6, H2(3,3,3-tet); 7, H4(3,3,3-tet); 8, (CMP)2H6(3,3,3-tet); 9, (CMP)H4(3,3,3-tet); 10, (CMP)H3(3,3,3-tet); CCMPs0.015 M; C3,3,3-tets0.015 M. Table 1 Equilibrum constants (log Ke), overall stability constants (log b) and equilibrium of adducts formation in NMP/PA systems; NMPsAMP, CMP; PAsPut, Spd, 3,3-tri, 3,3,3-tet Reaction
Log b
Log Ke
AMPqH2Put|(AMP)H2(Put) AMPqH4Spd|(AMP)H4(Spd) AMPqH3Spd|(AMP)H3(Spd) HAMPqH3(3,3-tri)|(AMP)H4(3,3-tri) AMPqH3(3,3-tri)|(AMP)H3(3,3-tri) AMPqH2(3,3-tri)|(AMP)H2(3,3-tri) AMPqH4(3,3,3-tet)|(AMP)H4(3,3,3-tet) AMPqH3(3,3,3-tet)|(AMP)H3(3,3,3-tet) AMPqH2(3,3,3-tet)|(AMP)H2(3,3,3-tet)
22.25 (4) 37.44 (2) 31.87 (1) 36.25 (1) 30.65 (1) 22.25 (1) 39.21 (3) 31.52 (4) 22.38 (3)
1.74 1.40 2.26 1.79 2.62 2.03 2.82 2.51 2.00
CMPqH2Put|(CMP)H2(Put) HCMPqH3Spd|(CMP)H4(Spd) CMPqH3Spd|(CMP)H3(Spd) HCMPqH3(3,3-tri)|(CMP)H4(3,3-tri) CMPqH3(3,3-tri)|(CMP)H3(3,3-tri) CMPqH2(3,3-tri)|(CMP)H2(3,3-tri) 2HCMPqH4(3,3,3-tet)|(CMP)2H6(3,3,3-tet) CMPqH4(3,3,3-tet)|(CMP)H4(3,3,3-tet) CMPqH3(3,3,3-tet)|(CMP)H3(3,3,3-tet)
22.09 (6) 37.31 (4) 31.70 (2) 36.28 (2) 30.34 (3) 22.26 (3) 54.26 (2) 39.52 (1) 31.58 (3)
1.58 2.07 2.26 1.79 2.62 2.03 5.05 2.70 1.97
Molecular complexes of both triamines, 3,3-tri or Spd, with the nucleotides as well as 3,3,3-tet complexes with CMP start forming from pH of about 3 (Fig. 1). The protonation degree indicates that, in this range of pH, the only effective interaction centers are the –NHxq groups of PA and the phosphate group of the nucleotide (the nitrogen atoms from the purine or pyrimidine rings are protonated) which suggests the occurrence of weak interactions involving both groups. It is confirmed below in the section concerning NMR studies. The equilibrium constants of adduct formation (the reactions from Table 1) are higher than the analogous values for nucleosides [13,14] 1, which confirms the involve1 The values of log Ke, missing in Refs. [13,14], for the reaction of complex formation of (Ado)H4(3,3,3-tet), (Ado)H3(3,3,3-tet) and (Ado)H2(3,3,3-tet) are 2.80, 2.45 and 2.10, respectively, while for the reaction of (Cyd)H4(3,3,3-tet) and (Cyd)H3(3,3,3-tet) formation they are 2.70 and 1.97, respectively [26].
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Fig. 2. Distribution diagrams for the AMP/3,3,3-tet system: 1, (AMP)H4(3,3,3-tet); 2, (AMP)H3(3,3,3-tet); 3, (AMP)H2(3,3,3-tet); 4, H2AMP; 5, HAMP; 6, AMP; 7, H4(3,3,3-tet); 8, H3(3,3,3-tet); 9, H2(3,3,3tet); 10, H(3,3,3-tet); 11, 3,3,3-tet; CAMPs0.015 M; C3,3,3-tets0.015 M.
ment of the phosphate group in the interactions. The exception is the system AMP/3,3,3-tet. Molecular complexes of 3,3,3-tet with AMP (Fig. 2) as well as those of putrescine with AMP or CMP start forming from pH;4. The equilibrium constants of formation of AMP complexes with 3,3,3tet (Table 1) are similar to those obtained for the adducts with Ado, which suggests that the phosphate group from the nucleotide does not take part in the interactions (at least to any considerable degree). On the other hand, the values of the equilibrium constants of adducts formation (Ado)H2(Put), log Kes1.53, and (AMP)H2(Put), log Kes1.74, indicate the involvement of the phosphate group in the interactions. However, a relatively small difference between these values points to only minor participation of the group in the reaction. The problem of interplay of the phosphate group was resolved on the basis of the spectral studies discussed below. 3.2. NMR studies of metal-free systems Analysis of the differences between the 13C NMR spectra of the systems NMP/PA and those of free ligands in the pH range from 2 to 10 indicates that the interaction centers in all cases are the atoms N(1) and N(7) from AMP, N(3) from
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Table 2 13 C NMR signal positions for the AMP/3,3-tri system and their changes in relation to single ligands (ppm, in parentheses) a pD 2 4 6 8 10 a
C(6)
C(2)
C(4)
C(8)
C(5)
C(19)
C(49)
C(39)
C(29)
C(59)
C(3)
C(1)
C(2)
150.673 (0.002) 156.312 (1.294) 156.002 (0.159) 156.134 (0.083) 156.291 (0.061)
149.070 (0.004) 152.750 (3.581) 153.357 (0.059) 153.416 (0.261) 156.486 (0.030)
145.182 (0.000) 148.660 (0.003) 145.694 (0.005) 149.740 (0.015) 149.752 (0.002)
143.112 (0.020) 140.785 (0.190) 140.709 (0.076) 140.848 (0.028) 141.149 (0.258)
119.283 (0.001) 119.903 (0.696) 119.103 (0.169) 119.189 (0.093) 119.398 (0.113)
88.890 (0.007) 87.953 (0.037) 87.745 (0.016) 87.632 (0.010) 87.630 (0.015)
85.092 (0.009) 84.754 (0.018) 85.224 (0.108) 85.612 (0.101) 85.920 (0.290)
75.511 (0.019) 75.225 (0.063) 75.218 (0.053) 75.321 (0.108) 76.923 (0.605)
71.120 (0.002) 71.234 (0.005) 71.414 (0.097) 71.527 (0.011) 72.734 (0.223)
65.214 (0.004) 65.477 (0.264) 64.430 (0.508) 64.317 (0.501) 65.202 (0.687)
45.568 (0.002) 45.557 (y0.107) 45.533 (y0.067) 46.450 (y0.201) 47.158 (y0.618)
37.452 (y0.014) 37.422 (y0.110) 37.491 (y0.098) 38.715 (y0.222) 39.602 (y0.476)
24.635 (0.003) 24.644 (y0.094) 24.892 (y0.143) 28.129 (y0.071) 30.578 (y0.329)
Negative sign stands for a high field shift, positive sign stands for a low field shift.
