J Solution Chem

DOI 10.1007/s10953-010-9532-y

Equilibrium Study of the Mixed Complexes of Copper(II)

with Adenine and Amino Acids in Aqueous Solution
R.A. Ammar E. Al-Mutiri M.A. Abdalla

Received: 27 November 2009 / Accepted: 27 December 2009

Springer Science+Business Media, LLC 2010

Abstract Solution equilibria of the systems Cu(II)-adenine (A)-amino acids (L) have been
studied pH-metrically. The formation constants of the resulting mixed ligand complexes
have been calculated at 25 C and ionic strength 0.1 moldm3 NaNO3 . Ternary complexes
are formed by simultaneous reactions. The relative stability of each ternary complex was
compared with that of the corresponding binary complexes in terms of  log10 K values.
The concentration distribution of the complexes in solution was evaluated.
Keywords Stability constant Adenine Amino acids

1 Introduction
Adenine (A) is one of the most common naturally occurring purine derivatives; it is found in
almost all living tissues as components of nucleic acids, many antibiotics and various coenzyme systems. It is also found in all the living cells as the mono-, di- and triphosphate of
A-nucleosides, viz. adenosine monophosphate (AMP), adenosine diphosphate (ADP) and
adenosine triphosphate (ATP) which play vital roles in many metabollic processes. Adenine has been reported to possess chelating properties, and also increased anticancer activity
when administered as metal complexes [15]. In living systems, almost all the biochemical processes are known to proceed mostly in the solution phase where several metal ions
are present in trace quantities. Most of the physiological activities regarding nucleic acid
interactions are promoted by metal ions through the formation of ternary (mixed-ligand)
complexes [68]. Whenever a metal ion exists in solution together with two or more different ligands, the formation of various simple as well as ternary (mixed-ligand) complexes
is always possible, depending on the pH of the system. The actual complex formation depends on the affinity of the metal ion towards the various ligands present, and the relative
concentrations thereof. The present paper describes the potentiometric study of the binary
and ternary complexes involving copper(II), adenine (A), and other amino acids and related
R.A. Ammar () E. Al-Mutiri M.A. Abdalla
Department of Chemistry, Faculty of Science, King Saud University, Riyadh, Saudi Arabia
e-mail: Dr_reda06@yahoo.com

J Solution Chem

compounds. The concentration distribution relations of the various complex species will be
evaluated.

2 Experimental
2.1 Materials
Adenine was obtained from Aldrich Chem. Co. The amino acids and related compounds:
glycine, alanine, valine, proline, -phenyl-alanine, methionine, threonine, methylamineHCl,
S-methylcysteine, histidineHCl, histamine2HCl, imidazole, penicillamine, lysine,
ornithineHCl, mercaptoethylamine, ethanolamineHCl, cysteine and iso-leucine were obtained from Sigma Chemical U.S.A. All these chemical were used as received without
any further purification, their purities ranged from 9999.9%. For the stability constant
determination, a solution of adenine was prepared with two equivalents of nitric acid.
Cu(NO3 )2 2H2 O and NaNO3 were provided by BDH-Biochemicals Ltd. Poole, England.
The copper content of solutions was determined by complexometric EDTA titrations [9].
Carbonate-free NaOH (titrant) was prepared and standardized against potassium hydrogen
phthalate solution. All solutions were prepared with deionized water.
2.2 Apparatus and Measuring Techniques
The pH-measurements were performed with a Metrohm 686 titroprocessor equipped with a
665 Dosimat (Switzerland-Herisau). The titroprocessor and electrode were calibrated with
standard buffer solutions, prepared according to NBS specification [10]. The pH meter
readings were converted into hydrogen concentration by titrating a standard acid solution
(0.05 moldm3 ), the ionic strength of which was adjusted to 0.1 moldm3 (NaNO3 ), with
a standard base solution (0.05 moldm3 ) at 25 C. The pKw of water was calculated at the
ionic strength of 0.1 moldm3 to be 13.87 (0.05).
The acid dissociation constants of the ligands were determined potentiometrically by
titrating the ligand (40 cm3 ) solution (1.25 103 moldm3 ) of constant ionic strength
0.1 moldm3 , adjusted with NaNO3 . The stability constants of the binary complexes were
determined by titrating 40 cm3 of a solution mixture of metal ion (1.25 103 moldm3 ),
the ligand (2.5 103 moldm3 ) and 0.1 moldm3 NaNO3 . The stability constants of
mixed ligand complexes were determined by titrating 40 cm3 of solution containing Cu(II),
A and amino acids, all of concentration (1.25 103 moldm3 ) and 0.1 moldm3 NaNO3 .
All titrations were performed in a purified N2 atmosphere, using aqueous 0.05 moldm3
NaOH as titrant.
The general four component equilibrium can be written as follows (charges are omitted
for simplicity).
lM + pA + qL + rH  Ml Ap Lq Hr
lpqr =

