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The interaction of BMXD and its copper(II) complexes with glycine, aspartic acid, and histidine
Inorganica Chimica Acta 286 (1999) 55 – 61
The interaction of BMXD and its copper(II) complexes with glycine, aspartic acid, and histidine Eric Ross, Ramunas J. Motekaitis, Arthur E. Martell * Department of Chemistry, Texas A&M Uni6ersity, College Station, TX 77842 -3012, USA Received 26 May 1998; accepted 9 September 1998
Abstract The macrocycle, 3,6,9,17,20,23-hexaazatricyclo[23.3.1.111,15]triaconta-1(29)11(30),12,14,25,27-hexaene, BMXD, is shown to recognize three amino acids, glycine, aspartic acid, and histidine, to form binary species. The mono- and dinuclear copper(II) complexes are also shown to host these amino acids. The stability constants for the binary complexes of the amino acids with the macrocycle, and of the ternary complexes containing amino acid, copper(II) and macrocycle, are reported, and binding schemes are suggested for the recognition of glycine, and for the dinuclear ternary species with histidine and glycine. Aspartic acid is found to form the most stable complexes, both with and without the presence of copper(II) ion. © 1999 Elsevier Science S.A. All rights reserved. Keywords: Copper complexes; Amino acid complexes; Macrocyclic complexes
1. Introduction Macrocyclic polyamines are versatile chelating agents capable of forming many different complexes depending on the protonation state of the amine. In its protonated form, the positively charged macrocycle can bind anionic species through hydrogen bonds and electrostatic forces [1–3]. The ligand can form mono- and dinuclear metal complexes with several different metal ions. These species have been shown to further selectively bind anionic species to form tertiary cascade complexes [2–5]. The hexabasic macrocycle BMXD (3,6,9,17,20,23hexaazatricyclo[23.3.1.111,15]triaconta-1(29)11(30),12,14, 25,27-hexaene) (1), has been shown to form stable mono- and dinuclear complexes with the copper(II) ion [6]. These complexes are the subject of a recent study involving biologically interesting inorganic phosphates [7]. Another study involving OBISDIEN, the ether bridged analog of BMXD, reported the stability constants for the ligand and its copper(II) complexes with * Corresponding author. Tel.: +1-409-8452011; fax: + 1-4098454719.
various peptides [8]. This work is related to the metal ion promoted hydrolysis of peptides and related compounds. It is of interest to investigate the interaction of other biological molecules with macrocycles and their metal complexes. In the present work, the ability of BMXD and its copper(II) complexes to host three amino acids, glycine, aspartic acid, and histidine, is investigated. Thus, the three types of a-amino acids are considered in this study: equal numbers of carboxylate and amino groups (glycine), more carboxylate than amino groups (aspartic acid) and more amino groups than carboxylate groups (histidine). The stability constants for the
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E. Ross et al. / Inorganica Chimica Acta 286 (1999) 55–61
complex species formed are reported and used to prepare distribution diagrams showing the relative concentrations of the complexes formed between these amino acids and the macrocycles.
2. Experimental
2.1. Materials Reagent grade inorganic materials were used throughout the experiments without further purification. The KOH solution was made from Baker ‘Dilutit’ carbonate-free sealed ampoules diluted as per directions. The base was stored under a sealed inert atmosphere at all times and was checked periodically for carbonate content by the use of a Gran’s plot [9]. The carbonate content was never greater than 1.5%. The KOH was standardized by potassium acid phthalate titration [9]. All aqueous solutions were prepared with distilled water. The copper(II) chloride solution was standardized by EDTA titration [9]. Several grams of the ligand as its hexahydrochloride salt were kindly donated by David Nation.
2.2. Potentiometric equilibrium measurements For p[H] determination, a Corning Research model 150 pH meter was used with glass and calomel reference electrodes. A 10 ml piston burette was used to deliver the KOH solution. Titrations were performed in a temperature regulated cell that was sealed and purged continuously with purified and humidified argon. The pH meter was calibrated with a standard dilute acid solution (HCl) before each titration to read the H + -ion concentration directly so that the p[H] is defined as − log [H + ]. The ionic strength of the solution was adjusted to 0.100 M by addition of 1.00 M KCl, and so served as the supporting electrolyte.
2.3. Computations Stability constants were calculated with the program [9]. Species distribution curves were calculated from the stability constants with the program SPE and plotted with SPEPLOT [9]. The value of Kw at the given ionic strength was calculated to be 10 − 13.78. All titrations contained approximately 0.002 M ligand and appropriate ratios of other constituents. The protonation constants of the three amino acids were determined experimentally under the conditions employed here for use in the calculations. All mixed systems titrations contained at least 80 points of data between p[H] 2.0 and 11.1. All backward titrations were carried out three or more times to ensure accuracy and reproducibility. Each ternary system was titrated at
BEST
Table 1 Protonation and formation constants of BMXD and Cu(II) complexes (m= 0.10 (KCl), 25.0°C; Mx =BMXD, OH = OH−, H= H+, and Cu=Cu2+) Quotient K
Log K a
[MxH]/[Mx][H] [MxH2]/[MxH][H] [MxH3]/[MxH2][H] [MxH4]/[MxH3][H] [MxH5]/[MxH4][H] [MxH6]/[MxH5][H] [MxCu]/[Mx][Cu] [MxCuH]/[MxCu][H] [MxCuH2]/[MxCuH][H] [MxCuH3]/[MxCuH2][H] [MxCu(OH)][H]/[MxCu] [MxCu2]/[MxCu][Cu] [MxCu2(OH)][H]/[MxCu2] [MxCu2(OH)2][H]/[MxCu(OH)] [MxCu2(OH)3][H]/[MxCu(OH)2]
9.43 8.71 7.98 7.11 3.80 3.39 13.45 8.38 7.45 3.87 −9.17 11.24 −7.87 −8.78 −11.52
Log K (1) (1) (1) (1) (1) (1) (3) (4) (3) (1) (6) (3) (3) (5) (9)
9.49b 8.73b 8.03b 7.29b 3.64b 3.45b 13.63c 8.40c 7.20c 3.68c −8.93c 10.86c −7.83c −8.74c −11.70c
a
Estimated error in parentheses based on error propagation. Ref. [6]. c Ref. [7]. b
least twice. The computation of stability constants was based on the minimization of the sigma fit, however, error estimates were performed by a propagation of errors analysis. Thus the errors of each input variable were originally estimated and their affects on the calculated result were then tabulated together with the new stability constants in Tables 1–8.