CMP and all nitrogen atoms from the polyamines (Tables 2– 4). For example, in the system AMP/3,3-tri at pH 4, the shifts of the signals from C(6), C(2), C(8) and C(5) are 1.294, 3.581, 0.190 and 0.696 ppm, respectively. Moreover, the shift of the signal from the C(4) atom which is far from reaction centers, is only 0.003 ppm. The changes in the signal positions of the carbon atoms C(1), C(2) and C(3) from 3,3-tri are y0.110, y0.094 and y0.107 ppm, respectively. No significant shifts of the bioligand signals are observed at pH 2, at which the adducts do not form (Table 2). Analysis of the 13C and 31P NMR spectra suggests the involvement of the phosphate groups in the interactions in all systems except AMP/ 3,3,3-tet, which corresponds very well to the results of the equilibrium studies. For example, in the 31P NMR spectra of NMP/Put at pH 6, the shifts of the phosphorus atom signals in AMP or CMP are 0.291 and 0.208 ppm, respectively (Table 5), which is strong evidence for the involvement of the phosphate group from the nucleotide in the interaction with Put. In the system AMP/3,3,3-tet, in which the phosphate group is not involved in the adduct formation, the analogous changes are only 0.008 and 0.006 ppm, respectively, for C(59) and P (Tables 4 and 5). These observations confirm a difference in the character of tetramines relative to the shorter polyamines (it was also unexpectedly found earlier for the systems with spermine [13]). Unlike the systems of AMP with tetramines (Spm or 3,3,3-tet) in which no interactions with the phosphate group were found, in the systems of these tetramines with CMP, the phosphate group from the nucleotide takes part in formation of molecular complexes (it was also deduced from analysis of NMR signal changes). 3.3. Equilibrium studies of the Cu(II)/nucleotide/polyamine systems Table 6 presents values of the overall stability constants of the complexes formed in the systems of Cu(II) with nucleotides and polyamines. The results for the ternary systems show the formation of molecular complexes ML-L9 (with complex–ligand non-covalent interaction), protonated com-
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plexes MHxL and mixed-ligand compounds of the MLL9 type. 3.3.1. Cu/AMP/Put, Cu/AMP/3,3-tri, Cu/AMP/Spd Mixed-ligand complexes are not formed in the Cu/AMP/ Put system. Taking into account the degree of protonation of the nucleotide and polyamine [13,27], it should be assumed that in the protonated Cu(AMP)H2(3,3-tri) complex which exists in the pH range from about 4.5 to 7 (Fig. 3(a)) only one nitrogen atom of 3,3-tri is involved in coordination. The hypothetical reaction of the complex formation is described by Eq. (2): Cu(AMP)qH 2 (3,3-tri)|Cu(AMP)H 2 (3,3-tri)
(2)
The equilibrium constant for reaction 2, calculated from the overall stability constants and the protonation constants, log K eslog b Cu(AMP)H 2 (3,3-t r i)ylog b Cu AMP ylog b H 2 (3,3-t r i) is 6.72. The value of log Ke for reaction 3 of the protonated binary complex formation is 5.00: CuqHPut|CuHPut
(3)
The increase of log Ke for reaction 2 relative to the value for reaction 3 suggests additional stabilization of the mixedligand complexes. In the first approach it should be assumed that the additional stabilization is related to the presence of non-covalent intramolecular interactions [28]. In the system Cu/AMP/Spd, as mentioned in Section 2, a precipitate starts to appear from a pH of about 5.5 (formation of barely soluble ternary complexes is under study). 3.3.2. CuCMP/Put, Cu/CMP/(3,3-tri), Cu(CMP)/Spd A similar mode of interaction as in Section 3.3.1 is met for the complexes Cu(CMP)H(Put) (Fig. 3(b)) which exists from a pH of about 5.5, and binds 30% of the metal at a pH of about 7 and for the Cu(CMP)H2(3,3-tri) complex formed from a pH of about 5, binding about 15% of metal ions at pH 6. The hypothetical reactions of formation of these complexes are described by Eqs. (4) and (5):
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150.762 (0.089) 154.865 (1.002) 156.029 (0.132) 156.058 (0.159) 156.186 (0.044)
C(6) 149.047 (0.019) 149.344 (0.175) 153.367 (0.149) 153.396 (0.241) 153.460 (0.302)
C(2) 145.668 (0.486) 151.797 (0.310) 149.536 (0.133) 149.553 (0.172) 149.703 (0.047)
C(4) 143.002 (0.091) 140.940 (0.017) 140.673 (0.040) 140.776 (0.100) 140.897 (0.006)
C(8) 119.262 (0.026) 119.262 (0.055) 119.145 (0.127) 119.137 (0.145) 119.250 (0.035)
C(5) 88.823 (0.007) 88.990 (0.951) 87.762 (0.000) 87.710 (0.088) 87.661 (0.046)
C(19) 85.047 (0.009) 84.808 (0.005) 85.202 (0.104) 85.449 (0.183) 85.526 (0.102)
C(49) 75.514 (0.016) 75.254 (0.025) 75.223 (0.048) 75.222 (0.089) 75.336 (0.018)
C(39) 71.118 (0.000) 71.172 (0.057) 71.375 (0.048) 71.457 (0.081) 71.536 (0.025)
C(29) 65.157 (0.012) 65.412 (0.199) 64.666 (0.272) 64.417 (0.099) 64.277 (0.362)
C(59) 47.931 (0.004) 47.936 (y0.004) 47.856 (y0.063) 47.904 (y0.498) 49.612 (y0.692)
C(4)
10
8
6
4
2
pD
151.537 (0.003) 155.171 (0.634) 155.977 (0.301) 154.831 (1.356) 156.284 (0.067)
C(6) 149.189 (0.012) 152.149 (2.419) 153.359 (0.176) 152.682 (0.723) 153.556 (0.012)
C(2) 146.787 (0.007) 148.482 (0.190) 149.493 (0.060) 149.394 (0.281) 149.770 (0.003)
C(4) 142.743 (0.009) 140.950 (0.040) 140.649 (0.138) 141.063 (0.137) 140.961 (0.107)
C(8) 119.304 (0.014) 119.161 (0.000) 119.081 (0.136) 119.223 (0.100) 119.263 (0.068)
C(5) 88.729 (0.002) 88.021 (0.010) 87.849 (0.003) 87.081 (0.000) 87.644 (0.013)
C(19) 85.011 (0.011) 84.390 (0.040) 85.019 (0.019) 84.795 (0.010) 85.645 (0.002)
C(49) 75.484 (0.013) 75.246 (0.002) 75.155 (0.003) 75.250 (0.002) 75.329 (0.010)
C(39) 71.079 (0.007) 71.172 (0.010) 71.270 (0.052) 71.141 (0.020) 71.528 (0.000)
C(29)
37.385 (y0.008) 37.349 (y0.002) 37.348 (y0.038) 37.344 (y0.179) 38.682 (y0.217)
24.654 (0.002) 24.670 (y0.046) 24.696 (y0.052) 24.940 (y1.180) 27.517 (y2.994)
C(6)
45.436 (0.005) 45.407 (y0.018) 45.376 (y0.079) 46.410 (y0.123) 46.328 (y0.145)
C(3)
24.825 (0.003) 24.832 (y0.004) 24.829 (y0.013) 25.137 (y2.869) 29.450 (y3.049)
C(2)
C(1)
37.471 (0.009) 37.468 (y0.015) 37.446 (y0.028) 37.605 (y1.089) 38.590 (y0.997)
C(1)
24.588 (y0.011) 23.520 (y0.025) 23.563 (y0.023) 23.519 (y0.301) 25.546 (y0.066)
C(2)
39.699 (y0.004) 39.695 (y0.013) 39.648 (y0.052) 39.692 (y0.511) 40.515 (y0.816)
C(7)
23.519 (0.002) 24.590 (y0.018) 24.608 (y0.010) 24.585 (y0.172) 28.075 (y0.123)
C(5)
45.408 (y0.005) 45.412 (y0.006) 45.364 (y0.043) 45.483 (y0.881) 46.568 (y0.515)
C(3)
65.154 (0.002) 65.196 (0.010) 64.843 (0.032) 65.249 (0.008) 64.493 (0.007)
C(59)
Table 4 13 C NMR signal positions for the AMP/3,3,3-tet system and their changes in relation to single ligands (ppm, in parentheses)
10
8
6
4
2
pD
Table 3 C NMR signal positions for the AMP/Spd system and their changes in relation to single ligands (ppm, in parentheses)
13
45.561 (y0.008) 45.540 (y0.006) 45.486 (y0.075) 46.090 (y0.215) 48.487 (y0.032)
C(4)
23.656 (y0.004) 23.665 (y0.011) 23.644 (0.000) 23.891 (y1.495) 25.764 (y1.121)
C(5)
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Table 5 31 P NMR signal positions for the AMP/PA and CMP/PA systems and their changes in relation to single ligands (ppm, in parentheses) pD
2 4 6 8 10
AMP/PA
CMP/PA
Put
Spd
3,3-Tri
3,3,3-Tet
Put
Spd
3,3-Tri
3,3,3-Tet
0.181 (0.010) 0.338 (0.017) 1.587 (0.291) 4.080 (0.263) 3.986 (0.093)
0.164 (0.027) 0.220 (0.101) 2.250 (0.372) 4.101 (0.284) 3.772 (0.121)
0.174 (0.017) 0.209 (0.112) 2.193 (0.315) 4.120 (0.303) 3.680 (0.213)
0.193 (0.002) 0.317 (0.004) 1.879 (0.001) 3.811 (0.006) 3.888 (0.005)
0.030 (0.007) 0.184 (0.062) 2.193 (0.208) 3.955 (0.184) 3.801 (0.046)
0.034 (0.011) 0.194 (0.072) 2.179 (0.194) 3.553 (0.218) 3.879 (0.032)
0.028 (0.005) 0.250 (0.128) 2.299 (0.314) 3.534 (0.237) 3.979 (0.132)
0.031 (0.009) 0.296 (0.174) 2.281 (0.297) 3.538 (0.283) 3.728 (0.119)
Table 6 Overall stability constants (log b) of Cu(II) complexes with AMP (or CMP) and polyamines Cu(II)/AMP/PA
Cu(II)/CMP/PA
Species
Log b
Species
Log b
CuAMP Cu(AMP)(OH)
3.02 (8) y3.82 (5)
CuCMP Cu(CMP)(OH)
2.71 (6) y4.26 (8)
CuH(Put) Cu(Put) Cu(Put)2 Cu(Put)2(OH)
15.83 (37) 8.62 (13) 13.40 (18) 0.065 (16)
CuH(Put) Cu(Put) Cu(Put)2 Cu(Put)2(OH) Cu(CMP)H(Put)
15.83 (37) 8.62 (13) 13.40 (18) 0.065 (16) 19.02 (8)
CuH(3,3-tri) Cu(3,3-tri) Cu(3,3-tri)2 Cu(3,3-tri)(OH) Cu(AMP)H2(3,3-tri)
18.87 (18) 13.71 (3) 18.48 (5) 3.14 (5) 29.96 (15)
CuH(3,3-tri) Cu(3,3-tri) Cu(3,3-tri)2 Cu(3,3-tri)(OH) Cu(CMP)H2(3,3-tri) Cu(CMP)H4(3,3-tri)
18.87 (18) 13.71 (3) 18.48 (5) 3.14 (5) 28.40 (8) 39.71 (5)
CuH2(3,3,3-tet) CuH(3,3,3-tet) Cu(3,3,3-tet) Cu(AMP)(3,3,3-tet)
42.03 (7) 27.49 (7) 16.36 (4) 20.15 (4)
CuH2(3,3,3-tet) CuH(3,3,3-tet) Cu(3,3,3-tet) Cu(CMP)(3,3,3-tet)
42.03 (7) 27.49 (7) 16.36 (4) 20.62 (5)
Cu(CMP)qHPut|Cu(CMP)H(Put)
(4)
Cu(CMP)qH 2 (3,3-tri)|Cu(CMP)H 2 (3,3-tri)
(5)
The equilibrium constants calculated from the overall stability constants and the protonation constants (e.g. for reaction 4, log Keslog bCu(CMP)H(Put)ylog bCuCMPylog bHPut) for the above reactions are 5.48 and 5.47. The increase of log Ke for reactions 4 and 5 relative to the value for reaction 3 suggests additional stabilization of the mixed-ligand complexes. The same values of log Ke for the complexes Cu(CMP)H(Put) and Cu(CMP)H2(3,3-tri) indicate a similar character of interaction including only one nitrogen atom of PA. The mixed-ligand complexes are not formed in the Cu/ CMP/Spd system, which confirms a low tendency of spermidine to form stable ternary complexes.