[Ml Ap Lq Hr ]
[M]l [A]p [L]q [H]r

(1)
(2)

M, A, L, and H denote Cu(II), adenine, amino acids and the proton, respectively. The calculations were performed using the computer program [11] MINIQUAD-75 on an IBM computer. The stoichiometry and stability constants of the complexes formed were determined
by trying various possible composition models for the systems studied. The model selected

J Solution Chem

was that which gave the best statistical fit and which was chemically consistent with the
titration data without giving any systematic drifts in the magnitudes of various residuals, as
described elsewhere [11]. The concentration distribution diagrams were obtained with the
program SPECIES [12], taking into account the experimental conditions used. The results
obtained are shown in Table 1.

3 Results and Discussion

The acid dissociation constants of the ligands and the formation constants of their binary
complexes were determined under the experimental conditions t = 25 C and at constant
0.1 moldm3 ionic strength (adjusted with NaNO3 ), which were also used for determining
the stability constants of the Cu(II) complexes. The results obtained are in good agreement
with the literature data [13].
3.1 Copper (II)-Adenine Equilibria
The potentiometric titration curves of the Cu(II)-adenine system are given in Fig. 1. The
titration curve of the Cu(II)-adenine complex is lowered from that of the free adenine curve,
indicating formation of Cu(II) complexes by displacement of protons. The formation constants were determined by fitting potentiometric data on the basis of possible composition
models. The selected model, with the best statistical fit, was found to consist of CuA (adenine) (1100) and CuAH (1101) complexes. The stability constants of the complexes are
given in Table 1. After complete formation of the CuA complex, the titration curve drifts
due to the formation of hydroxo complexes. The data in this region are fitted considering
the formation of CuA(OH)n species, where n = 1 and 2. The concentration distribution diagram of the Cu(II)A system is shown in Fig. 2. The concentration of the CuA(OH) species
increase with increasing pH and attains a maximum concentration of 65.8% at pH = 7.8.
Further increase in pH is accompanied by a decrease in the concentration of the CuA(OH)
species and an increase in the concentration of the CuA(OH2 ) species. Therefore, the species
CuA(OH) predominates in the physiological pH range. For clarity, the species with concentrations less than 5% were neglected in the concentration distribution plot.
3.2 Copper (II)Amino Acids Equilibria
It is noteworthy that previous studies on complexes of Cu(II) with amino acids were rather
ambiguous and were carried out under very different conditions, which did not allow meaningful comparison of the reported stability constants and definite stoichiometries. For example, it was argued that Cu(II) might form a tris complex in stepwise formation, but biscomplexes were considered in a number of literature reports [14]. In the present investigation, it was planned to redetermine the formation constants of Cu(II)amino acid complexes
under carefully specified conditions used to study the respective ternary complexes.
3.3 Ternary Complex Formation Equilibria
The ternary complex formation may proceed either through a stepwise or simultaneous
mechanism depending on the chelating potentiality of adenine and other ligands. The formation constants of 1:1 cupper(II) complexes with adenine or amino acids are of the same order
of magnitude, Table 1. Consequently the ligation of adenine and amino acid will proceed

