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 withglycine, 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 rec-ognize 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 themacrocycle, and of the ternary complexes containing amino acid, copper(II) and macrocycle, are reported, and binding schemesare suggested for the recognition of glycine, and for the dinuclear ternary species with histidine and glycine. Aspartic acid is foundto form the most stable complexes, both with and without the presence of copper(II) ion. © 1999 Elsevier Science S.A. All rightsreserved.

Keywords: Copper complexes; Amino acid complexes; Macrocyclic complexes

1. Introduction

Macrocyclic polyamines are versatile chelating agentscapable of forming many different complexes depend-ing on the protonation state of the amine. In its proto-nated form, the positively charged macrocycle can bindanionic species through hydrogen bonds and electro-static forces [1–3]. The ligand can form mono- anddinuclear metal complexes with several different metalions. These species have been shown to further selec-tively bind anionic species to form tertiary cascadecomplexes [2–5].

The hexabasic macrocycle BMXD (3,6,9,17,20,23-hexaazatricyclo[23.3.1.111,15]triaconta-1(29)11(30),12,14,25,27-hexaene) (1), has been shown to form stablemono- and dinuclear complexes with the copper(II) ion[6]. These complexes are the subject of a recent studyinvolving biologically interesting inorganic phosphates[7]. Another study involving OBISDIEN, the etherbridged analog of BMXD, reported the stability con-stants for the ligand and its copper(II) complexes with

various peptides [8]. This work is related to the metalion promoted hydrolysis of peptides and relatedcompounds.

It is of interest to investigate the interaction of otherbiological molecules with macrocycles and their metalcomplexes. In the present work, the ability of BMXDand 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 inthis study: equal numbers of carboxylate and aminogroups (glycine), more carboxylate than amino groups(aspartic acid) and more amino groups than carboxy-late groups (histidine). The stability constants for the

* Corresponding author. Tel.: +1-409-8452011; fax: +1-409-8454719.

0020-1693/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.

PII: S 0 0 2 0 -1693 (98 )00380 -6

E. Ross et al. / Inorganica Chimica Acta 286 (1999) 55–6156

complex species formed are reported and used to pre-pare distribution diagrams showing the relative concen-trations of the complexes formed between these aminoacids and the macrocycles.

2. Experimental

2.1. Materials

Reagent grade inorganic materials were usedthroughout the experiments without further purifica-tion. The KOH solution was made from Baker ‘Dilut-it’ carbonate-free sealed ampoules diluted as perdirections. The base was stored under a sealed inertatmosphere at all times and was checked periodicallyfor 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 phtha-late titration [9]. All aqueous solutions were preparedwith distilled water. The copper(II) chloride solutionwas standardized by EDTA titration [9]. Several gramsof the ligand as its hexahydrochloride salt were kindlydonated by David Nation.

2.2. Potentiometric equilibrium measurements

For p[H] determination, a Corning Research model150 pH meter was used with glass and calomel refer-ence electrodes. A 10 ml piston burette was used todeliver the KOH solution. Titrations were performed ina temperature regulated cell that was sealed and purgedcontinuously with purified and humidified argon. ThepH meter was calibrated with a standard dilute acidsolution (HCl) before each titration to read the H+-ionconcentration directly so that the p[H] is defined as− log [H+]. The ionic strength of the solution wasadjusted to 0.100 M by addition of 1.00 M KCl, and soserved as the supporting electrolyte.

2.3. Computations

Stability constants were calculated with the programBEST [9]. Species distribution curves were calculatedfrom the stability constants with the program SPE andplotted with SPEPLOT [9]. The value of Kw at the givenionic strength was calculated to be 10−13.78.