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In the Cu/CMP/3,3-tri system, the presence of the complex Cu(CMP)H4(3,3-tri) was also found, which starts forming from a pH of about 3 and reaches a maximum concentration at a pH about 5, at which it binds 30% of the metal ions. The number of the protonated ligand donor groups in this complex suggests formation of a molecular complex in which the polyamine remains out of the inner coordination sphere.
3.3.3. Cu/AMP/3,3,3-tet, Cu/CMP/3,3,3-tet Mixed-ligand complexes of the MLL9 type form only in the systems of nucleotides with tetramine-Cu(AMP)(3,3,3tet) or Cu(CMP)(3,3,3-tet). These complexes start forming from a pH of about 5 and dominate from a pH of about 6 binding 85% of the metal (Fig. 3(c)).
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Fig. 3. Distribution diagrams for the systems: (a) Cu(II)/AMP/3,3-tri: 1, Cu2q; 2, Cu(AMP)H2(3,3-tri); 3, Cu(3,3-tri); 4, CuAMP; CAMPs0.0025 M; C3,3-tris0.0025 M; CCus0.0016 M; (b) Cu(II)/CMP/Put: 1, Cu2q; 2, Cu(CMP)H(Put); 3, CuCMP; 4, Cu(CMP)OH; 5, CuH(Put); 6, CuPut; CCMPs 0.0025 M; C3,3-tris0.0025 M; CCus0.0016 M; (c) Cu(II)/AMP/3,3,3-tet: 1, Cu2q; 2, Cu(AMP)(3,3,3-tet); 3, CuH(3,3,3-tet); 4, Cu(3,3,3-tet); 5, CuAMP; 6, CuH2(3,3,3-tet); CAMPs0.0025 M; C3,3,3-tets0.0025 M; CCus0.0016 M.
3.4. Spectral studies of the Cu(II)/nucleotide/polyamine systems 3.4.1. Cu/AMP/Put, Cu/CMP/Put Mixed-ligand complexes are not formed in the Cu/AMP/ Put system (at least in detectable amounts). Analysis of the electron spectra UV–Vis and EPR (combined with the results of equilibrium studies) in the system Cu/CMP/Put suggests that only one nitrogen atom from polyamine and the oxygen atoms from the phosphate group take part in the metallation. The position of the absorption band is lmaxs736 nm (pH 6.5), which corresponds to the coordination mode of the N1O type, as has been deduced from the data published in [29,32]. The EPR spectral parameters are g≤s2.2473, A≤s186.5 at pH 6.5 (Table 7). It has been established that with the increasing number of coordinated donor atoms the g≤ value decreases while A≤ increases, which has been observed for Cu–Nx (xs1 to 6) and Cu– NxOy (xs0 to 4, ys0 to 4) chromophores [30,31]. Analysis of the values of these parameters and their comparison with
the results reported for similar systems indicate metallation involving one nitrogen atom from the polyamine and oxygen atoms from the phosphate group. The equilibrium constant (log Ke) of the Cu(CMP)H(Put) complex formation (reaction 4) is 5.48. The value for the CuHPut (reaction 3) is 5.00 and for the Cu(CMP) complex formation (with N1O coordination) is 2.71 [13,17]. The comparison of the values testifies to the involvement of one nitrogen atom from Put into the inner coordination sphere. The earlier discussed stabilization of the mixed-ligand complex relative to that formed in the binary system is a result of non-covalent interactions. The protonated amine group from the polyamine is involved in weak interplay with the donor centers of the pyrimidine base. For all systems of Cu(II), CMP and PA, the involvement of the nucleotide carbonyl group in coordination was excluded. In the IR spectra of metal/ligand systems, the position of the band corresponding to the stretching vibrations of the carbonyl group at 1652 cmy1 was the same as for metalfree systems.
Table 7 EPR and absorption spectral data for the Cu/NMP/PA systems System
pH
lmax (nm)
´ (dm3/(mol cm))
gH
g≤
A≤ (10y4 cmy1)
Cu/CMP/Put Cu/AMP/(3,3-tri) Cu/CMP/(3,3-tri)
6.5 6.0 4.5 5.8 6.5 6.5
736 725 798 618 578 587
27.3 28.5 31.8 16.4 19.2 19.8
2.0554 2.0577 2.0678 2.0530 2.0577 2.0517
2.2473 2.2446 2.3382 2.2333 2.2232 2.2115
186.5 163.0 161.3 185.0 175.0 190.4
Cu/AMP/(3,3,3-tet) Cu/CMP/(3,3,3-tet)
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3.4.2. Cu/AMP/3,3-tri, Cu/CMP/3,3-tri Similar to the above discussed system with Put, the results of the UV–Vis and EPR study (including equilibrium data) for both the Cu/AMP/3,3-tri and Cu/CMP/3,3-tri systems point to the metallation through one of the nitrogen atoms from 3,3-tri and the oxygen atoms from the phosphate group (Table 7). Equilibrium studies indicate additional stabilization in the Cu(AMP)H2(3,3-tri) as well as in Cu(CMP)H2(3,3-tri) species (discussed in Section 3.3). The protonated nitrogen atoms from the polyamine in both complexes take part in the intramolecular interactions with the donor atoms from the base, which is manifested by the increase in complex stability (log Ke for reaction 2 or 5 comparing to reaction 3). Moreover, formation of the molecular complex Cu(CMP)H4(3,3-tri) was detected in the Cu/CMP/3,3-tri system. At pH 4.5, when this species is the only complex, lmaxs798 nm, while the EPR parameters are g≤s2.3382, A≤s161.3, Table 7, which suggests the coordination of copper ions only through the oxygen atoms of the phosphate group (Cu–O chromophore). In the pH range below 5 (where the Cu(CMP)H4(3,3-tri) dominates), polyamine is completely protonated and all donor groups in the amine are blocked for metallation. The ligand remaining outside the inner coordination sphere is involved in non-covalent interaction with donor groups of the nucleotide. The conclusion concerning formation of the ML-L9 type complex has also been supported by the results of equilibrium studies. Analogous interactions were observed earlier in the system Cu/ AMP/Spm [13]. 3.4.3. Cu/AMP/3,3,3-tet, Cu/CMP/3,3,3-tet A different mode of coordination was observed in the systems of Cu(II) with the nucleotides and tetramine-3,3,3-tet. UV–Vis and EPR results (Table 7) indicate coordination with the N4O chromophore. At the pH range in which the MLL9 type complexes dominate (Fig. 3(c)), the absorption band maxima are at 578 and 587 nm for the systems with AMP and CMP, and the EPR parameters are g≤s2.2232 and
A≤s175.0 as well as g≤s2.2115 and A≤s190.0, respectively. Moreover, NMR results suggest that all nitrogen atoms from the polyamine and the oxygen atoms from the nucleotide phosphate group are involved in the metallation (Fig. 4). For instance, at pH about 6, in the 13C and 31P NMR spectra of the Cu(AMP)(3,3,3-tet) complex, the signals from AMP C(59) as well as P (0.303, 0.098 ppm, respectively) and from carbon atoms of the tetramine are shifted: C(1), C(2), C(3), C(4) and C(5) by y1.014, y0.827, y0.695, y1.101 and y0.739 ppm, respectively. The shifts of the nucleotides signals assigned to C(2), C(6), C(5) and C(8) from the close neighborhood of the atoms N(1) and N(7) are only 0.019, 0.025, 0.016 and 0.017 ppm, respectively. In order to minimize the broadening of the NMR signals related to the paramagnetic character of Cu(II) ions, the spectra were taken at low metal concentrations (on the distribution diagrams, pH ranges of particular complex domination are practically the same as in the systems for equilibrium studies with higher concentrations of ligands). Similar to that observed earlier when studying analogous systems [13,15,16,18,19], significant changes in the chemical shift were noted only in the pH ranges in which complex formation takes place, established on the basis of potentiometric studies. The NMR technique was also used to determine the coordination mode in similar systems with paramagnetic species [33,34]. Fig. 5 (referring to the Cu/CMP/3,3,3-tet system) presents an exemplary 13C NMR spectrum of the ligand obtained by the decoupling technique, and the spectrum of its complex with Cu(II), besides the spectra taken under the conditions in which complexes are not formed. The mode of coordination in both systems of Cu(II) with 3,3,3-tet and both nucleotides studied is substantially different to that in the analogous systems with another tetraminespermine, described in [13,17]. The results of this work prove that even small structural changes of the polyamine influence the mode of coordination, and confirm the role of the structural factor in the interactions of this group of bioligands. This factor also explains the significantly different properties of biogenic amines from
Fig. 4. Changes in 13C and 31P NMR signal positions (NDdN) as a function of pH for the Cu/AMP/3,3,3-tet system (in relation to free ligands): (a) AMP; (b) 3,3,3-tet; CAMPsC3,3,3-tets0.05 M; CCus0.0005 M.