3. Results and discussion The protonation constants of the ligand BMXD and its Cu(II) binding constants have been reported previously [6,7]. They were re-determined at the experimental conditions employed in this work, and the results and comparisons are located in Table 1. The protonation constants and Cu(II) binding constants of the three amino acids were also re-evaluated at the experimental conditions used in this research. Their values and comparisons to literature values [10] are listed in Table 2. The difference in the constants in Tables 1 and 2, between the values determined in the present work and Table 2 Protonation constants of substrates (m= 0.10 (KCl), 25.0°C)a Substrate
Log K 1H
Log K 2H
Log K 3H
Glycine Aspartic acid Histidine
9.55 (9.58) 9.68 (9.65) 9.11 (9.09)
2.22 (2.34) 3.68 (3.70) 6.03 (6.04)
1.83 (2.00) 1.65 (1.7)
a Values in parentheses, Ref. [10]. Error estimates: glycine 9 0.01; aspartic acid 9 0.01; histidine 9 0.01.
E. Ross et al. / Inorganica Chimica Acta 286 (1999) 55–61 Table 3 Stability constants for the BMXD–glycine system (m= 0.10 (KCl), 25.0°C; Mx = BMXD, Gl = Glycinate, and H =H+) Stepwise quotient K
Stoichiometry Mx
Gl
H
1 1 1 1 1
1 1 1 1 1
1 2 3 4 5
a
[MxGlH]/[MxH][Gl] [MxGlH2]/[MxH2][Gl] [MxGlH3]/[MxH3][Gl] [MxGlH4]/[MxH4][Gl] [MxGlH5]/[MxH5][Gl]
Log K a
3.11 3.48 4.33 4.98 7.91
Table 5 Stability constants for the BMXD–aspartic acid system (m= 0.10 (KCl), 25.0°C; Mx =BMXD, As =aspartate, and H =H+) Stoichiometry
(4) (3) (3) (2) (2)
Estimated error in parentheses.
Mx
As
H
1 1 1 1 1 1 1
1 1 1 1 1 1 1
1 2 3 4 5 6 7
a
the values determined by others, are minor and about what would be expected from the difference in conditions employed. In the absence of copper ions, the recognition of the amino acids was found to be quite poor (Fig. 1). The order of increasing abundance of various binary species (of MXBD–amino acid) was found to be histidine B 10%, glycineB 25% and aspartic acidB 40%. In the presence of copper the relative recognition for amino acids is increased considerably. Thus histidine (Fig. 2) and glycine approach 50% maximum formation as dicopper–BMXD – amino acid complexes while aspartic acid approaches 100% formation (Fig. 3).
3.1. Glycine recognition by BMXD The equilibrium constants calculated for the binding of glycine to protonated forms of BMXD are listed in Table 3. It was found that five-protonated ligandglycine species are formed between p[H] 2.8 and 12. Stability of the species decreases as the degree of protonation decreases. This is in accordance with the coulombic effect in that increased positive charge of the ligand nitrogens more strongly coordinate the anionic portion of the glycine zwitterion, the negative carboxylate group through hydrogen bonding. The strongest method of binding in solution probably involves the glycine amino nitrogen hydrogen bonded to an amino group of the ligand. A suggested binding scheme of the Table 4 Stability constants for the BMXD–Cu(II)–glycine system (m= 0.10 (KCl), 25.0°C; Mx =BMXD, Gl =Glycinate, Cu = Cu2+ and H = H+) Stoichiometry Mx
Cu
Gl
H
1 1 1
2 2 2
1 2 2
0 0 1
a
Stepwise quotient K
Log K a
[MxAsH]/[MxH][As] [MxAsH2]/[MxH2][As] [MxAsH3]/[MxH3][As] [MxAsH4]/[MxH4][As] [MxAsH5]/[MxH5][As] [MxAsH6]/[MxH6][As] [MxAsH7]/[MxH6][AsH]
3.21 3.73 4.64 5.45 8.68 9.14 3.16
(5) (3) (4) (4) (4) (4) (3)
Estimated error in parentheses.
MxGlH5 species is illustrated in Fig. 2. The titration curve of the system indicates a buffered region between a values, zero and two by a break, with the remainder of the curve buffered at higher p[H]. The first buffer region corresponds to the deprotonation of the macrocycle to the tetra-protonated state. The following buffer region is the formation of successive deprotonated states of the ligand. In 2 a glycine unit in its neutral zwitterion form is shown to bind the tetra-protonated macrocycle through hydrogen bonding. The presence of binary species MxGlH with the fully deprotonated ligand is consistent with the primary binding of the glycine being through the hydrogen bond formed by the amino acid nitrogen. The increasing stability of the more highly protonated species involving the positively charged macrocycle indicates that the negative carboxylate group is involved in the binding. Further protonation of the hepta-protonated binary species may not be possible because the positively charged diprotonated glycine molecules would not be attracted to the positively charged macrocyclic ligand. Support for such intermolecular hydrogen bonding can be found in the crystallographic determination of CH3NH2 –H– + NH2CH3 [11]. In this system as well as in the aspartic acid and histidine systems it is difficult to uniquely quantify the relative contributions of pure electrostatic attractions relative to hydrogen bonding forces in the carboxylate affinity for protonated amines since the ammonium groups by definition all possess a potentially bindable extra proton [1].
