**Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules**

Ahmed A. El-Sherif<sup>∗</sup>

*Department of Chemistry, Faculty of Science, Cairo University, Cairo, Egypt* 

### **1. Introduction**

*Cis*platin, *cis*-Diamminedichloroplatinum (II), is one of the most effective anticancer agents (Rosenberg, 1969). It has demonstrated a remarkable chemotherapeutic potential in a large variety of human solid cancers, such as, testicular, ovarian, bladder, lung and stomach carcinomas (Wong and Giandomenico, 1999; Guo and Sadler, 2000). The successful use of platinum (II) complexes as potent anticancer drugs has attracted the interest of many scientists. It was observed that the nature and arrangement of the ligands can affect the mode of action and metabolism of the drug while crossing the cell membrane and inside the cell. Despite the widespread use of *cis*-platin as an anticancer drug there is still scope for improvement, with respect to: i) reduced toxicity; ii) increased clinical effectiveness; iii) broader spectrum of action; iv) elimination of side effects (e.g., nausea, hearing loss, vomiting, etc); v) increased solubility and vi) ability to use them in combination with other drugs, limited by severe toxicities so far. Replacement of the chloro ligands by carboxylate groups in carboplatin, *cis*-diamine(1,1cyclobutanedicarboxylate)platinum(II), is a widely used second-generation platinum anticancer drug showing less side effects than *cis*-platin. The development of several new anticancer platinum drugs including Carboplatin, Nedaplatin, Lobaplatin and Oxaliplatin (Scheme 1) still have draw-backs and offer no more clinical advantages over the existing cisplatin (Gill, 1984; Galanski et al., 2005; Momekov et al., 2005). Furthermore, the development of acquired resistance to cis-platin is frequently observed during chemotherapy (Heim, 1993).

There is also much interest in Pd(II) analogues because they are usually isostructural with those of Pt(II), which show a very similar coordination process and geometry. However, Pd(II) systems attain equilibrium much more quickly than Pt(II) systems (~104-105 faster kinetics). The slow formation kinetics for Pt(II) complexes generally rules out the determination of stability constants. Therefore, Pd(II) complexes are frequently used as model complexes to study the interaction of Pt(II) with DNA and to mimic the binding properties of various platinum(II) species (Tercero-Moreno et al., 1996). It was also suggested that the faster aquation of palladium(II) compared with platinum(II) *in vitro*, makes the former a better model for studying Pt(II) reactions *in vivo* (Nelson, et al.,1976) with biological molecules, since these reactions always start with the aquation of the platinum(II) complexes. Several palladium complexes have been reported (Gill, 1984) with

<sup>∗</sup> Corresponding Author

Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 81

In addition, other experimental techniques are sometimes available. For example, if the metal ion or the ligand is coloured, and the colour changes (in intensity and/or frequency) upon complexation, spectrophotometry can be used. If the metal ion is diamagnetic, or if the ligand contains a suitable nucleus, nuclear magnetic resonance (NMR) is a striking method. This latter method, which ideally gives one separated signal for each unique chemical surrounding, can provide information not only on the free metal ion or ligand concentration, but also on the number of species and their respective concentration for a given analytical composition. Furthermore, since the positions of these signals are susceptible to protonation/deprotonation reactions, they can also be used to gain information on acid/base reactions of ligands and their complexes. Stopped-flow technique

The solution equilibria between metal ions and ligands may be described by two wordcontinuous competitions: The proton and a range of metal ions compete for a range of donor sites, the contest being ruled by concentration and pH conditions. The determination of equilibrium constants is an important process for many branches of chemistry (Motekaitis and Martell, 1988) Equilibrium constants can be determined from potentiometric data and/or spectrophotometric data. Developments in the field of computation of equilibrium constants from experimental data were reviewed by Leggett (Legget, 1985) and Meloun et al.[ (Meloum, et al., 1994). Since then, many more programs have been published, mainly so as to be able to use microcomputers for the computations. The most commonly used programs for solution equilibrium constant determination are given in Table 1 (Motekaitis and Martell, 1988; Sabatini et al., 1974; Gans et al.,1976; Gans et al.,1985; Zekany and Nagypal, 1985; Sabatini et al., 1992; Gordon, 1982; Chandler et al., 1984; Perrin and Stunzi, 1985 ; Beltrán et al., 1993; Frassineti et al., 1995; Gampp et al.,1985; Tauler, et al., 1991). All of these programs use least-square refinements to reduce the differences between calculated and experimental data to get the best model from the best fit. The sum of square of residuals between experimental and calculated values are normally very small, it is typically between 10-6-10-9. Potentiometry generally used for measurements of formation constants of metal complexes is based on pH-metric titration of the ligand in absence and presence of metal ions. The formation constants derived by the least squares analysis of potentiometric data can describe completely the solution equilibria. The measurements are usually carried out at a constant ionic strength higher than the metal ion concentration. Therefore, no appreciable change in the ionic strength of the solution medium occurs. In general for the reaction:

