The nitrate salt of Na⁺, Ca²⁺, Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺ (all pro analysis) were from Merck, Darmstadt, Germany. MNPPAP was synthesized by G. Bagherzadeh according to published procedures [10]. The purification was carried out carefully and was about 98%. All the starting materials were of pro analysis grade and used without further purification. Potassium hydrogen phthalate and standard solutions of sodium hydroxide (titrasol), nitric acid, EDTA and of the buffer solutions of pH 4.0, 7.0 and 9.0 were all from Merck. All solutions were prepared with de-ionized water. Water was purified by Milil-Q water purification system, deionized and distillated.

Carbonate-free sodium hydroxide 0.03 M was prepared and standardized against sodium hydrogen phthalate and a standard solution of nitric acid 0.5 mM. M (П) nitrate solution (0.03 M) was prepared by dissolving the above substance in water and was standardized with standard solution of EDTA 0.1 M (triplex).

All pH titrations were performed using a Metrohm 794 basic automatic titrator (Titrino), coupled with a thermostating bath Hero at (25˚C ± 0.1˚C) and a Metrohm combined glass electrode (Ag/AgCl). The pH meter was calibrated with Merck standard buffer solutions (4.0, 7.0 and 9.0).

For the determination of acid dissociation constants of the ligand MNPPAP, an aqueous solution (0.3 mM) of the protonated ligand was titrated with 0.03 M NaOH at 25˚C under nitrogen atmosphere and ionic strength of 0.1 M, NaNO₃. For the determination of binary (a ligand and M²⁺) system, the ratios used were 1:1, M (П): MNPPAP, 0.3 mM. This solution was titrated with 0.03 M NaOH under the same conditions mentioned above. Each titration was repeated seven times in order to check the reproducibility of the data.

The acid dissociation constants and for H₂(MNPPAP) were calculated by an algebraic method [11,12]. The equilibrium involved in the formation of 1:1 complex of MNPPAP and a divalent metal ion may be expressed as Equations (1) and (2).

MNPPAP ion (MNPPAP^-), , is a one-basic species, and thus it can accept one proton on the carboxyl side. On the other hand MNPPAP—releases another proton, for which the following de-protonation equilibriums are hold:

The two protons in H₂(MNPPAP) are released from RNHCH(CH₃)CO₂H according to equilibriums (1) and (2). It is also closed to the de-protonation of acetate groups which occurs at the terminal acetate groups of related amino acids [11,12]. MNPPAP can release the first proton from the terminal acetate group. Hence, here due addition to equilibrium (1) should be considered, which takes place near at pH ≈ 3 (Table 1).

If we abbreviate for simplicity associating of Ca²⁺, Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺ with M²⁺, then one may write the following two equlibriums of (3) and (4):

The experimental data of the potentiometric pH titrations may be completed by considering the above-mentioned equilibria (1) through (4), if the evaluation thereof is not carried into the pH range, where hydroxo complex formation occurs.

Table 1. Negative logarithm of the acidity constants of H₂(MNPPAP) at 25˚C, 0.1 M, NaNO₃, Equations (1) and (2).

^*The given errors are three times the standard error of the mean value or the sum of the probable systematic errors.

Table 2. Logarithm of the stability constants of binary complexes of M²⁺ at 25˚C, 0.1 M, NaNO₃^*, Equation (4).

^*The given errors are three times the standard error of the mean value or the sum of the probable systematic errors.

The results of all potentiometric pH-titration, i.e. acidity and stability constants, are summarized in Tables 1 and 2. The de-protonated MNPPAP^2- can accept two protons, to give the acid H₂(MNPPAP). The first one of these two protons of carboxylate residue is released; its pKa is »3. However, MNPPAP^- can release one more proton, which is pH ≈ 5.2. The measured first acidity constants in this work show good agreement with the same value received by other authors for related amino acids [11,13,14]. However, the carboxyl group is a far stronger acid than residue group [15].

The stability constants of the binary complexes of M (MNPPAP) were refined separately using the titration data of this system in a 1:1, ligand: M²⁺ ratio in the same conditions of temperature and ionic strength (according Equations (3) and (4)), as they were not in agreement with reported value for related amino acids [11,14]. We didn’t receive reasonable results for. All the stability constants of Table 2 show approximately the usual trend. The obtained order is Ca²⁺ < Mg²⁺ > Mn²⁺ < Co²⁺ < Ni²⁺ < Cu²⁺ > Zn²⁺. The observed stability order for MNPPAP follows approximately the Irving-Williams sequence (except Mg²⁺) [16] (Figure 2). Based on the HSAB Principle: Hard acids prefer to coordinate to hard bases, and soft acids prefer to coordinate soft bases. This means that metal ions like Mg²⁺, Mn²⁺ prefer to coordinate on carboxyl site and the other above-mentioned

metal ions (Table 2) (as borderline metal ions) show tendency for both carboxyl group and for pyrimidine group and can be coordinate bidentate [17].

Another interesting point is the tentative and simplified structure for the macrochelated outer-sphere isomer. In the case of hard-metals such as Mg²⁺, and Mn²⁺ can be observed the outer-sphere complex bilding. It should be noted that the term outer-sphere is used here with regard to the M²⁺/N(3)-pyrimidine ring coordination. If an intramolecular direct M²⁺/N(3)-pyrimidine ring coordination occurs, then it is strictly more favorable to have a water molecule between M²⁺ and N(3)-pyrimidine ring. With an outer-sphere N(3)-pyrimidine ring binding can increase the stability of binary complexes.

Now we are able to compare the stability constants of two species M (Am) and M (MNPPAP) (see Table 3). Am represents the amino acids such as Leucine, Valine, Alanine, Glycine, and Methionine. It could easily distinguish that these constants of M (MNPPAP) are not (within the propabable systematic errors) the same of the corresponding M (Am) species. These mean that we can not distinguish a reasonable increased stability in case of M (MNPPAP).

This is an interesting point, because interaction of hard metals such as Mg²⁺ with MNPPAP is considerable, which we can use from results (Table 2).

Based on the results of this work we can draw the conclusion, that hard metal ions just with different stability constants could not have similarly interaction with MNPPAP. The metal ions such as Co²⁺, Ni²⁺, Cu²⁺ and Zn²⁺ are able to have additional interactions with MNPPAP (Figure 3).

Amino acids contain a primary amine group, but in MNPPAP there is a secondary amine. As we see from the results, there is a large difference between the second acidity constants of amino acids and MNPPAP. This means a difference of about four log units (Table 1). We also see big differences between stability constants of amino acids and MNPPAP. In previous work it has shown that amino acids bind simultaneously bi-dentate to metal ions (on carboxyland amine-group), which then

Table 3. Logarithm of the stability constants of binary complexes of M²⁺ with L at 25˚C, 0.1 M, NaNO₃^*, Equation (4). L: Trp (tryptophan), and related compounds such as Met (methionine), Ala (Alanine), Leu (Leucine), Val (Valine), Gly (Glycine), and MNPPAP.

a) From [11,12]; b) From [12]; c) From [11]. ^*The given errors are three times the standard error of the mean value or the sum of the probable systematic errors.

leads to a larger stability. But in the case of MNPPAP, metal ions coordinate on carboxyl-group and N(3) form of prymidine ring. This shows a smaller stability.