J Solution Chem
Table 1 Stability constants of the ternary species in the Cu(II)-A-amino acid systems and proton-association
constants and their binary stability constants
System
Adenine (A)

Glycine

Alanine

Valine

-Phenylalanine

Proline

Threonine

ra

log10 b

9.29 (0.01)

13.32 (0.02)

6.44 (0.07)

12.99 (0.02)

9.18 (0.02)

12.23 (0.02)

2.23 (0.02)

13.37 (0.04)

9.62 (0.02)

11.90 (0.01)

8.11 (0.02)

Sc
7.4 108
9.6 107
3.2 109

1.6 107

15.07 (0.03)

17.15 (0.02)

1.2 108

9.69 (0.01)

9.3 108

11.88 (0.02)

8.00 (0.03)

14.64 (0.01)

16.75 (0.07)

6.3 108

9.57 (0.02)

9.9 108

11.70 (0.01)

8.16 (0.02)

14.99 (0.02)

17.23 (0.02)

9.0 108

9.12 (0.01)

2.0 108

11.01 (0.03)

7.86 (0.01)

14.85 (0.01)

17.43 (0.03)

7.4 108

10.49 (0.00)

2.0 108

12.03 (0.03)

8.74 (0.02)

14.45 (0.05)

17.66 (0.02)

7.4 108

9.06 (0.01)

7.4 108

11.03 (0.02)

8.22 (0.01)

14.90 (0.02)

17.20 (0.01)

6.43 (0.02)

0.26

4.3 108

0.39

4.3 108

0.11

2.7 108

0.43

3.1 108

0.14

3.0 107

 log10 K

6.2 108
9.4 108

0.20

J Solution Chem
Table 1 (Continued)
System

ra

S-methylcysteine

8.25 (0.01)

8.85 (0.02)

17.92 (0.01)

4.4 108

9.12 (0.01)

8.7 108

11.10 (0.04)

8.86 (0.01)

Methionine

Methylamine

Histidine

Histamine

Imidazole

Lysine

Ethanolamine

log10 b

Sc

 log10 K

3.0 107

0.11

6.6 108

3.6 108

14.60 (0.01)

17.11 (0.02)

6.3 108

10.55 (0.01)

1.7 108

6.67 (0.02)

11.66 (0.05)

1.9 107

13.23 (0.01)

21.55 (0.05)

9.53 (0.01)

15.81 (0.03)

17.81 (0.06)

10.89 (0.01)

18.23 (0.04)

19.77 (0.03)

25.90 (0.02)

9.85 (0.01)

16.05 (0.05)

10.52 (0.02)

2.62

6.1 108
1.8 107

0.30

4.7 108
5.8 108
2.4 108

1.01

2.8 108

17.56 (0.01)

18.69 (0.03)

22.45 (0.02)

7.04 (0.01)

1.7 108

4.50 (0.01)

7.66 (0.05)

3.0 107

11.62 (0.032)

14.33 (0.06)

10.44 (0.01)

19.66 (0.01)

10.81 (0.05)

18.89 (0.07)

19.52 (0.02)

24.23 (0.04)

7.94 (0.01)

5.10 (0.09)

12.21 (0.01)

5.23 (0.06)

0.93

3.9 108
2.06

8.3 108
6.3 109

0.47

5.3 108
7.2 108
5.5 108
7.8 107
7.2 108

2.07

J Solution Chem
Table 1 (Continued)
System

ra

Ornithine

10.58 (0.00)

19.43 (0.02)

Mercaptoethylamine

Pencillamine

iso-Leucine

Cysteine

log10 b

21.39 (0.02)

11.90 (0.05)

16.30 (0.06)

18.88 (0.02)

23.57 (0.01)

10.03 (0.04)

18.64 (0.02)

11.61 (0.05)

18.89 (0.07)

18.45 (0.02)

25.89 (0.03)

10.10 (0.01)

17.97 (0.02)

11.45 (0.03)

Sc

 log10 K

1.0 108

2.20

1.6 107
6.4 108
3.7 108
2.0 108
6.8 108
3.0 108

19.52 (0.05)