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Fig. 5. 13C NMR spectra. (a) Top: Cu(II)/CMP/3,3,3-tet system; bottom: metal-free system; pH 2, CCMPsC3,3,3-tets0.05 M; CCus0.0005 M (no changes in the signal positions in relation to the metal-free system); (b) Top: Cu(II)/CMP/3,3,3-tet system; bottom: metal-free system; pH 8, CCMPsC3,3,3-tets0.05 M; CCus0.0005 M (changes in the signal positions observed for: C(29) y8 and C(59) y9 from CMP; C(3) y10, C(4) y11, C(1) y12, C(2) y13, C(5) y14 from 3,3,3-tet).
those of their biologically inactive analogs. Moreover, metal ions interfere with the reactions of bioligands. The similar properties of spermine and 3,3,3-tet in binary metal-free systems are substantially changed in systems with Cu(II). Significant differences in coordination character were also observed in the ternary systems with triamines, spermidine and 3,3-tri, which also testifies to a different behavior of biogenic amines.
Acknowledgements This work was supported by Grant No. 3 T09A 092 12 of the Polish State Committee for Scientific Research.
References [1] C.W. Tabor, H. Tabor, Annu. Rev. Biochem. 53 (1984) 749. [2] V. Zappia, A.E. Pegg (Eds.), Progress in Polyamine Research, Plenum, New York, 1988. [3] L. Lomozik, Wiad. Chem. (in Polish) 48 (1994) 439. [4] L. Lomozik, in: G. Berthon (Ed.), Handbook of Metal–Ligand Interactions in Biological Fluids, vol. 1, Bioinorganic Chemistry, Marcel Dekker, New York, 1995, p. 686. [5] O.P. Shukla, J. Sci. Ind. Res. India 49 (1990) 263. [6] F. Schuber, Biochem. J. 260 (1989) 1. [7] H.D. Bratek-Wiewiorowska, M. Alejska, M. Figlerowicz, J. Barciszewski, M. Wiewiorowski, M. Jaskolski, W. Zielenkiewicz, A. Zielenkiewicz, M. Kaminski, Pure Appl. Chem. 59 (1987) 407. [8] S.S. Cohen, Introduction to the Polyamines, Prentice-Hall, Englewood Cliffs, NJ, 1979. [9] J. Janne, H. Poso, A. Raina, Biochim. Biophys. Acta 473 (1978) 241. [10] P.M. Vertino, R.J. Bergeron, P.F. Cavanaugh Jr., C.W. Porter, Biopolymers 26 (1987) 691.
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[11] B.G. Feuerstein, N. Pattabiraman, L.J. Marton, Nucleic Acids Res. 18 (1990) 1271. [12] G.E. Plum, P.G. Arscott, V.A. Bloomfield, Biopolymers 30 (1990) 631. [13] L. Lomozik, A. Gasowska, J. Inorg. Biochem. 72 (1998) 37. [14] L. Lomozik, A. Gasowska, L. Bolewski, J. Chem. Soc., Perkin Trans. 2 (1997) 1161. [15] L. Lomozik, A. Gasowska, J. Inorg. Biochem. 62 (1996) 103. [16] L. Lomozik, A. Gasowska, L. Bolewski, J. Inorg. Biochem. 63 (1996) 191. [17] A. Gasowska, L. Lomozik, Pol. J. Chem. 73 (1999) 465. [18] L. Lomozik, A. Gasowska, Monatsh. Chem. 124 (1993) 109. [19] A. Gasowska, L. Lomozik, Monatsh. Chem. 126 (1995) 13. [20] L. Lomozik, Monatsh. Chem. 115 (1984) 261. [21] H.M. Irwing, M.G. Miles, L.D. Pettit, Anal. Chim. Acta 38 (1967) 475. [22] P. Gans, A. Sabatini, A. Vacca, J. Chem. Soc., Dalton Trans. (1985) 1195. [23] N. Ingri, W. Kakolowicz, L.G. Sillen, B. Warqvist, Talanta 14 (1967) 1261. ´ [24] L. Lomozik, M. Jaskolski, A. Wojciechowska, Pol. J. Chem. 65 (1991) 1797. [25] P.K. Glasoe, F.A. Long, J. Phys. Chem. 64 (1960) 188. [26] A. Gasowska, L. Lomozik, unpublished data. [27] A. Wojciechowska, L. Bolewski, L. Lomozik, Monatsh. Chem. 122 (1991) 131. [28] H. Sigel, in: D. Banerjea (Ed.), Coordination Chemistry, vol. 20, Pergamon, Oxford, 1980, p. 27. [29] H. Gampp, H. Sigel, A.D. Zuberbuhler, Inorg. Chem. 21 (1982) 1190. [30] R. Barbucci, M.J.M. Cambell, Inorg. Chim. Acta 16 (1976) 113. [31] L. Lomozik, L. Bolewski, R. Dworczak, J. Coord. Chem. 41 (1997) 261. [32] L. Lomozik, L. Bolewski, R. Bregier-Jarzebowska, Pol. J. Chem. 69 (1995) 197. [33] G. Kotowycz, O. Suzuki, Biochemistry 12 (1973) 5325. [34] U. Weser, G.J. Strobel, H. Rupp, W. Voelter, Eur. J. Biochem. 50 (1974) 91.
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Mixed-ligand complexes of copper(II) ions with AMP and CMP in the systems with polyamines and non-covalent interaction between bioligands Anna Gasowska, Lechoslaw Lomozik *, Renata Jastrzab Faculty of Chemistry, A. Mickiewicz University, 60-780 Poznan, Poland Received 15 April 1999; received in revised form 26 November 1999; accepted 30 November 1999
Abstract The occurrence of non-covalent interactions and formation of molecular complexes between adenosine 59-monophosphate (AMP) or cytidine 59-monophosphate (CMP) and the polyamines, putrescine, 1,7-diamino-4-azaheptane (3,3-tri), spermidine and 1,11-diamino-4,8diazaundecane (3,3,3-tet), were detected in metal-free systems. The stoichiometric composition of the adducts and their stability constants were determined on the basis of computer analysis of the titration data, taking into account the fact that the acid–base properties of the system change as a result of these interactions. Spectral analysis allowed an identification of the interaction centers in the adducts as protonated amine groups of polyamines, phosphate groups as well as nitrogen atoms of high electron density from nucleotides. Unexpectedly, no participation of the phosphate group from AMP in the formation of molecular complexes with tetramine-3,3,3-tet was detected. The stoichiometric composition and stability constants of mixed-ligand complexes in the systems of Cu(II) with AMP or CMP and polyamines were obtained. Analysis of the results of equilibrium studies and 13C, 31P NMR, UV–Vis, IR and EPR data permitted determination of the mode of coordination. In the systems with metal ions, the formation of molecular complexes Cu(CMP)H4(3,3-tri) was found, apart from heteroligand complexes of the MLL9 and MLL9Hx type. In protonated complexes the occurrence of non-covalent interactions leading to stabilization of the coordination compounds was observed. The differences in the character of coordination biogenic amines and their biologically inactive analogs were identified. q2000 Elsevier Science Inc. All rights reserved. Keywords: Copper(II) complexes; Adenosine 59-monophosphate; Cytidine 59-monophosphate; Polyamines; Bioligands
1. Introduction Aliphatic polyamines (PAs), putrescine (Put; NH2(CH2)4NH2), spermidine (Spd; NH2(CH3)3NH(CH2)4NH2) and spermine (Spm; NH2(CH2)3NH(CH2)4NH(CH2)3NH2), belong to the fundamental components of living cells and play a very important role in many biological processes. [1–7]. These compounds occur practically in all types of cells [8] and their elevated level is observed in young cells, including cancer cells [9]. PAs in physiological fluid occur in the protonated form and mainly in such a form they interact with other bioligands. Of particular importance are the interactions with nucleic acids, which lead to structural changes in the latter on a few levels of organization. These reactions determine the role of PA in the transfer of genetic information [10–12]. The ligand–ligand interactions are * Corresponding author. Fax: q48-61-865-8008; e-mail: lomozik@ chem.amu.edu.pl
observed in the range of pH in which the polyamine is protonated while the nucleoside or nucleotide is deprotonated. The tendency towards formation of molecular complexes depends not only on the number of –NHxq groups but also on the length of the polyamine methyl chain [13,14]. The model of electrostatic interactions sometimes suggested does not explain the high specificity of certain processes. Moreover, the centers of interactions between bioligands are also the potential sites of metal ion bonding, which influence the process of formation of molecular complexes [13]. It has already been established that the presence of putrescine in the systems with Cu(II) and adenosine leads to the disappearance of the coordination dichotomy observed in the binary systems. On the other hand, the presence of spermidine or spermine extends the range of this dichotomy [15,16]. Relative to the reactions taking place in binary systems, the process of complexation in ternary systems (in particular in the systems with nucleotides) occurs at lower pH, and bio-
0162-0134/00/$ - see front matter q2000 Elsevier Science Inc. All rights reserved. PII S 0 1 6 2 - 0 1 3 4 ( 9 9 ) 0 0 2 2 3 - 8
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ligands occur in the complexed form under physiological conditions [13,17–19]. This paper reports results of the study on interactions in the systems of Cu(II) ions with adenosine 59-monophosphate (AMP) or cytidine 59-monophosphate (CMP), as well as the polyamines, 1,4-diaminobutane (putrescine; Put), 1,7diamino-4-azaheptane (3,3-tri), 1,8-diamino-4-azaoctane (Spd), and 1,11-diamino-4,8-diazaundecane (3,3,3-tet), and interactions between bioligands in metal-free systems.