All titrations contained approximately 0.002 M lig-and and appropriate ratios of other constituents. Theprotonation constants of the three amino acids weredetermined experimentally under the conditions em-ployed here for use in the calculations. All mixedsystems titrations contained at least 80 points of databetween p[H] 2.0 and 11.1. All backward titrations werecarried out three or more times to ensure accuracy andreproducibility. Each ternary system was titrated at

Table 1Protonation and formation constants of BMXD and Cu(II) com-plexes (m=0.10 (KCl), 25.0°C; Mx=BMXD, OH=OH−, H=H+,and Cu=Cu2+)

Quotient K Log Ka Log K

[MxH]/[Mx][H] 9.43 (1) 9.49b

8.73b[MxH2]/[MxH][H] 8.71 (1)7.98 (1)[MxH3]/[MxH2][H] 8.03b

[MxH4]/[MxH3][H] 7.11 (1) 7.29b

[MxH5]/[MxH4][H] 3.80 (1) 3.64b

3.39 (1)[MxH6]/[MxH5][H] 3.45b

13.63c[MxCu]/[Mx][Cu] 13.45 (3)8.38 (4)[MxCuH]/[MxCu][H] 8.40c

7.45 (3)[MxCuH2]/[MxCuH][H] 7.20c

3.68c3.87 (1)[MxCuH3]/[MxCuH2][H]−9.17 (6)[MxCu(OH)][H]/[MxCu] −8.93c

11.24 (3)[MxCu2]/[MxCu][Cu] 10.86c

−7.83c−7.87 (3)[MxCu2(OH)][H]/[MxCu2]−8.78 (5)[MxCu2(OH)2][H]/[MxCu(OH)] −8.74c

−11.52 (9)[MxCu2(OH)3][H]/[MxCu(OH)2] −11.70c

a Estimated error in parentheses based on error propagation.b Ref. [6].c Ref. [7].

least twice. The computation of stability constants wasbased on the minimization of the sigma fit, however,error estimates were performed by a propagation oferrors analysis. Thus the errors of each input variablewere originally estimated and their affects on the calcu-lated result were then tabulated together with the newstability constants in Tables 1–8.

3. Results and discussion

The protonation constants of the ligand BMXD andits Cu(II) binding constants have been reported previ-ously [6,7]. They were re-determined at the experimen-tal conditions employed in this work, and the resultsand comparisons are located in Table 1. The protona-tion constants and Cu(II) binding constants of the threeamino acids were also re-evaluated at the experimentalconditions used in this research. Their values and com-parisons 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 2Protonation constants of substrates (m=0.10 (KCl), 25.0°C)a

Log K1H Log K2

HSubstrate Log K3H

9.55 (9.58)Glycine 2.22 (2.34)9.68 (9.65) 1.83 (2.00)3.68 (3.70)Aspartic acid9.11 (9.09) 6.03 (6.04)Histidine 1.65 (1.7)

a Values in parentheses, Ref. [10]. Error estimates: glycine90.01;aspartic acid90.01; histidine90.01.

E. Ross et al. / Inorganica Chimica Acta 286 (1999) 55–61 57

Table 3Stability constants for the BMXD–glycine system (m=0.10 (KCl),25.0°C; Mx=BMXD, Gl=Glycinate, and H=H+)

Stepwise quotient K Log KaStoichiometry

Gl HMx

[MxGlH]/[MxH][Gl]11 3.11 (4)11 2 [MxGlH2]/[MxH2][Gl] 3.48 (3)1

3 [MxGlH3]/[MxH3][Gl]1 1 4.33 (3)[MxGlH4]/[MxH4][Gl]4 4.98 (2)11

1 5 [MxGlH5]/[MxH5][Gl] 7.91 (2)1

a Estimated error in parentheses.

Table 5Stability constants for the BMXD–aspartic acid system (m=0.10(KCl), 25.0°C; Mx=BMXD, As=aspartate, and H=H+)

Log KaStepwise quotient KStoichiometry

Mx As H

1 1 [MxAsH]/[MxH][As] 3.21 (5)11 21 [MxAsH2]/[MxH2][As] 3.73 (3)

[MxAsH3]/[MxH3][As]3 4.64 (4)111 41 [MxAsH4]/[MxH4][As] 5.45 (4)

1 1 5 [MxAsH5]/[MxH5][As] 8.68 (4)9.14 (4)1 1 6 [MxAsH6]/[MxH6][As]

1 71 [MxAsH7]/[MxH6][AsH] 3.16 (3)

a Estimated error in parentheses.the values determined by others, are minor and aboutwhat would be expected from the difference in condi-tions employed.