3.2. Aspartic acid recognition by BMXD
a
Quotient K
Log K
[MxCu2Gl]/[MxCu2][Gl] [MxCu2Gl2]/[MxCu2Gl][Gl] [MxCu2Gl2H]/[MxCu2Gl2][H]
6.27 (3) 4.68 (3) 8.74 (4)
Estimated error in parentheses.
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The binding constants determined for the binary species of BMXD and aspartic acid are listed in Table 5. In comparison with the glycine binary systems, BMXD is shown to recognize aspartic acid with greater affinity. Seven different protonated binary species are seen to form, necessarily incorporating a number of binding schemes.
E. Ross et al. / Inorganica Chimica Acta 286 (1999) 55–61
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Table 6 Stability constants for the BMXD–Cu(II)–aspartic acid system (m= 0.10 (KCl), 25.0°C; Mx =BMXD, As =aspartic acid, Cu =Cu2+ and H = H+) Stoichiometry Mx
Cu
As
H
1 1 1 1 1
1 2 2 2 2
1 1 1 2 1
4 0 1 0 −1
a
Quotient K
Log K a
[MxCuAsH4]/[MxCuH3][AsH] [MxCu2As]/[MxCu2][As] [MxCu2AsH]/[MxCu2][AsH] [MxCu2As2]/[MxCu2As][As] [MxCu2As(OH)]/[MxCu2(OH)][As]
2.86 8.52 4.75 3.30 5.58
(8) (6) (3) (7) (3)
Estimated errors in parentheses.
3.3. Histidine recognition by BMXD
A dramatic increase in stability between the tetraand penta-protonated binary species might be due to the incorporation of carboxylate hydrogen bonds into the bonding mode. A hepta-protonated species is present from low p[H] up to p[H] 4. The only possible bonding mode for this species is hydrogen bonding between the most acidic carboxylate groups and protonated amino groups on the macrocyclic ligand. The monoprotonated binary species is the result of a hydrogen bond formed between the protonated amino group of the amino acid and a deprotonated amino group of the macrocycle. The additional carboxylic acid group on the aspartic acid results in an increased negative charge on the species throughout most of the pH range. This accounts for the increased bonding to the positively charged protonated macrocycle and accounts for the existence of highly protonated binary systems.
Table 7 Stability constants for the BMXD–histidine system (m = 0.10 (KCl), 25.0°C; Mx = BMXD, Hi = histidinate, and H = H+) Stoichiometry Mx
Hi
H
1 1
1 1
6 7
a
Stepwise quotient K
Log K a
[MxHiH6]/[MxH6][Hi] [MxHiH7]/[MxH6][HiH]
9.47 (2) 4.52 (2)
Estimated errors in parentheses.
The two binding constants for the binary species formed from histidine and protonated BMXD are listed in Table 7. The hepta- and hexa-protonated ternary species are shown to form between p[H] 5 and 10. It is suggested that the imidizole groups on the histidine molecule form hydrogen bonds in addition to those formed by the primary amine group in the amino acid. When the imidizole group is protonated at p[H] values below its pKa value, the ternary species no longer form. The coordination of the imidizole nitrogen is also suggested in the MxHiH6 species. Its concurrent formation with the tetra-protonated free ligand, and its stability compared to the binary system formed between the tetra-protonated macrocycle and glycine, indicate the involvement of imidizole groups. However it cannot be ignored that the degree of complex formation with BMXD is the smallest of the three amino acids studied here.
3.4. Binding of glycine by Cu(II) complexes of BMXD The equilibrium constants determined for the copper(II) complexes of the BMXD macrocycle and glycine are listed in Table 4. The dinucleating tendency of the macrocycle is shown in the 1:1:1 titration of BMXD, Cu(II), and glycine, as the dinuclear mono-glycinated species exists over a wide p[H] range (4–10) and as one of the principal species in solution. The glycine nitrogen is deprotonated at p[H] as low as 3.5, indicating its importance in the formation of the ternary species. The monoprotonated complex exists only as a minor species at lower p[H], so the predominant factor in the binding is the glycine amine–Cu(II) coordinate bond. In the titration with two equivalents of Cu(II) and glycine, the protonated species appears to the extent of around 10%, so the carboxylate group is capable of forming weak bonds with the macrocycle if the stoichiometry does not favor other species, namely the mononuclear di- and tri-protonated macrocycle, as in the 1:1:1 system.
E. Ross et al. / Inorganica Chimica Acta 286 (1999) 55–61
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Table 8 Stability constants for the BMXD–Cu(II)–histidine system (m= 0.10 (KCl), 25.0°C; Mx =BMXD, Hi = histidinate, Cu =Cu2+ and H =H+) Stoichiometry Mx
Cu
Hi
H
1 1 1 1 1 1 1
1 1 1 1 2 2 2
1 1 1 1 1 2 2
0 1 2 3 0 0 −1
a
Quotient K
Log K a
[MxCuHi]/[MxCu][Hi] [MxCuHiH]/[MxCuH][Hi] [MxCuHiH2]/[MxCuH2][Hi] [MxCuHiH3]/[MxCuH3][Hi] [MxCu2Hi]/[MxCu2][Hi] [MxCu2Hi2]/[MxCuHi][Hi] [MxCu2Hi2(OH)][H]/[MxCu2Hi2]
4.92 5.54 6.21 8.49 6.15 4.60 −9.06
(4) (3) (3) (8) (5) (5) (6)
Estimated errors in parentheses.
carboxylic acids in this p[H] range, and so its binding to an amino nitrogen of the macrocycle is indicated. In the solution containing two equivalents of copper and one equivalent of aspartic acid, MxAsHCu2 and MxAsCu2 are found to be the dominant species, each involving nearly 100% of the ligand in solution. The aspartic acid amino group is deprotonated at p[H] 7, indicating the tendency of the amino group of aspartic acid toward Cu(II) coordination.