L(M) + p(L1) + q(L2) + r(H) [(M)l(L1)p(L2)q(H)r] (1)

[L1]p[L2]q[H]r (2)

**2.1 Determination of stability constants of mixed ligand complexes** 

The overall stability constant, βlpqr, can be calculated from:

(charges are omitted for simplicity)

respectively. For OH-

βlpqr = [(M)l(L1)p(L2)q(H)r]/[M]l

the coefficient (r) for H = -1.

where M, L1, L2 and H stand for [Pd(diamine)(H2O)2]2+ ion, ligand(1), ligand(2) and proton,

can be applied for fast reactions.

bidentate amine ligands which have shown anticancer activity comparable to or greater than cisplatin. Moreover, a series of labile Pd(II) complexes have proved to be useful as models to obtain a reasonable picture of the thermodynamics of the reactions for closely related Pt(II) complexes. It has also been suggested that these palladium complexes may be useful for the treatment of tumors of the gastrointestinal region where cisplatin fails. Mono-dentate ligands can bind in both cis- and *trans* arrangements around the metal and the isomers stability depend on several factors. Consequently, bidentate ligands are more reliable for the preparation of *cis*-complexes, in particular with palladium(II) and platinum(II) (Misra et al., 1998; Byabartta et al., 2001; Santra et al., 1999; Pal, et al., 2000; Rauth, et al., 2001; Roy et al., 1996; Das, et al., 1997; Das, et al., 1998). The reaction of DNA bases with Pd(II)/Pt(II) complexes of chelating N,N' -donors having *cis*-MCl2 configuration constitutes a model system which may allow for exploration of the mechanism of the anti-tumor activity of cisplatin. Considering the importance of palladium complexes as potential anticancer drugs, we report here the coordination chemistry of mixed-ligand palladium complexes of bidentate amines with biologically active ligands.

Scheme 1. Platinum-based drugs currently in clinical use.

### **2. Methods used for detection of aqueous solution complexes**

Ion-selective electrodes were used for determination of the position of dynamic equilibrium system and the most common one is the glass electrode or hydrogen gas electrode which can be used for hydrogen ion measurements. Metal-ion selective electrodes or metalamalgam electrodes can also be used for certain metal ions, but they are seldom as precise or convenient as the hydrogen ion electrode.

A great advantage with the use of ion-selective electrode measurements is that series of data can be easily collected through a titration procedure. From an initial analytical composition, stepwise changes with a burette are made with intervening electrode recordings. The elapsed time between these changes must be certained to be sufficient for equilibrium to be attained. A good method to check for this pre-requisite is to make repeated high-resolution electrode readings at predetermined time intervals, since this will make sluggish attainments of equilibrium clearly visible.

bidentate amine ligands which have shown anticancer activity comparable to or greater than cisplatin. Moreover, a series of labile Pd(II) complexes have proved to be useful as models to obtain a reasonable picture of the thermodynamics of the reactions for closely related Pt(II) complexes. It has also been suggested that these palladium complexes may be useful for the treatment of tumors of the gastrointestinal region where cisplatin fails. Mono-dentate ligands can bind in both cis- and *trans* arrangements around the metal and the isomers stability depend on several factors. Consequently, bidentate ligands are more reliable for the preparation of *cis*-complexes, in particular with palladium(II) and platinum(II) (Misra et al., 1998; Byabartta et al., 2001; Santra et al., 1999; Pal, et al., 2000; Rauth, et al., 2001; Roy et al., 1996; Das, et al., 1997; Das, et al., 1998). The reaction of DNA bases with Pd(II)/Pt(II)

system which may allow for exploration of the mechanism of the anti-tumor activity of cisplatin. Considering the importance of palladium complexes as potential anticancer drugs, we report here the coordination chemistry of mixed-ligand palladium complexes of