2. Experimental Adenosine 59-monophosphate (AMP) and cytidine 59monophosphate (CMP) were purchased from Sigma. 1,4Diaminobutane (Put), 1,7-diamino-4-azaheptane (3,3-tri), 1,8-diamino-4-azaoctane (Spd), 1,11-diamino-4,8-diazaundecane (3,3,3-tet) and their hydrochlorides, PutP2HCl, 3,3-triP3HCl, SpdP3HCl, were purchased from Sigma; 3,3,3-tetP4HCl was obtained in the reaction of HCl solution with 3,3,3-tet in methanol. Results of elemental analyses (%C, %N, %H) were consistent with the calculated ones ("0.5%). The source of Cu(II) ions was Cu(NO3)2 prepared in the following way. About 10 g of CuO was wetted with H2O, then concentrated HNO3 was added at about 10% excess of CuO. At the moment when the pH reached a value of 5, the unreacted oxide was removed. After evaporation of the filtrate to about 50% of the initial volume, the crystallized salt was filtered off, washed with ether and dried over P4O10. The method of determination of the Cu(II) concentration was described earlier [20]. The potentiometric measurements were carried out using a Multi-Titrator DTS 800 Radiometer with a GK-2401c electrode calibrated according to the procedure from [21]. The concentrations of AMP, CMP, PutP2HCl, 3,3-triP3HCl, SpdP3HCl and 3,3,3tetP4HCl were in the range from 1.5=10y3 to 3=10y3 M, and the metal-to-ligand ratio was from 1:2 to 1:6.5. Titrations of the metal-free systems were carried out at the ligand-toligand molar ratio of 1:1 (ligand concentration was 1=10y2 M). All measurements were performed in helium atmosphere at an ionic strength ms0.1 (KNO3), Ts(20"1) 8C, using as a titrant CO2-free NaOH at a concentration of 0.2280 or 0.5418 M. In the Cu/AMP/Put system a precipitate appeared at pH;6 and in the Cu/CMP/Put system at pH;9.5. The precipitate dissolved at pH;10.7 in both systems. In the systems, Cu/AMP(CMP)/Spd, Cu/AMP(CMP)/(3,3-tri) and Cu/AMP/(3,3,3-tet), the precipitate appeared at pH;5.5, 8 and 7.5, respectively. The selection of the models and the determination of the stability constants of the complexes were made using the SUPERQUAD program [22], whereas the distribution of particular forms was determined by the HALTFALL program [23]. The criteria used for verification of results are given in [24]. The calculations were performed using 100–350 points for each job taking into account only this part of the titration curve, when there was no precipitate in the solution.
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Samples to be studied by NMR and IR were obtained by dissolving AMP, CMP, Cu(II) and the polyamine hydrochlorides in D2O. The pH values of the samples were corrected according to the formula pDspH meter readings q0.4 [25]. The concentration of ligands in the samples was 0.05 M, and the metal-to-ligand ratio was 1:2 (IR), and 1:100 or 1:200 (NMR). The 13C NMR spectra were recorded on a NMR Gemini 300VT Varian spectrometer using dioxane as an internal standard. The positions of 13C NMR signals were given in the TMS scale. Measurements of 31P NMR were performed on a NMR Unity-300 Varian spectrometer using H3PO4 as a standard. IR spectra were recorded on a Bruker IFS-113v instrument using a KRS5 cell. The UV–Vis spectra were taken on a UV 160 Shimadzu spectrometer at the ligand concentration of 0.1 M and at the metal:ligand ratio of 1:3.5. EPR spectra were recorded on a Radiopan SE/X 2547 instrument (CCus0.001 M or 0.005 M).
3. Results and discussion 3.1. Potentiometric studies of metal-free systems Non-covalent interactions between polyamines and fragments of nucleic acids change the acid–base properties of the system components. The formation of molecular complexes described by the formula: H x PAqH y NMP|(NMP)H(xqyyn)(PA)qn Hq
(1)
was analyzed on the basis of results of the potentiometric studies (for the sake of simplicity for most of the species, charges have been omitted). The procedure of measurements has been described earlier in [13,14,24]. The formulae of the bioligands studied are given in Scheme 1. Table 1 presents the overall stability constants b and the equilibrium constants Ke of the reaction of molecular complex formation. In the system with the diamine-putrescine and AMP or CMP, only molecular complexes of the type (NMP)H2(Put) are formed, while the tetramine 3,3,3-tet forms three adducts with each of the nucleotides studied.
Scheme 1.
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Fig. 1. Distribution diagrams for the systems: (a) AMP/3,3-tri: 1, (AMP)H4(3,3-tri); 2, (AMP)H3(3,3-tri); 3, (AMP)H2(3,3-tri); 4, H2AMP; 5, HAMP; 6, AMP; 7, H3(3,3-tri); 8, H2(3,3-tri); 9, H(3,3-tri); 10, 3,3-tri; CAMPs0.015 M; C3,3-tris0.015 M; (b) CMP/3,3,3-tet: 1, H2CMP; 2, HCMP; 3, CMP; 4, H4(3,3,3-tet); 5, H3(3,3,3-tet); 6, H2(3,3,3-tet); 7, H4(3,3,3-tet); 8, (CMP)2H6(3,3,3-tet); 9, (CMP)H4(3,3,3-tet); 10, (CMP)H3(3,3,3-tet); CCMPs0.015 M; C3,3,3-tets0.015 M. Table 1 Equilibrum constants (log Ke), overall stability constants (log b) and equilibrium of adducts formation in NMP/PA systems; NMPsAMP, CMP; PAsPut, Spd, 3,3-tri, 3,3,3-tet Reaction
Log b
Log Ke
AMPqH2Put|(AMP)H2(Put) AMPqH4Spd|(AMP)H4(Spd) AMPqH3Spd|(AMP)H3(Spd) HAMPqH3(3,3-tri)|(AMP)H4(3,3-tri) AMPqH3(3,3-tri)|(AMP)H3(3,3-tri) AMPqH2(3,3-tri)|(AMP)H2(3,3-tri) AMPqH4(3,3,3-tet)|(AMP)H4(3,3,3-tet) AMPqH3(3,3,3-tet)|(AMP)H3(3,3,3-tet) AMPqH2(3,3,3-tet)|(AMP)H2(3,3,3-tet)
22.25 (4) 37.44 (2) 31.87 (1) 36.25 (1) 30.65 (1) 22.25 (1) 39.21 (3) 31.52 (4) 22.38 (3)
1.74 1.40 2.26 1.79 2.62 2.03 2.82 2.51 2.00
CMPqH2Put|(CMP)H2(Put) HCMPqH3Spd|(CMP)H4(Spd) CMPqH3Spd|(CMP)H3(Spd) HCMPqH3(3,3-tri)|(CMP)H4(3,3-tri) CMPqH3(3,3-tri)|(CMP)H3(3,3-tri) CMPqH2(3,3-tri)|(CMP)H2(3,3-tri) 2HCMPqH4(3,3,3-tet)|(CMP)2H6(3,3,3-tet) CMPqH4(3,3,3-tet)|(CMP)H4(3,3,3-tet) CMPqH3(3,3,3-tet)|(CMP)H3(3,3,3-tet)
22.09 (6) 37.31 (4) 31.70 (2) 36.28 (2) 30.34 (3) 22.26 (3) 54.26 (2) 39.52 (1) 31.58 (3)
1.58 2.07 2.26 1.79 2.62 2.03 5.05 2.70 1.97
Molecular complexes of both triamines, 3,3-tri or Spd, with the nucleotides as well as 3,3,3-tet complexes with CMP start forming from pH of about 3 (Fig. 1). The protonation degree indicates that, in this range of pH, the only effective interaction centers are the –NHxq groups of PA and the phosphate group of the nucleotide (the nitrogen atoms from the purine or pyrimidine rings are protonated) which suggests the occurrence of weak interactions involving both groups. It is confirmed below in the section concerning NMR studies. The equilibrium constants of adduct formation (the reactions from Table 1) are higher than the analogous values for nucleosides [13,14] 1, which confirms the involve1 The values of log Ke, missing in Refs. [13,14], for the reaction of complex formation of (Ado)H4(3,3,3-tet), (Ado)H3(3,3,3-tet) and (Ado)H2(3,3,3-tet) are 2.80, 2.45 and 2.10, respectively, while for the reaction of (Cyd)H4(3,3,3-tet) and (Cyd)H3(3,3,3-tet) formation they are 2.70 and 1.97, respectively [26].