The lack of formation of the MxGlCu species reflects the coordination saturation of Cu(II) by the six amino groups of the BMXD. The di-glycinated dinuclear species exists as a significant (60%) species in its stoichiometrically favored solution. No other species were found to form at higher p[H] values.
3.5. Binding of aspartic acid by Cu(II) complexes of BMXD Constants determined for the 1:1:1 and 1:2:2 Cu(II)– aspartic acid–BMXD systems are presented in Table 6. It is evident from the stability constants listed that aspartic acid forms more stable ternary species than does glycine. Above neutral p[H] the aspartic acid is fully deprotonated, and the aspartic acid complexes formed resemble the glycine species with respect to the approximate p[H] range over which the species exist. The reason for the increased stability is probably due to the additional donor group, the carboxylate group, and the increased basicity of the aspartic acid amino group. The presence of MxCuAsH4 probably results from the addition of a carboxylate hydrogen bond in addition to the amino nitrogen Cu(II) coordinate bond. This species probably resembles the mononucleated glycine species (Fig. 2). The species MxHAsCu2 exists from p[H] 3 to 8, it is unlikely that the proton resides on one of the
Fig. 1. Differences in molecular recognition by BMXD for the amino acids: (a) glycine; (b) histidine; (c) aspartic acid. In each case the total concentration of each component is 2.0 mmol at 25.0°C and m= 0.1 M (KCl), Gl = glycine, Aa =histidine, and As = aspartic acid.
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E. Ross et al. / Inorganica Chimica Acta 286 (1999) 55–61
Fig. 3. Species distribution showing very strong interaction between aspartic acid and the BMXD – Cu2 chelates at 25.0°C and m =0.100 M (KCl). Total BMXD and aspartic acid concentrations are each 0.00200 M. The two unlabeled small peaks just above pH 3 are species MxH5As and MxH6As.
Fig. 2. (a) Percentage of species formed from 1 mmol copper(II) and (b) 2 mmol of copper relative to 1.0 mmol in both BMXD and histidine at 25.0°C and m= 0.100 M (KCl). Aa = histidine.
3.6. Binding of histidine by Cu(II) complexes of BMXD The binding constants determined for the mononuclear and dinuclear Cu(II) complexes of the macrocycle with histidine are listed in Table 8. It is seen in the 1:1:1 system that histidine forms four ternary species. None of them are dinuclear species, as are formed with glycine or aspartic acid under equivalent conditions. With the mononuclear species it is difficult to decide where the protons might reside since there are many logical choices for protonation with the diethylenetriamine moiety relatively free, and many amino nitrogens in the ternary species have similar pKa values. Histidine’s increased basicity compared with glycine is evident in the more stable bonds it forms with the copper(II) ion. This is the reason for the increased stability of the mononuclear deprotonated ternary species. It is evident that addition of the imidizole group creates stable bonds involving both coordination to the copper(II) ion and formation of hydrogen bonds involving the imidizole amino groups. These effects reduce the dinucleating tendency of the macrocycle sufficiently to make
such species negligible in a solution of 1:1:1 stoichiometry. The stabilities of the mononuclear species result in their presence in the system containing a 1:2:1 ratio of macrocycle–Cu(II)–histidine. Formation of the dinuclear ternary species is less favorable than in the corresponding glycine system. This is probably a result of steric hindrance of the imidizole group. When the imidizole is deprotonated, and the histidine has two available amino groups for bonding, the stability of the ternary species is not high enough compared to the glycine ternary species to indicate a binding contribution from the imidizole moiety. In fact, the constant is smaller, indicating a weaker bond, probably due to steric interference. In the light of this argument, the MxHHiCu2 and MxHiCu2 species differ only by the imidizole proton, they cross at the imidizole pKa, and are therefore most likely identically bonded, resembling the MxGlCu2 structure, with the imidizole oriented away from the ligand, with coordination of the alpha amino group of histidine to one Cu(II) ion and the carboxylate group coordinated to the other metal cation. A suggested binding mode for these species is shown in 3. Support for the proposed binding modes will be obtained in the future by analysis of the visible spectrum as a function of pH.
Acknowledgements The authors express thanks to NSF for support of E.R. in the Undergraduate Summer Research Program (NSF-REU) and to The Robert A. Welch Foundation (Grant no. A-0259).
E. Ross et al. / Inorganica Chimica Acta 286 (1999) 55–61
References [1] Q. Lu, R.J. Motekaitis, A.E. Martell, Inorg. Chem. 34 (1995) 4958. [2] A. Llobet, J.H. Reibenspies, A.E. Martell, Inorg. Chem. 33 (1994) 5946. [3] R.J. Motekaitis, A.E. Martell, Inorg. Chem. 31 (1992) 5534. [4] L. Fabbrizzi, P. Pallavicini, L. Parodi, A. Perotti, A. Taglietti, J. Chem. Soc., Chem. Commun. (1995) 2439. [5] P.E. Jurek, A.E. Martell, R.J. Motekaitis, R.D. Hancock, Inorg. Chem. 34 (1995) 1823.