18.26 (0.04)

25.25 (0.03)

9.76 (0.00)

12.22 (0.01)

9.23 (0.003)

15.27 (0.004)

17.37 (0.02)

5.3 108

9.77 (0.03)

3.4 107

17.67 (0.07)

17.03 (0.004)

18.24 (0.02)

24.57 (0.01)

2.37

1.3 108

8.98 (0.003)

2.34

1.9 108
3.4 107

1.04

3.2 1010

0.17

3.2 1010
5.3 108

a l, p, q and r are the stoichiometric coefficient corresponding to Cu(II), A, amino acids and H+ , respectively
b Standard deviations are given in parentheses
c Sum of square of residuals

simultaneously. The titration data of the ternary complexes with amino acids and adenine fit
satisfactorily with formation of the species: Cu(A), Cu(A)2 Cu(L), Cu(L)2 , Cu(A)(L) and
Cu(A)(LH). The formation of mixed-ligand complex by simultaneous mechanisms may be
confirmed by comparing the theoretical curve, conducted based on the calculated formation
constants and the experimental titration data points, Fig. 3, for the CuAvaline system,
taken as a representative. The good fit obtained is indicative of the validity of the complex
formation model.

J Solution Chem
Fig. 1 Potentiometric titration
curves of the Cu(II)-A system

Fig. 2 The distribution of Cu2+ (1000), CuA (adenine) (1100), CuA(OH) (1101) and CuA(OH)2 (1102)
as a function of pH in 0.1 moldm3 NaNO3 at 25 C

The formation constants of amino acid complexes are higher than those of the corresponding monodentate methylamine and imidazole complexes, as seen in Table 1. This indicates that amino acids function as bidentate ligands coordinating through the amino and
carboxylate groups.
Histidine is potentially a tridentate ligand with amino, imidazole, and carboxylate groups
as metal ion binding sites. The stability constant of the histidine complex is higher than that
of -amino acids and close to that of histamine (see Table 1). This indicates that histidine is
coordinating in the histamine-like way. Histidine is shown to form both protonated (1111)
and deprotonated (1110) complex species.

J Solution Chem

Fig. 3 Potentiometric titration curve of the Cu(II)-A-valine system

The acid dissociation constant of the protonated species is given by the following Eq. 3.
pK H = log 1111 log 1110

(3)

The pKa for the histidine complex amounts to 6.13, being lower than that of the protonated amino group NH+
3 (pKa = 9.53), but closer to that of the protonated imidazole group
(pKa = 6.28), suggesting the proton in the protonated complex would be located mainly on
the imidazole group.
Threonine has an extra binding center on the -alcoholato-group. It forms, in addition to the previously mentioned complexes, the Cu(A)(LH1 ) species. This complex is
formed through induced ionization of the -alcohol group, as mentioned in the literature [15]. The pKa value of the -alcoholato-group incorporated in the Cu(II) complex
(log 1110 log 1111 ) is 10.77. This is in good agreement with that reported in literature
for the Cuthreonine complex [16]. Also, ethanolamine form the complex species 1110 and
111-1. The log 1110 value for ethanolamine is smaller than those of amino acids. This may
be attributed to the weaker coordinating tendency of an alcohol group compared to a carboxylate group. Charge effect will also be important since the alcohol is neutral where as
the carboxylate group is negatively charged. The pKa value of coordinated alcohol group in
ethanolamine complex (6.43) is considerably smaller than that of threonine complex. This
is consistent with the scheme where the alcohol group in ethanolamine is coordinated to the
copper centre whereas the OH group in threonine remains attached prior to deprotonation.
Due to the donation of the electron pair on oxygen to the metal centre, the OH bond can be
considerably weakened and the ionization of a proton occurs at fairly low pH.
Lysine and ornithine may bind to Cu(II) ion as -amino acid (N,O-donor set) or by and -amino groups (N,N-donor set). The stability constants of their ternary complexes are
higher than those of -amino acids, indicating that lysine and ornithine ligating by the two
amino groups.
S-methylcysteine has the lowest pKa value (8.25) among the amino acids studied. Its
complex has a higher stability constant than that for amino acids such as glycine. This may
be taken as evidence that the sulfur atom participates in the complex formation process.
Also, S-methylcysteine forms a more stable complex than methionine, plausibly due to the