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Fig. 2. Distribution diagrams for the AMP/3,3,3-tet system: 1, (AMP)H4(3,3,3-tet); 2, (AMP)H3(3,3,3-tet); 3, (AMP)H2(3,3,3-tet); 4, H2AMP; 5, HAMP; 6, AMP; 7, H4(3,3,3-tet); 8, H3(3,3,3-tet); 9, H2(3,3,3tet); 10, H(3,3,3-tet); 11, 3,3,3-tet; CAMPs0.015 M; C3,3,3-tets0.015 M.
ment of the phosphate group in the interactions. The exception is the system AMP/3,3,3-tet. Molecular complexes of 3,3,3-tet with AMP (Fig. 2) as well as those of putrescine with AMP or CMP start forming from pH;4. The equilibrium constants of formation of AMP complexes with 3,3,3tet (Table 1) are similar to those obtained for the adducts with Ado, which suggests that the phosphate group from the nucleotide does not take part in the interactions (at least to any considerable degree). On the other hand, the values of the equilibrium constants of adducts formation (Ado)H2(Put), log Kes1.53, and (AMP)H2(Put), log Kes1.74, indicate the involvement of the phosphate group in the interactions. However, a relatively small difference between these values points to only minor participation of the group in the reaction. The problem of interplay of the phosphate group was resolved on the basis of the spectral studies discussed below. 3.2. NMR studies of metal-free systems Analysis of the differences between the 13C NMR spectra of the systems NMP/PA and those of free ligands in the pH range from 2 to 10 indicates that the interaction centers in all cases are the atoms N(1) and N(7) from AMP, N(3) from
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Table 2 13 C NMR signal positions for the AMP/3,3-tri system and their changes in relation to single ligands (ppm, in parentheses) a pD 2 4 6 8 10 a
C(6)
C(2)
C(4)
C(8)
C(5)
C(19)
C(49)
C(39)
C(29)
C(59)
C(3)
C(1)
C(2)
150.673 (0.002) 156.312 (1.294) 156.002 (0.159) 156.134 (0.083) 156.291 (0.061)
149.070 (0.004) 152.750 (3.581) 153.357 (0.059) 153.416 (0.261) 156.486 (0.030)
145.182 (0.000) 148.660 (0.003) 145.694 (0.005) 149.740 (0.015) 149.752 (0.002)
143.112 (0.020) 140.785 (0.190) 140.709 (0.076) 140.848 (0.028) 141.149 (0.258)
119.283 (0.001) 119.903 (0.696) 119.103 (0.169) 119.189 (0.093) 119.398 (0.113)
88.890 (0.007) 87.953 (0.037) 87.745 (0.016) 87.632 (0.010) 87.630 (0.015)
85.092 (0.009) 84.754 (0.018) 85.224 (0.108) 85.612 (0.101) 85.920 (0.290)
75.511 (0.019) 75.225 (0.063) 75.218 (0.053) 75.321 (0.108) 76.923 (0.605)
71.120 (0.002) 71.234 (0.005) 71.414 (0.097) 71.527 (0.011) 72.734 (0.223)
65.214 (0.004) 65.477 (0.264) 64.430 (0.508) 64.317 (0.501) 65.202 (0.687)
45.568 (0.002) 45.557 (y0.107) 45.533 (y0.067) 46.450 (y0.201) 47.158 (y0.618)
37.452 (y0.014) 37.422 (y0.110) 37.491 (y0.098) 38.715 (y0.222) 39.602 (y0.476)
24.635 (0.003) 24.644 (y0.094) 24.892 (y0.143) 28.129 (y0.071) 30.578 (y0.329)
Negative sign stands for a high field shift, positive sign stands for a low field shift.
CMP and all nitrogen atoms from the polyamines (Tables 2– 4). For example, in the system AMP/3,3-tri at pH 4, the shifts of the signals from C(6), C(2), C(8) and C(5) are 1.294, 3.581, 0.190 and 0.696 ppm, respectively. Moreover, the shift of the signal from the C(4) atom which is far from reaction centers, is only 0.003 ppm. The changes in the signal positions of the carbon atoms C(1), C(2) and C(3) from 3,3-tri are y0.110, y0.094 and y0.107 ppm, respectively. No significant shifts of the bioligand signals are observed at pH 2, at which the adducts do not form (Table 2). Analysis of the 13C and 31P NMR spectra suggests the involvement of the phosphate groups in the interactions in all systems except AMP/ 3,3,3-tet, which corresponds very well to the results of the equilibrium studies. For example, in the 31P NMR spectra of NMP/Put at pH 6, the shifts of the phosphorus atom signals in AMP or CMP are 0.291 and 0.208 ppm, respectively (Table 5), which is strong evidence for the involvement of the phosphate group from the nucleotide in the interaction with Put. In the system AMP/3,3,3-tet, in which the phosphate group is not involved in the adduct formation, the analogous changes are only 0.008 and 0.006 ppm, respectively, for C(59) and P (Tables 4 and 5). These observations confirm a difference in the character of tetramines relative to the shorter polyamines (it was also unexpectedly found earlier for the systems with spermine [13]). Unlike the systems of AMP with tetramines (Spm or 3,3,3-tet) in which no interactions with the phosphate group were found, in the systems of these tetramines with CMP, the phosphate group from the nucleotide takes part in formation of molecular complexes (it was also deduced from analysis of NMR signal changes). 3.3. Equilibrium studies of the Cu(II)/nucleotide/polyamine systems Table 6 presents values of the overall stability constants of the complexes formed in the systems of Cu(II) with nucleotides and polyamines. The results for the ternary systems show the formation of molecular complexes ML-L9 (with complex–ligand non-covalent interaction), protonated com-
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plexes MHxL and mixed-ligand compounds of the MLL9 type. 3.3.1. Cu/AMP/Put, Cu/AMP/3,3-tri, Cu/AMP/Spd Mixed-ligand complexes are not formed in the Cu/AMP/ Put system. Taking into account the degree of protonation of the nucleotide and polyamine [13,27], it should be assumed that in the protonated Cu(AMP)H2(3,3-tri) complex which exists in the pH range from about 4.5 to 7 (Fig. 3(a)) only one nitrogen atom of 3,3-tri is involved in coordination. The hypothetical reaction of the complex formation is described by Eq. (2): Cu(AMP)qH 2 (3,3-tri)|Cu(AMP)H 2 (3,3-tri)
(2)
The equilibrium constant for reaction 2, calculated from the overall stability constants and the protonation constants, log K eslog b Cu(AMP)H 2 (3,3-t r i)ylog b Cu AMP ylog b H 2 (3,3-t r i) is 6.72. The value of log Ke for reaction 3 of the protonated binary complex formation is 5.00: CuqHPut|CuHPut
(3)
The increase of log Ke for reaction 2 relative to the value for reaction 3 suggests additional stabilization of the mixedligand complexes. In the first approach it should be assumed that the additional stabilization is related to the presence of non-covalent intramolecular interactions [28]. In the system Cu/AMP/Spd, as mentioned in Section 2, a precipitate starts to appear from a pH of about 5.5 (formation of barely soluble ternary complexes is under study). 3.3.2. CuCMP/Put, Cu/CMP/(3,3-tri), Cu(CMP)/Spd A similar mode of interaction as in Section 3.3.1 is met for the complexes Cu(CMP)H(Put) (Fig. 3(b)) which exists from a pH of about 5.5, and binds 30% of the metal at a pH of about 7 and for the Cu(CMP)H2(3,3-tri) complex formed from a pH of about 5, binding about 15% of metal ions at pH 6. The hypothetical reactions of formation of these complexes are described by Eqs. (4) and (5):