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[6] R. Menif, A.E. Martell, P. Squattrito, A. Clearfield, J. Am. Chem. Soc. 29 (1990) 4723. [7] D.A. Nation, A.E. Martell, R.I. Carroll, A. Clearfield, Inorg. Chem. 35 (1996) 7246. [8] M.B. Luiz, B. Szpoganicz, M. Rizzoto, A.E. Martell, M.G. Basallote, Inorg. Chim. Acta 254 (1997) 345. [9] A.E. Martell, R.J. Motekaitis, Determination and Use of Stability Constants, 2nd ed., VCH, New York, 1992. [10] R.M. Smith, A.E. Martell, NIST Critically Selected Constants: Version 5.0, 1998. [11] H. Brock, T. Vaupel, H. Schodel, J. Prakt. Chem. 339 (1997) 26.
The interaction of BMXD and its copper(II) complexes with glycine, aspartic acid, and histidine Eric Ross, Ramunas J. Motekaitis, Arthur E. Martell * Department of Chemistry, Texas A&M Uni6ersity, College Station, TX 77842 -3012, USA Received 26 May 1998; accepted 9 September 1998
Abstract The macrocycle, 3,6,9,17,20,23-hexaazatricyclo[23.3.1.111,15]triaconta-1(29)11(30),12,14,25,27-hexaene, BMXD, is shown to recognize three amino acids, glycine, aspartic acid, and histidine, to form binary species. The mono- and dinuclear copper(II) complexes are also shown to host these amino acids. The stability constants for the binary complexes of the amino acids with the macrocycle, and of the ternary complexes containing amino acid, copper(II) and macrocycle, are reported, and binding schemes are suggested for the recognition of glycine, and for the dinuclear ternary species with histidine and glycine. Aspartic acid is found to form the most stable complexes, both with and without the presence of copper(II) ion. © 1999 Elsevier Science S.A. All rights reserved. Keywords: Copper complexes; Amino acid complexes; Macrocyclic complexes
1. Introduction Macrocyclic polyamines are versatile chelating agents capable of forming many different complexes depending on the protonation state of the amine. In its protonated form, the positively charged macrocycle can bind anionic species through hydrogen bonds and electrostatic forces [1–3]. The ligand can form mono- and dinuclear metal complexes with several different metal ions. These species have been shown to further selectively bind anionic species to form tertiary cascade complexes [2–5]. The hexabasic macrocycle BMXD (3,6,9,17,20,23hexaazatricyclo[23.3.1.111,15]triaconta-1(29)11(30),12,14, 25,27-hexaene) (1), has been shown to form stable mono- and dinuclear complexes with the copper(II) ion [6]. These complexes are the subject of a recent study involving biologically interesting inorganic phosphates [7]. Another study involving OBISDIEN, the ether bridged analog of BMXD, reported the stability constants for the ligand and its copper(II) complexes with * Corresponding author. Tel.: +1-409-8452011; fax: + 1-4098454719.
various peptides [8]. This work is related to the metal ion promoted hydrolysis of peptides and related compounds. It is of interest to investigate the interaction of other biological molecules with macrocycles and their metal complexes. In the present work, the ability of BMXD and its copper(II) complexes to host three amino acids, glycine, aspartic acid, and histidine, is investigated. Thus, the three types of a-amino acids are considered in this study: equal numbers of carboxylate and amino groups (glycine), more carboxylate than amino groups (aspartic acid) and more amino groups than carboxylate groups (histidine). The stability constants for the
0020-1693/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved. PII: S 0 0 2 0 - 1 6 9 3 ( 9 8 ) 0 0 3 8 0 - 6
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E. Ross et al. / Inorganica Chimica Acta 286 (1999) 55–61
complex species formed are reported and used to prepare distribution diagrams showing the relative concentrations of the complexes formed between these amino acids and the macrocycles.
2. Experimental
2.1. Materials Reagent grade inorganic materials were used throughout the experiments without further purification. The KOH solution was made from Baker ‘Dilutit’ carbonate-free sealed ampoules diluted as per directions. The base was stored under a sealed inert atmosphere at all times and was checked periodically for carbonate content by the use of a Gran’s plot [9]. The carbonate content was never greater than 1.5%. The KOH was standardized by potassium acid phthalate titration [9]. All aqueous solutions were prepared with distilled water. The copper(II) chloride solution was standardized by EDTA titration [9]. Several grams of the ligand as its hexahydrochloride salt were kindly donated by David Nation.
2.2. Potentiometric equilibrium measurements For p[H] determination, a Corning Research model 150 pH meter was used with glass and calomel reference electrodes. A 10 ml piston burette was used to deliver the KOH solution. Titrations were performed in a temperature regulated cell that was sealed and purged continuously with purified and humidified argon. The pH meter was calibrated with a standard dilute acid solution (HCl) before each titration to read the H + -ion concentration directly so that the p[H] is defined as − log [H + ]. The ionic strength of the solution was adjusted to 0.100 M by addition of 1.00 M KCl, and so served as the supporting electrolyte.