J Solution Chem

Fig. 4 The distribution of Cu2+ (1000), CuA (adenine) (1100), CuL (ornithine) (1010), CuL2 (1020),
CuALH (1111) and CuAL (1110) as a function of pH in 0.1 moldm3 NaNO3 at 25 C

fact that the five-membered chelate ring in the former complex is energetically more favored
than six-membered chelate ring in the latter complex.
Penicillamine has three binding sites, carboxylic, amino and sulfhydryl groups. It forms
the complexes 1110 and 1111. The stability constant of the 1110 complex is in fair agreement with that of mercaptoethylamine (where the binding sites are the amino and sulfhydryl
groups) and higher than those for -amino acids (where the binding sites are the amino and
carboxylate groups). This indicates that penicillamine interacts with Cu(II) ion by the amino
and deprotonated-SH groups.
Ornithine was found to form more stable complex than -amino acids. The stability constant of its complex is higher than those of -amino acids. This may be taken to indicate that
ornithine most likely chelates through the two amino groups. The species distribution for ornithine, taken as representative amino acid, is given in Fig. 4. The protonated 1111 complex
species predominates with a formation degree amounting to 90% at pH 8.4, whereas the
deprotonated species 1110 attains a maximum concentration of 98% at pH 11.2. Therefore,
the species 1111 predominates in the physiological pH range.
The parameter log10 K values are used to indicate the relative stability of the ternary
complexes as compared to the binary ones as in the following equations:
Cu(A) + Cu(L)  Cu(A)(L) + Cu


Cu(A)
Cu
Cu
log10 Cu(A)
+ log10 Cu(L)
 log10 K = log10 Cu(A)L

(4)
(5)

The  log10 K values for the ternary complexes studied in this paper are listed in Table 1.
The theoretical  log10 K value for a square-planer copper(II) complex is 0.9 [17]. The
tendency to form ternary complexes was compared with this value, so that if  log10 K is
greater than 0.9, this should be taken as an indication that the ternary complex is favored.
The calculated  log10 K values of the -amino acids are less negative than the theoretical
value (0.90). This may be considered as evidence for the occurrence of enhanced stabilities
involving -back-donation from the negatively charged amino acid to the -system of the
adenine.
The positive  log10 K value for the phenylalanine mixed-ligand complex (1110) indicate that the ternary complexes are more stable than the corresponding binary ones [18].

J Solution Chem

This may be attributed to intramolecular aromatic-ring stacking, hydrogen bond, and a

cooperative effect between ligands.

4 Conclusion
The present investigation describes the formation equilibria of copper(II), adenine (A), and
amino acids. The ligands include N-donor, O-donor and S-donor. The complexes are formed
in a simultaneous mechanism, whereby the formation constants of Cu(II) complexes with
adenine and the amino acids under investigation have been found to be of the same order.
The stability constants of complexes in solution have been calculated and their concentration
distribution are evaluated. It has been found that amino acids function as bidentate ligands
and coordinate through amino and carboxylate group. The imidazole group of histidine increases the stability of the complex due to the high affinity of Cu(II) for the nitrogen donor
group. The thioether group in S-methylcysteine increases the stability of its complex as a
result of the stronger donor properties of the sulfur atom. Sulfur ligands form protonated
and deprotonated complexes with Cu(II) ion.
Furthermore, in the case of (A), metalation occurs indirectly at N7 involving the possible
contribution from C(6)NH2 in binary systems [19], while in ternary complexes (A) coordinates only through N7 to metal ions [20] as is evident from the higher values of equilibrium
constants of the ternary complexes, which is probably favored by the electrostatic interaction
between the amino hydrogen of (A) and the chelation of amino acids.
Acknowledgement

The author is indebted to Prof M.M. Shoukry for his help and interest.

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