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150.762 (0.089) 154.865 (1.002) 156.029 (0.132) 156.058 (0.159) 156.186 (0.044)
C(6) 149.047 (0.019) 149.344 (0.175) 153.367 (0.149) 153.396 (0.241) 153.460 (0.302)
C(2) 145.668 (0.486) 151.797 (0.310) 149.536 (0.133) 149.553 (0.172) 149.703 (0.047)
C(4) 143.002 (0.091) 140.940 (0.017) 140.673 (0.040) 140.776 (0.100) 140.897 (0.006)
C(8) 119.262 (0.026) 119.262 (0.055) 119.145 (0.127) 119.137 (0.145) 119.250 (0.035)
C(5) 88.823 (0.007) 88.990 (0.951) 87.762 (0.000) 87.710 (0.088) 87.661 (0.046)
C(19) 85.047 (0.009) 84.808 (0.005) 85.202 (0.104) 85.449 (0.183) 85.526 (0.102)
C(49) 75.514 (0.016) 75.254 (0.025) 75.223 (0.048) 75.222 (0.089) 75.336 (0.018)
C(39) 71.118 (0.000) 71.172 (0.057) 71.375 (0.048) 71.457 (0.081) 71.536 (0.025)
C(29) 65.157 (0.012) 65.412 (0.199) 64.666 (0.272) 64.417 (0.099) 64.277 (0.362)
C(59) 47.931 (0.004) 47.936 (y0.004) 47.856 (y0.063) 47.904 (y0.498) 49.612 (y0.692)
C(4)
10
8
6
4
2
pD
151.537 (0.003) 155.171 (0.634) 155.977 (0.301) 154.831 (1.356) 156.284 (0.067)
C(6) 149.189 (0.012) 152.149 (2.419) 153.359 (0.176) 152.682 (0.723) 153.556 (0.012)
C(2) 146.787 (0.007) 148.482 (0.190) 149.493 (0.060) 149.394 (0.281) 149.770 (0.003)
C(4) 142.743 (0.009) 140.950 (0.040) 140.649 (0.138) 141.063 (0.137) 140.961 (0.107)
C(8) 119.304 (0.014) 119.161 (0.000) 119.081 (0.136) 119.223 (0.100) 119.263 (0.068)
C(5) 88.729 (0.002) 88.021 (0.010) 87.849 (0.003) 87.081 (0.000) 87.644 (0.013)
C(19) 85.011 (0.011) 84.390 (0.040) 85.019 (0.019) 84.795 (0.010) 85.645 (0.002)
C(49) 75.484 (0.013) 75.246 (0.002) 75.155 (0.003) 75.250 (0.002) 75.329 (0.010)
C(39) 71.079 (0.007) 71.172 (0.010) 71.270 (0.052) 71.141 (0.020) 71.528 (0.000)
C(29)
37.385 (y0.008) 37.349 (y0.002) 37.348 (y0.038) 37.344 (y0.179) 38.682 (y0.217)
24.654 (0.002) 24.670 (y0.046) 24.696 (y0.052) 24.940 (y1.180) 27.517 (y2.994)
C(6)
45.436 (0.005) 45.407 (y0.018) 45.376 (y0.079) 46.410 (y0.123) 46.328 (y0.145)
C(3)
24.825 (0.003) 24.832 (y0.004) 24.829 (y0.013) 25.137 (y2.869) 29.450 (y3.049)
C(2)
C(1)
37.471 (0.009) 37.468 (y0.015) 37.446 (y0.028) 37.605 (y1.089) 38.590 (y0.997)
C(1)
24.588 (y0.011) 23.520 (y0.025) 23.563 (y0.023) 23.519 (y0.301) 25.546 (y0.066)
C(2)
39.699 (y0.004) 39.695 (y0.013) 39.648 (y0.052) 39.692 (y0.511) 40.515 (y0.816)
C(7)
23.519 (0.002) 24.590 (y0.018) 24.608 (y0.010) 24.585 (y0.172) 28.075 (y0.123)
C(5)
45.408 (y0.005) 45.412 (y0.006) 45.364 (y0.043) 45.483 (y0.881) 46.568 (y0.515)
C(3)
65.154 (0.002) 65.196 (0.010) 64.843 (0.032) 65.249 (0.008) 64.493 (0.007)
C(59)
Table 4 13 C NMR signal positions for the AMP/3,3,3-tet system and their changes in relation to single ligands (ppm, in parentheses)
10
8
6
4
2
pD
Table 3 C NMR signal positions for the AMP/Spd system and their changes in relation to single ligands (ppm, in parentheses)
13
45.561 (y0.008) 45.540 (y0.006) 45.486 (y0.075) 46.090 (y0.215) 48.487 (y0.032)
C(4)
23.656 (y0.004) 23.665 (y0.011) 23.644 (0.000) 23.891 (y1.495) 25.764 (y1.121)
C(5)
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Table 5 31 P NMR signal positions for the AMP/PA and CMP/PA systems and their changes in relation to single ligands (ppm, in parentheses) pD
2 4 6 8 10
AMP/PA
CMP/PA
Put
Spd
3,3-Tri
3,3,3-Tet
Put
Spd
3,3-Tri
3,3,3-Tet
0.181 (0.010) 0.338 (0.017) 1.587 (0.291) 4.080 (0.263) 3.986 (0.093)
0.164 (0.027) 0.220 (0.101) 2.250 (0.372) 4.101 (0.284) 3.772 (0.121)
0.174 (0.017) 0.209 (0.112) 2.193 (0.315) 4.120 (0.303) 3.680 (0.213)
0.193 (0.002) 0.317 (0.004) 1.879 (0.001) 3.811 (0.006) 3.888 (0.005)
0.030 (0.007) 0.184 (0.062) 2.193 (0.208) 3.955 (0.184) 3.801 (0.046)
0.034 (0.011) 0.194 (0.072) 2.179 (0.194) 3.553 (0.218) 3.879 (0.032)
0.028 (0.005) 0.250 (0.128) 2.299 (0.314) 3.534 (0.237) 3.979 (0.132)
0.031 (0.009) 0.296 (0.174) 2.281 (0.297) 3.538 (0.283) 3.728 (0.119)
Table 6 Overall stability constants (log b) of Cu(II) complexes with AMP (or CMP) and polyamines Cu(II)/AMP/PA
Cu(II)/CMP/PA
Species
Log b
Species
Log b
CuAMP Cu(AMP)(OH)
3.02 (8) y3.82 (5)
CuCMP Cu(CMP)(OH)
2.71 (6) y4.26 (8)
CuH(Put) Cu(Put) Cu(Put)2 Cu(Put)2(OH)
15.83 (37) 8.62 (13) 13.40 (18) 0.065 (16)
CuH(Put) Cu(Put) Cu(Put)2 Cu(Put)2(OH) Cu(CMP)H(Put)
15.83 (37) 8.62 (13) 13.40 (18) 0.065 (16) 19.02 (8)
CuH(3,3-tri) Cu(3,3-tri) Cu(3,3-tri)2 Cu(3,3-tri)(OH) Cu(AMP)H2(3,3-tri)
18.87 (18) 13.71 (3) 18.48 (5) 3.14 (5) 29.96 (15)
CuH(3,3-tri) Cu(3,3-tri) Cu(3,3-tri)2 Cu(3,3-tri)(OH) Cu(CMP)H2(3,3-tri) Cu(CMP)H4(3,3-tri)
18.87 (18) 13.71 (3) 18.48 (5) 3.14 (5) 28.40 (8) 39.71 (5)
CuH2(3,3,3-tet) CuH(3,3,3-tet) Cu(3,3,3-tet) Cu(AMP)(3,3,3-tet)
42.03 (7) 27.49 (7) 16.36 (4) 20.15 (4)
CuH2(3,3,3-tet) CuH(3,3,3-tet) Cu(3,3,3-tet) Cu(CMP)(3,3,3-tet)
42.03 (7) 27.49 (7) 16.36 (4) 20.62 (5)
Cu(CMP)qHPut|Cu(CMP)H(Put)
(4)
Cu(CMP)qH 2 (3,3-tri)|Cu(CMP)H 2 (3,3-tri)
(5)
The equilibrium constants calculated from the overall stability constants and the protonation constants (e.g. for reaction 4, log Keslog bCu(CMP)H(Put)ylog bCuCMPylog bHPut) for the above reactions are 5.48 and 5.47. The increase of log Ke for reactions 4 and 5 relative to the value for reaction 3 suggests additional stabilization of the mixed-ligand complexes. The same values of log Ke for the complexes Cu(CMP)H(Put) and Cu(CMP)H2(3,3-tri) indicate a similar character of interaction including only one nitrogen atom of PA. The mixed-ligand complexes are not formed in the Cu/ CMP/Spd system, which confirms a low tendency of spermidine to form stable ternary complexes.
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In the Cu/CMP/3,3-tri system, the presence of the complex Cu(CMP)H4(3,3-tri) was also found, which starts forming from a pH of about 3 and reaches a maximum concentration at a pH about 5, at which it binds 30% of the metal ions. The number of the protonated ligand donor groups in this complex suggests formation of a molecular complex in which the polyamine remains out of the inner coordination sphere.
3.3.3. Cu/AMP/3,3,3-tet, Cu/CMP/3,3,3-tet Mixed-ligand complexes of the MLL9 type form only in the systems of nucleotides with tetramine-Cu(AMP)(3,3,3tet) or Cu(CMP)(3,3,3-tet). These complexes start forming from a pH of about 5 and dominate from a pH of about 6 binding 85% of the metal (Fig. 3(c)).