2.3. Computations Stability constants were calculated with the program [9]. Species distribution curves were calculated from the stability constants with the program SPE and plotted with SPEPLOT [9]. The value of Kw at the given ionic strength was calculated to be 10 − 13.78. All titrations contained approximately 0.002 M ligand and appropriate ratios of other constituents. The protonation constants of the three amino acids were determined experimentally under the conditions employed here for use in the calculations. All mixed systems titrations contained at least 80 points of data between p[H] 2.0 and 11.1. All backward titrations were carried out three or more times to ensure accuracy and reproducibility. Each ternary system was titrated at
BEST
Table 1 Protonation and formation constants of BMXD and Cu(II) complexes (m= 0.10 (KCl), 25.0°C; Mx =BMXD, OH = OH−, H= H+, and Cu=Cu2+) Quotient K
Log K a
[MxH]/[Mx][H] [MxH2]/[MxH][H] [MxH3]/[MxH2][H] [MxH4]/[MxH3][H] [MxH5]/[MxH4][H] [MxH6]/[MxH5][H] [MxCu]/[Mx][Cu] [MxCuH]/[MxCu][H] [MxCuH2]/[MxCuH][H] [MxCuH3]/[MxCuH2][H] [MxCu(OH)][H]/[MxCu] [MxCu2]/[MxCu][Cu] [MxCu2(OH)][H]/[MxCu2] [MxCu2(OH)2][H]/[MxCu(OH)] [MxCu2(OH)3][H]/[MxCu(OH)2]
9.43 8.71 7.98 7.11 3.80 3.39 13.45 8.38 7.45 3.87 −9.17 11.24 −7.87 −8.78 −11.52
Log K (1) (1) (1) (1) (1) (1) (3) (4) (3) (1) (6) (3) (3) (5) (9)
9.49b 8.73b 8.03b 7.29b 3.64b 3.45b 13.63c 8.40c 7.20c 3.68c −8.93c 10.86c −7.83c −8.74c −11.70c
a
Estimated error in parentheses based on error propagation. Ref. [6]. c Ref. [7]. b
least twice. The computation of stability constants was based on the minimization of the sigma fit, however, error estimates were performed by a propagation of errors analysis. Thus the errors of each input variable were originally estimated and their affects on the calculated result were then tabulated together with the new stability constants in Tables 1–8.
3. Results and discussion The protonation constants of the ligand BMXD and its Cu(II) binding constants have been reported previously [6,7]. They were re-determined at the experimental conditions employed in this work, and the results and comparisons are located in Table 1. The protonation constants and Cu(II) binding constants of the three amino acids were also re-evaluated at the experimental conditions used in this research. Their values and comparisons to literature values [10] are listed in Table 2. The difference in the constants in Tables 1 and 2, between the values determined in the present work and Table 2 Protonation constants of substrates (m= 0.10 (KCl), 25.0°C)a Substrate
Log K 1H
Log K 2H
Log K 3H
Glycine Aspartic acid Histidine
9.55 (9.58) 9.68 (9.65) 9.11 (9.09)
2.22 (2.34) 3.68 (3.70) 6.03 (6.04)
1.83 (2.00) 1.65 (1.7)
a Values in parentheses, Ref. [10]. Error estimates: glycine 9 0.01; aspartic acid 9 0.01; histidine 9 0.01.
E. Ross et al. / Inorganica Chimica Acta 286 (1999) 55–61 Table 3 Stability constants for the BMXD–glycine system (m= 0.10 (KCl), 25.0°C; Mx = BMXD, Gl = Glycinate, and H =H+) Stepwise quotient K
Stoichiometry Mx
Gl
H
1 1 1 1 1
1 1 1 1 1
1 2 3 4 5
a
[MxGlH]/[MxH][Gl] [MxGlH2]/[MxH2][Gl] [MxGlH3]/[MxH3][Gl] [MxGlH4]/[MxH4][Gl] [MxGlH5]/[MxH5][Gl]
Log K a
3.11 3.48 4.33 4.98 7.91
Table 5 Stability constants for the BMXD–aspartic acid system (m= 0.10 (KCl), 25.0°C; Mx =BMXD, As =aspartate, and H =H+) Stoichiometry
(4) (3) (3) (2) (2)
Estimated error in parentheses.
Mx
As
H
1 1 1 1 1 1 1
1 1 1 1 1 1 1
1 2 3 4 5 6 7
a
the values determined by others, are minor and about what would be expected from the difference in conditions employed. In the absence of copper ions, the recognition of the amino acids was found to be quite poor (Fig. 1). The order of increasing abundance of various binary species (of MXBD–amino acid) was found to be histidine B 10%, glycineB 25% and aspartic acidB 40%. In the presence of copper the relative recognition for amino acids is increased considerably. Thus histidine (Fig. 2) and glycine approach 50% maximum formation as dicopper–BMXD – amino acid complexes while aspartic acid approaches 100% formation (Fig. 3).
3.1. Glycine recognition by BMXD The equilibrium constants calculated for the binding of glycine to protonated forms of BMXD are listed in Table 3. It was found that five-protonated ligandglycine species are formed between p[H] 2.8 and 12. Stability of the species decreases as the degree of protonation decreases. This is in accordance with the coulombic effect in that increased positive charge of the ligand nitrogens more strongly coordinate the anionic portion of the glycine zwitterion, the negative carboxylate group through hydrogen bonding. The strongest method of binding in solution probably involves the glycine amino nitrogen hydrogen bonded to an amino group of the ligand. A suggested binding scheme of the Table 4 Stability constants for the BMXD–Cu(II)–glycine system (m= 0.10 (KCl), 25.0°C; Mx =BMXD, Gl =Glycinate, Cu = Cu2+ and H = H+) Stoichiometry Mx
Cu
Gl
H
1 1 1
2 2 2
1 2 2
0 0 1
a
Stepwise quotient K
Log K a
[MxAsH]/[MxH][As] [MxAsH2]/[MxH2][As] [MxAsH3]/[MxH3][As] [MxAsH4]/[MxH4][As] [MxAsH5]/[MxH5][As] [MxAsH6]/[MxH6][As] [MxAsH7]/[MxH6][AsH]
3.21 3.73 4.64 5.45 8.68 9.14 3.16
(5) (3) (4) (4) (4) (4) (3)
Estimated error in parentheses.