In the absence of copper ions, the recognition of theamino acids was found to be quite poor (Fig. 1). Theorder of increasing abundance of various binary species(of MXBD–amino acid) was found to be histidineB10%, glycineB25% and aspartic acidB40%.

In the presence of copper the relative recognition foramino acids is increased considerably. Thus histidine(Fig. 2) and glycine approach 50% maximum formationas dicopper–BMXD–amino acid complexes while as-partic acid approaches 100% formation (Fig. 3).

3.1. Glycine recognition by BMXD

The equilibrium constants calculated for the bindingof glycine to protonated forms of BMXD are listed inTable 3. It was found that five-protonated ligand-glycine species are formed between p[H] 2.8 and 12.Stability of the species decreases as the degree of proto-nation decreases. This is in accordance with the cou-lombic effect in that increased positive charge of theligand nitrogens more strongly coordinate the anionicportion of the glycine zwitterion, the negative carboxy-late group through hydrogen bonding. The strongestmethod of binding in solution probably involves theglycine amino nitrogen hydrogen bonded to an aminogroup of the ligand. A suggested binding scheme of the

MxGlH5 species is illustrated in Fig. 2. The titrationcurve of the system indicates a buffered region betweena values, zero and two by a break, with the remainderof the curve buffered at higher p[H]. The first bufferregion corresponds to the deprotonation of the macro-cycle to the tetra-protonated state. The following bufferregion is the formation of successive deprotonatedstates of the ligand. In 2 a glycine unit in its neutralzwitterion form is shown to bind the tetra-protonatedmacrocycle through hydrogen bonding. The presence ofbinary species MxGlH with the fully deprotonated lig-and is consistent with the primary binding of theglycine being through the hydrogen bond formed by theamino acid nitrogen. The increasing stability of themore highly protonated species involving the positivelycharged macrocycle indicates that the negative carboxy-late group is involved in the binding. Further protona-tion of the hepta-protonated binary species may not bepossible because the positively charged diprotonatedglycine molecules would not be attracted to the posi-tively charged macrocyclic ligand. Support for suchintermolecular hydrogen bonding can be found in thecrystallographic determination of CH3NH2–H–+NH2CH3 [11]. In this system as well as in the asparticacid and histidine systems it is difficult to uniquelyquantify the relative contributions of pure electrostaticattractions relative to hydrogen bonding forces in thecarboxylate affinity for protonated amines since theammonium groups by definition all possess a poten-tially bindable extra proton [1].

3.2. Aspartic acid recognition by BMXD

The binding constants determined for the binaryspecies of BMXD and aspartic acid are listed in Table5. In comparison with the glycine binary systems,BMXD is shown to recognize aspartic acid with greateraffinity. Seven different protonated binary species areseen to form, necessarily incorporating a number ofbinding schemes.

Table 4Stability constants for the BMXD–Cu(II)–glycine system (m=0.10(KCl), 25.0°C; Mx=BMXD, Gl=Glycinate, Cu=Cu2+ and H=H+)

Quotient KStoichiometry Log Ka

HGlCuMx

2 0 [MxCu2Gl]/[MxCu2][Gl] 6.27 (3)1 11 2 [MxCu2Gl2]/[MxCu2Gl][Gl] 4.68 (3)2 0

2 8.74 (4)[MxCu2Gl2H]/[MxCu2Gl2][H]121

a Estimated error in parentheses.


Table 6Stability constants for the BMXD–Cu(II)–aspartic acid system (m=0.10 (KCl), 25.0°C; Mx=BMXD, As=aspartic acid, Cu=Cu2+ andH=H+)

Stoichiometry Quotient K Log Ka

Mx Cu As H

[MxCuAsH4]/[MxCuH3][AsH]4 2.86 (8)11 10 [MxCu2As]/[MxCu2][As]1 2 8.52 (6)11 [MxCu2AsH]/[MxCu2][AsH]1 2 1 4.75 (3)

[MxCu2As2]/[MxCu2As][As]0 3.30 (7)21 2[MxCu2As(OH)]/[MxCu2(OH)][As] 5.58 (3)1 2 1 −1

a Estimated errors in parentheses.