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Fig. 3. Distribution diagrams for the systems: (a) Cu(II)/AMP/3,3-tri: 1, Cu2q; 2, Cu(AMP)H2(3,3-tri); 3, Cu(3,3-tri); 4, CuAMP; CAMPs0.0025 M; C3,3-tris0.0025 M; CCus0.0016 M; (b) Cu(II)/CMP/Put: 1, Cu2q; 2, Cu(CMP)H(Put); 3, CuCMP; 4, Cu(CMP)OH; 5, CuH(Put); 6, CuPut; CCMPs 0.0025 M; C3,3-tris0.0025 M; CCus0.0016 M; (c) Cu(II)/AMP/3,3,3-tet: 1, Cu2q; 2, Cu(AMP)(3,3,3-tet); 3, CuH(3,3,3-tet); 4, Cu(3,3,3-tet); 5, CuAMP; 6, CuH2(3,3,3-tet); CAMPs0.0025 M; C3,3,3-tets0.0025 M; CCus0.0016 M.
3.4. Spectral studies of the Cu(II)/nucleotide/polyamine systems 3.4.1. Cu/AMP/Put, Cu/CMP/Put Mixed-ligand complexes are not formed in the Cu/AMP/ Put system (at least in detectable amounts). Analysis of the electron spectra UV–Vis and EPR (combined with the results of equilibrium studies) in the system Cu/CMP/Put suggests that only one nitrogen atom from polyamine and the oxygen atoms from the phosphate group take part in the metallation. The position of the absorption band is lmaxs736 nm (pH 6.5), which corresponds to the coordination mode of the N1O type, as has been deduced from the data published in [29,32]. The EPR spectral parameters are g≤s2.2473, A≤s186.5 at pH 6.5 (Table 7). It has been established that with the increasing number of coordinated donor atoms the g≤ value decreases while A≤ increases, which has been observed for Cu–Nx (xs1 to 6) and Cu– NxOy (xs0 to 4, ys0 to 4) chromophores [30,31]. Analysis of the values of these parameters and their comparison with
the results reported for similar systems indicate metallation involving one nitrogen atom from the polyamine and oxygen atoms from the phosphate group. The equilibrium constant (log Ke) of the Cu(CMP)H(Put) complex formation (reaction 4) is 5.48. The value for the CuHPut (reaction 3) is 5.00 and for the Cu(CMP) complex formation (with N1O coordination) is 2.71 [13,17]. The comparison of the values testifies to the involvement of one nitrogen atom from Put into the inner coordination sphere. The earlier discussed stabilization of the mixed-ligand complex relative to that formed in the binary system is a result of non-covalent interactions. The protonated amine group from the polyamine is involved in weak interplay with the donor centers of the pyrimidine base. For all systems of Cu(II), CMP and PA, the involvement of the nucleotide carbonyl group in coordination was excluded. In the IR spectra of metal/ligand systems, the position of the band corresponding to the stretching vibrations of the carbonyl group at 1652 cmy1 was the same as for metalfree systems.
Table 7 EPR and absorption spectral data for the Cu/NMP/PA systems System
pH
lmax (nm)
´ (dm3/(mol cm))
gH
g≤
A≤ (10y4 cmy1)
Cu/CMP/Put Cu/AMP/(3,3-tri) Cu/CMP/(3,3-tri)
6.5 6.0 4.5 5.8 6.5 6.5
736 725 798 618 578 587
27.3 28.5 31.8 16.4 19.2 19.8
2.0554 2.0577 2.0678 2.0530 2.0577 2.0517
2.2473 2.2446 2.3382 2.2333 2.2232 2.2115
186.5 163.0 161.3 185.0 175.0 190.4
Cu/AMP/(3,3,3-tet) Cu/CMP/(3,3,3-tet)
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3.4.2. Cu/AMP/3,3-tri, Cu/CMP/3,3-tri Similar to the above discussed system with Put, the results of the UV–Vis and EPR study (including equilibrium data) for both the Cu/AMP/3,3-tri and Cu/CMP/3,3-tri systems point to the metallation through one of the nitrogen atoms from 3,3-tri and the oxygen atoms from the phosphate group (Table 7). Equilibrium studies indicate additional stabilization in the Cu(AMP)H2(3,3-tri) as well as in Cu(CMP)H2(3,3-tri) species (discussed in Section 3.3). The protonated nitrogen atoms from the polyamine in both complexes take part in the intramolecular interactions with the donor atoms from the base, which is manifested by the increase in complex stability (log Ke for reaction 2 or 5 comparing to reaction 3). Moreover, formation of the molecular complex Cu(CMP)H4(3,3-tri) was detected in the Cu/CMP/3,3-tri system. At pH 4.5, when this species is the only complex, lmaxs798 nm, while the EPR parameters are g≤s2.3382, A≤s161.3, Table 7, which suggests the coordination of copper ions only through the oxygen atoms of the phosphate group (Cu–O chromophore). In the pH range below 5 (where the Cu(CMP)H4(3,3-tri) dominates), polyamine is completely protonated and all donor groups in the amine are blocked for metallation. The ligand remaining outside the inner coordination sphere is involved in non-covalent interaction with donor groups of the nucleotide. The conclusion concerning formation of the ML-L9 type complex has also been supported by the results of equilibrium studies. Analogous interactions were observed earlier in the system Cu/ AMP/Spm [13]. 3.4.3. Cu/AMP/3,3,3-tet, Cu/CMP/3,3,3-tet A different mode of coordination was observed in the systems of Cu(II) with the nucleotides and tetramine-3,3,3-tet. UV–Vis and EPR results (Table 7) indicate coordination with the N4O chromophore. At the pH range in which the MLL9 type complexes dominate (Fig. 3(c)), the absorption band maxima are at 578 and 587 nm for the systems with AMP and CMP, and the EPR parameters are g≤s2.2232 and
A≤s175.0 as well as g≤s2.2115 and A≤s190.0, respectively. Moreover, NMR results suggest that all nitrogen atoms from the polyamine and the oxygen atoms from the nucleotide phosphate group are involved in the metallation (Fig. 4). For instance, at pH about 6, in the 13C and 31P NMR spectra of the Cu(AMP)(3,3,3-tet) complex, the signals from AMP C(59) as well as P (0.303, 0.098 ppm, respectively) and from carbon atoms of the tetramine are shifted: C(1), C(2), C(3), C(4) and C(5) by y1.014, y0.827, y0.695, y1.101 and y0.739 ppm, respectively. The shifts of the nucleotides signals assigned to C(2), C(6), C(5) and C(8) from the close neighborhood of the atoms N(1) and N(7) are only 0.019, 0.025, 0.016 and 0.017 ppm, respectively. In order to minimize the broadening of the NMR signals related to the paramagnetic character of Cu(II) ions, the spectra were taken at low metal concentrations (on the distribution diagrams, pH ranges of particular complex domination are practically the same as in the systems for equilibrium studies with higher concentrations of ligands). Similar to that observed earlier when studying analogous systems [13,15,16,18,19], significant changes in the chemical shift were noted only in the pH ranges in which complex formation takes place, established on the basis of potentiometric studies. The NMR technique was also used to determine the coordination mode in similar systems with paramagnetic species [33,34]. Fig. 5 (referring to the Cu/CMP/3,3,3-tet system) presents an exemplary 13C NMR spectrum of the ligand obtained by the decoupling technique, and the spectrum of its complex with Cu(II), besides the spectra taken under the conditions in which complexes are not formed. The mode of coordination in both systems of Cu(II) with 3,3,3-tet and both nucleotides studied is substantially different to that in the analogous systems with another tetraminespermine, described in [13,17]. The results of this work prove that even small structural changes of the polyamine influence the mode of coordination, and confirm the role of the structural factor in the interactions of this group of bioligands. This factor also explains the significantly different properties of biogenic amines from
Fig. 4. Changes in 13C and 31P NMR signal positions (NDdN) as a function of pH for the Cu/AMP/3,3,3-tet system (in relation to free ligands): (a) AMP; (b) 3,3,3-tet; CAMPsC3,3,3-tets0.05 M; CCus0.0005 M.
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A. Gasowska et al. / Journal of Inorganic Biochemistry 78 (2000) 139–147
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Fig. 5. 13C NMR spectra. (a) Top: Cu(II)/CMP/3,3,3-tet system; bottom: metal-free system; pH 2, CCMPsC3,3,3-tets0.05 M; CCus0.0005 M (no changes in the signal positions in relation to the metal-free system); (b) Top: Cu(II)/CMP/3,3,3-tet system; bottom: metal-free system; pH 8, CCMPsC3,3,3-tets0.05 M; CCus0.0005 M (changes in the signal positions observed for: C(29) y8 and C(59) y9 from CMP; C(3) y10, C(4) y11, C(1) y12, C(2) y13, C(5) y14 from 3,3,3-tet).
those of their biologically inactive analogs. Moreover, metal ions interfere with the reactions of bioligands. The similar properties of spermine and 3,3,3-tet in binary metal-free systems are substantially changed in systems with Cu(II). Significant differences in coordination character were also observed in the ternary systems with triamines, spermidine and 3,3-tri, which also testifies to a different behavior of biogenic amines.
Acknowledgements This work was supported by Grant No. 3 T09A 092 12 of the Polish State Committee for Scientific Research.
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