MxGlH5 species is illustrated in Fig. 2. The titration curve of the system indicates a buffered region between a values, zero and two by a break, with the remainder of the curve buffered at higher p[H]. The first buffer region corresponds to the deprotonation of the macrocycle to the tetra-protonated state. The following buffer region is the formation of successive deprotonated states of the ligand. In 2 a glycine unit in its neutral zwitterion form is shown to bind the tetra-protonated macrocycle through hydrogen bonding. The presence of binary species MxGlH with the fully deprotonated ligand is consistent with the primary binding of the glycine being through the hydrogen bond formed by the amino acid nitrogen. The increasing stability of the more highly protonated species involving the positively charged macrocycle indicates that the negative carboxylate group is involved in the binding. Further protonation of the hepta-protonated binary species may not be possible because the positively charged diprotonated glycine molecules would not be attracted to the positively charged macrocyclic ligand. Support for such intermolecular hydrogen bonding can be found in the crystallographic determination of CH3NH2 –H– + NH2CH3 [11]. In this system as well as in the aspartic acid and histidine systems it is difficult to uniquely quantify the relative contributions of pure electrostatic attractions relative to hydrogen bonding forces in the carboxylate affinity for protonated amines since the ammonium groups by definition all possess a potentially bindable extra proton [1].
3.2. Aspartic acid recognition by BMXD
a
Quotient K
Log K
[MxCu2Gl]/[MxCu2][Gl] [MxCu2Gl2]/[MxCu2Gl][Gl] [MxCu2Gl2H]/[MxCu2Gl2][H]
6.27 (3) 4.68 (3) 8.74 (4)
Estimated error in parentheses.
57
The binding constants determined for the binary species of BMXD and aspartic acid are listed in Table 5. In comparison with the glycine binary systems, BMXD is shown to recognize aspartic acid with greater affinity. Seven different protonated binary species are seen to form, necessarily incorporating a number of binding schemes.
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Table 6 Stability constants for the BMXD–Cu(II)–aspartic acid system (m= 0.10 (KCl), 25.0°C; Mx =BMXD, As =aspartic acid, Cu =Cu2+ and H = H+) Stoichiometry Mx
Cu
As
H
1 1 1 1 1
1 2 2 2 2
1 1 1 2 1
4 0 1 0 −1
a
Quotient K
Log K a
[MxCuAsH4]/[MxCuH3][AsH] [MxCu2As]/[MxCu2][As] [MxCu2AsH]/[MxCu2][AsH] [MxCu2As2]/[MxCu2As][As] [MxCu2As(OH)]/[MxCu2(OH)][As]
2.86 8.52 4.75 3.30 5.58
(8) (6) (3) (7) (3)
Estimated errors in parentheses.
3.3. Histidine recognition by BMXD
A dramatic increase in stability between the tetraand penta-protonated binary species might be due to the incorporation of carboxylate hydrogen bonds into the bonding mode. A hepta-protonated species is present from low p[H] up to p[H] 4. The only possible bonding mode for this species is hydrogen bonding between the most acidic carboxylate groups and protonated amino groups on the macrocyclic ligand. The monoprotonated binary species is the result of a hydrogen bond formed between the protonated amino group of the amino acid and a deprotonated amino group of the macrocycle. The additional carboxylic acid group on the aspartic acid results in an increased negative charge on the species throughout most of the pH range. This accounts for the increased bonding to the positively charged protonated macrocycle and accounts for the existence of highly protonated binary systems.
Table 7 Stability constants for the BMXD–histidine system (m = 0.10 (KCl), 25.0°C; Mx = BMXD, Hi = histidinate, and H = H+) Stoichiometry Mx
Hi
H
1 1
1 1
6 7
a
Stepwise quotient K
Log K a
[MxHiH6]/[MxH6][Hi] [MxHiH7]/[MxH6][HiH]
9.47 (2) 4.52 (2)
Estimated errors in parentheses.
The two binding constants for the binary species formed from histidine and protonated BMXD are listed in Table 7. The hepta- and hexa-protonated ternary species are shown to form between p[H] 5 and 10. It is suggested that the imidizole groups on the histidine molecule form hydrogen bonds in addition to those formed by the primary amine group in the amino acid. When the imidizole group is protonated at p[H] values below its pKa value, the ternary species no longer form. The coordination of the imidizole nitrogen is also suggested in the MxHiH6 species. Its concurrent formation with the tetra-protonated free ligand, and its stability compared to the binary system formed between the tetra-protonated macrocycle and glycine, indicate the involvement of imidizole groups. However it cannot be ignored that the degree of complex formation with BMXD is the smallest of the three amino acids studied here.
3.4. Binding of glycine by Cu(II) complexes of BMXD The equilibrium constants determined for the copper(II) complexes of the BMXD macrocycle and glycine are listed in Table 4. The dinucleating tendency of the macrocycle is shown in the 1:1:1 titration of BMXD, Cu(II), and glycine, as the dinuclear mono-glycinated species exists over a wide p[H] range (4–10) and as one of the principal species in solution. The glycine nitrogen is deprotonated at p[H] as low as 3.5, indicating its importance in the formation of the ternary species. The monoprotonated complex exists only as a minor species at lower p[H], so the predominant factor in the binding is the glycine amine–Cu(II) coordinate bond. In the titration with two equivalents of Cu(II) and glycine, the protonated species appears to the extent of around 10%, so the carboxylate group is capable of forming weak bonds with the macrocycle if the stoichiometry does not favor other species, namely the mononuclear di- and tri-protonated macrocycle, as in the 1:1:1 system.