3.3. Histidine recognition by BMXD

The two binding constants for the binary speciesformed from histidine and protonated BMXD are listedin Table 7. The hepta- and hexa-protonated ternaryspecies are shown to form between p[H] 5 and 10. It issuggested that the imidizole groups on the histidinemolecule form hydrogen bonds in addition to thoseformed by the primary amine group in the amino acid.When the imidizole group is protonated at p[H] valuesbelow its pKa value, the ternary species no longer form.The coordination of the imidizole nitrogen is also sug-gested in the MxHiH6 species. Its concurrent formationwith the tetra-protonated free ligand, and its stabilitycompared to the binary system formed between thetetra-protonated macrocycle and glycine, indicate theinvolvement of imidizole groups. However it cannot beignored that the degree of complex formation withBMXD is the smallest of the three amino acids studiedhere.

3.4. Binding of glycine by Cu(II) complexes of BMXD

The equilibrium constants determined for the cop-per(II) complexes of the BMXD macrocycle and glycineare listed in Table 4. The dinucleating tendency of themacrocycle is shown in the 1:1:1 titration of BMXD,Cu(II), and glycine, as the dinuclear mono-glycinatedspecies exists over a wide p[H] range (4–10) and as oneof the principal species in solution. The glycine nitrogenis deprotonated at p[H] as low as 3.5, indicating itsimportance in the formation of the ternary species. Themonoprotonated complex exists only as a minor speciesat lower p[H], so the predominant factor in the bindingis the glycine amine–Cu(II) coordinate bond. In thetitration with two equivalents of Cu(II) and glycine, theprotonated species appears to the extent of around10%, so the carboxylate group is capable of formingweak bonds with the macrocycle if the stoichiometrydoes not favor other species, namely the mononucleardi- and tri-protonated macrocycle, as in the 1:1:1system.

A dramatic increase in stability between the tetra-and penta-protonated binary species might be due tothe incorporation of carboxylate hydrogen bonds intothe bonding mode. A hepta-protonated species ispresent from low p[H] up to p[H] 4. The only possiblebonding mode for this species is hydrogen bondingbetween the most acidic carboxylate groups and proto-nated amino groups on the macrocyclic ligand. Themonoprotonated binary species is the result of a hydro-gen bond formed between the protonated amino groupof the amino acid and a deprotonated amino group ofthe macrocycle. The additional carboxylic acid groupon the aspartic acid results in an increased negativecharge on the species throughout most of the pH range.This accounts for the increased bonding to the posi-tively charged protonated macrocycle and accounts forthe existence of highly protonated binary systems.

Table 7Stability constants for the BMXD–histidine system (m=0.10 (KCl),25.0°C; Mx=BMXD, Hi=histidinate, and H=H+)

Stoichiometry Stepwise quotient K Log Ka

Mx HHi

1 9.47 (2)1 [MxHiH6]/[MxH6][Hi]64.52 (2)[MxHiH7]/[MxH6][HiH]71 1



Table 8Stability 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 Quotient K Log Ka

Mx Cu Hi H

[MxCuHi]/[MxCu][Hi]0 4.92 (4)11 11 [MxCuHiH]/[MxCuH][Hi]1 1 5.54 (3)12 [MxCuHiH2]/[MxCuH2][Hi]1 1 1 6.21 (3)

[MxCuHiH3]/[MxCuH3][Hi]3 8.49 (8)11 1[MxCu2Hi]/[MxCu2][Hi] 6.15 (5)1 2 1 0

0 [MxCu2Hi2]/[MxCuHi][Hi]1 2 2 4.60 (5)[MxCu2Hi2(OH)][H]/[MxCu2Hi2]−1 −9.06 (6)21 2


carboxylic acids in this p[H] range, and so its binding toan amino nitrogen of the macrocycle is indicated. In thesolution containing two equivalents of copper and oneequivalent of aspartic acid, MxAsHCu2 and MxAsCu2

are found to be the dominant species, each involvingnearly 100% of the ligand in solution. The aspartic acidamino group is deprotonated at p[H] 7, indicating thetendency of the amino group of aspartic acid towardCu(II) coordination.