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Table 8 Stability constants for the BMXD–Cu(II)–histidine system (m= 0.10 (KCl), 25.0°C; Mx =BMXD, Hi = histidinate, Cu =Cu2+ and H =H+) Stoichiometry Mx
Cu
Hi
H
1 1 1 1 1 1 1
1 1 1 1 2 2 2
1 1 1 1 1 2 2
0 1 2 3 0 0 −1
a
Quotient K
Log K a
[MxCuHi]/[MxCu][Hi] [MxCuHiH]/[MxCuH][Hi] [MxCuHiH2]/[MxCuH2][Hi] [MxCuHiH3]/[MxCuH3][Hi] [MxCu2Hi]/[MxCu2][Hi] [MxCu2Hi2]/[MxCuHi][Hi] [MxCu2Hi2(OH)][H]/[MxCu2Hi2]
4.92 5.54 6.21 8.49 6.15 4.60 −9.06
(4) (3) (3) (8) (5) (5) (6)
Estimated errors in parentheses.
carboxylic acids in this p[H] range, and so its binding to an amino nitrogen of the macrocycle is indicated. In the solution containing two equivalents of copper and one equivalent of aspartic acid, MxAsHCu2 and MxAsCu2 are found to be the dominant species, each involving nearly 100% of the ligand in solution. The aspartic acid amino group is deprotonated at p[H] 7, indicating the tendency of the amino group of aspartic acid toward Cu(II) coordination.
The lack of formation of the MxGlCu species reflects the coordination saturation of Cu(II) by the six amino groups of the BMXD. The di-glycinated dinuclear species exists as a significant (60%) species in its stoichiometrically favored solution. No other species were found to form at higher p[H] values.
3.5. Binding of aspartic acid by Cu(II) complexes of BMXD Constants determined for the 1:1:1 and 1:2:2 Cu(II)– aspartic acid–BMXD systems are presented in Table 6. It is evident from the stability constants listed that aspartic acid forms more stable ternary species than does glycine. Above neutral p[H] the aspartic acid is fully deprotonated, and the aspartic acid complexes formed resemble the glycine species with respect to the approximate p[H] range over which the species exist. The reason for the increased stability is probably due to the additional donor group, the carboxylate group, and the increased basicity of the aspartic acid amino group. The presence of MxCuAsH4 probably results from the addition of a carboxylate hydrogen bond in addition to the amino nitrogen Cu(II) coordinate bond. This species probably resembles the mononucleated glycine species (Fig. 2). The species MxHAsCu2 exists from p[H] 3 to 8, it is unlikely that the proton resides on one of the
Fig. 1. Differences in molecular recognition by BMXD for the amino acids: (a) glycine; (b) histidine; (c) aspartic acid. In each case the total concentration of each component is 2.0 mmol at 25.0°C and m= 0.1 M (KCl), Gl = glycine, Aa =histidine, and As = aspartic acid.
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E. Ross et al. / Inorganica Chimica Acta 286 (1999) 55–61
Fig. 3. Species distribution showing very strong interaction between aspartic acid and the BMXD – Cu2 chelates at 25.0°C and m =0.100 M (KCl). Total BMXD and aspartic acid concentrations are each 0.00200 M. The two unlabeled small peaks just above pH 3 are species MxH5As and MxH6As.
Fig. 2. (a) Percentage of species formed from 1 mmol copper(II) and (b) 2 mmol of copper relative to 1.0 mmol in both BMXD and histidine at 25.0°C and m= 0.100 M (KCl). Aa = histidine.
3.6. Binding of histidine by Cu(II) complexes of BMXD The binding constants determined for the mononuclear and dinuclear Cu(II) complexes of the macrocycle with histidine are listed in Table 8. It is seen in the 1:1:1 system that histidine forms four ternary species. None of them are dinuclear species, as are formed with glycine or aspartic acid under equivalent conditions. With the mononuclear species it is difficult to decide where the protons might reside since there are many logical choices for protonation with the diethylenetriamine moiety relatively free, and many amino nitrogens in the ternary species have similar pKa values. Histidine’s increased basicity compared with glycine is evident in the more stable bonds it forms with the copper(II) ion. This is the reason for the increased stability of the mononuclear deprotonated ternary species. It is evident that addition of the imidizole group creates stable bonds involving both coordination to the copper(II) ion and formation of hydrogen bonds involving the imidizole amino groups. These effects reduce the dinucleating tendency of the macrocycle sufficiently to make
such species negligible in a solution of 1:1:1 stoichiometry. The stabilities of the mononuclear species result in their presence in the system containing a 1:2:1 ratio of macrocycle–Cu(II)–histidine. Formation of the dinuclear ternary species is less favorable than in the corresponding glycine system. This is probably a result of steric hindrance of the imidizole group. When the imidizole is deprotonated, and the histidine has two available amino groups for bonding, the stability of the ternary species is not high enough compared to the glycine ternary species to indicate a binding contribution from the imidizole moiety. In fact, the constant is smaller, indicating a weaker bond, probably due to steric interference. In the light of this argument, the MxHHiCu2 and MxHiCu2 species differ only by the imidizole proton, they cross at the imidizole pKa, and are therefore most likely identically bonded, resembling the MxGlCu2 structure, with the imidizole oriented away from the ligand, with coordination of the alpha amino group of histidine to one Cu(II) ion and the carboxylate group coordinated to the other metal cation. A suggested binding mode for these species is shown in 3. Support for the proposed binding modes will be obtained in the future by analysis of the visible spectrum as a function of pH.
Acknowledgements The authors express thanks to NSF for support of E.R. in the Undergraduate Summer Research Program (NSF-REU) and to The Robert A. Welch Foundation (Grant no. A-0259).
E. Ross et al. / Inorganica Chimica Acta 286 (1999) 55–61
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