Fig. 1. Differences in molecular recognition by BMXD for the aminoacids: (a) glycine; (b) histidine; (c) aspartic acid. In each case the totalconcentration of each component is 2.0 mmol at 25.0°C and m=0.1M (KCl), Gl=glycine, Aa=histidine, and As=aspartic acid.

The lack of formation of the MxGlCu species reflectsthe coordination saturation of Cu(II) by the six aminogroups of the BMXD. The di-glycinated dinuclear spe-cies exists as a significant (60%) species in its stoichio-metrically favored solution. No other species werefound to form at higher p[H] values.

3.5. Binding of aspartic acid by Cu(II) complexes ofBMXD

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 thataspartic acid forms more stable ternary species thandoes glycine. Above neutral p[H] the aspartic acid isfully deprotonated, and the aspartic acid complexesformed resemble the glycine species with respect to theapproximate p[H] range over which the species exist.The reason for the increased stability is probably due tothe additional donor group, the carboxylate group, andthe increased basicity of the aspartic acid amino group.The presence of MxCuAsH4 probably results from theaddition of a carboxylate hydrogen bond in addition tothe amino nitrogen Cu(II) coordinate bond. This spe-cies probably resembles the mononucleated glycine spe-cies (Fig. 2). The species MxHAsCu2 exists from p[H] 3to 8, it is unlikely that the proton resides on one of the


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 andhistidine at 25.0°C and m=0.100 M (KCl). Aa=histidine.

Fig. 3. Species distribution showing very strong interaction betweenaspartic acid and the BMXD–Cu2 chelates at 25.0°C and m=0.100M (KCl). Total BMXD and aspartic acid concentrations are each0.00200 M. The two unlabeled small peaks just above pH 3 arespecies MxH5As and MxH6As.

such species negligible in a solution of 1:1:1stoichiometry.

The stabilities of the mononuclear species result intheir presence in the system containing a 1:2:1 ratioof macrocycle–Cu(II)–histidine. Formation of thedinuclear ternary species is less favorable than in thecorresponding glycine system. This is probably a re-sult of steric hindrance of the imidizole group. Whenthe imidizole is deprotonated, and the histidine hastwo available amino groups for bonding, the stabilityof the ternary species is not high enough comparedto the glycine ternary species to indicate a bindingcontribution from the imidizole moiety. In fact, theconstant is smaller, indicating a weaker bond, proba-bly due to steric interference. In the light of this ar-gument, the MxHHiCu2 and MxHiCu2 species differonly by the imidizole proton, they cross at the imidi-zole pKa, and are therefore most likely identicallybonded, resembling the MxGlCu2 structure, with theimidizole oriented away from the ligand, with coordi-nation of the alpha amino group of histidine to oneCu(II) ion and the carboxylate group coordinated tothe other metal cation. A suggested binding modefor these species is shown in 3. Support for the pro-posed binding modes will be obtained in the futureby analysis of the visible spectrum as a function ofpH.

Acknowledgements

The authors express thanks to NSF for support ofE.R. in the Undergraduate Summer Research Pro-gram (NSF-REU) and to The Robert A. WelchFoundation (Grant no. A-0259).

3.6. Binding of histidine by Cu(II) complexes ofBMXD

The binding constants determined for the mononu-clear and dinuclear Cu(II) complexes of the macrocy-cle with histidine are listed in Table 8. It is seen inthe 1:1:1 system that histidine forms four ternaryspecies. None of them are dinuclear species, as areformed with glycine or aspartic acid under equivalentconditions. With the mononuclear species it isdifficult to decide where the protons might residesince there are many logical choices for protonationwith the diethylenetriamine moiety relatively free,and many amino nitrogens in the ternary specieshave similar pKa values. Histidine’s increased basicitycompared with glycine is evident in the more stablebonds it forms with the copper(II) ion. This is thereason for the increased stability of the mononucleardeprotonated ternary species. It is evident that addi-tion of the imidizole group creates stable bonds in-volving both coordination to the copper(II) ion andformation of hydrogen bonds involving the imidizoleamino groups. These effects reduce the dinucleatingtendency of the macrocycle sufficiently to make


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the interaction of bmxd and its copper(ii) complexes with glycine, aspartic acid, and histidine

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