O

O

Ion-selective electrodes were used for determination of the position of dynamic equilibrium system and the most common one is the glass electrode or hydrogen gas electrode which can be used for hydrogen ion measurements. Metal-ion selective electrodes or metalamalgam electrodes can also be used for certain metal ions, but they are seldom as precise or

A great advantage with the use of ion-selective electrode measurements is that series of data can be easily collected through a titration procedure. From an initial analytical composition, stepwise changes with a burette are made with intervening electrode recordings. The elapsed time between these changes must be certained to be sufficient for equilibrium to be attained. A good method to check for this pre-requisite is to make repeated high-resolution electrode readings at predetermined time intervals, since this will make sluggish

O

O

Carboplatin

Pt

H3N

H3N


O

Pt

Nedaplatin

H3N

H3N

H2N

H2N

Lobaplatin

O

O

CH3

O

O

O

Pt

complexes of chelating N,N'

Cl

Cl Pt

H2N

H2N

Oxaliplatin

convenient as the hydrogen ion electrode.

attainments of equilibrium clearly visible.

Cisplatin

H3N

H3N

bidentate amines with biologically active ligands.

O

O

O

**2. Methods used for detection of aqueous solution complexes** 

Scheme 1. Platinum-based drugs currently in clinical use.

O

Pt

In addition, other experimental techniques are sometimes available. For example, if the metal ion or the ligand is coloured, and the colour changes (in intensity and/or frequency) upon complexation, spectrophotometry can be used. If the metal ion is diamagnetic, or if the ligand contains a suitable nucleus, nuclear magnetic resonance (NMR) is a striking method. This latter method, which ideally gives one separated signal for each unique chemical surrounding, can provide information not only on the free metal ion or ligand concentration, but also on the number of species and their respective concentration for a given analytical composition. Furthermore, since the positions of these signals are susceptible to protonation/deprotonation reactions, they can also be used to gain information on acid/base reactions of ligands and their complexes. Stopped-flow technique can be applied for fast reactions.

### **2.1 Determination of stability constants of mixed ligand complexes**

The solution equilibria between metal ions and ligands may be described by two wordcontinuous competitions: The proton and a range of metal ions compete for a range of donor sites, the contest being ruled by concentration and pH conditions. The determination of equilibrium constants is an important process for many branches of chemistry (Motekaitis and Martell, 1988) Equilibrium constants can be determined from potentiometric data and/or spectrophotometric data. Developments in the field of computation of equilibrium constants from experimental data were reviewed by Leggett (Legget, 1985) and Meloun et al.[ (Meloum, et al., 1994). Since then, many more programs have been published, mainly so as to be able to use microcomputers for the computations. The most commonly used programs for solution equilibrium constant determination are given in Table 1 (Motekaitis and Martell, 1988; Sabatini et al., 1974; Gans et al.,1976; Gans et al.,1985; Zekany and Nagypal, 1985; Sabatini et al., 1992; Gordon, 1982; Chandler et al., 1984; Perrin and Stunzi, 1985 ; Beltrán et al., 1993; Frassineti et al., 1995; Gampp et al.,1985; Tauler, et al., 1991). All of these programs use least-square refinements to reduce the differences between calculated and experimental data to get the best model from the best fit. The sum of square of residuals between experimental and calculated values are normally very small, it is typically between 10-6-10-9. Potentiometry generally used for measurements of formation constants of metal complexes is based on pH-metric titration of the ligand in absence and presence of metal ions. The formation constants derived by the least squares analysis of potentiometric data can describe completely the solution equilibria. The measurements are usually carried out at a constant ionic strength higher than the metal ion concentration. Therefore, no appreciable change in the ionic strength of the solution medium occurs. In general for the reaction:

$$\text{L(M)} + \text{p(L\_1)} + \text{q(L\_2)} + \text{r(H)} \xrightarrow{\Delta} \text{[(M)} \text{l(L\_1)}\_{\text{F}} \text{(L\_2)}\_{\text{q}} \text{(H)}\_{\text{r}} \text{]} \tag{1}$$

The overall stability constant, βlpqr, can be calculated from:

$$
\beta\_{\rm lpqr} = \left[ (\rm M)\_l (\rm L\_1)\_p (\rm L\_2)\_q (\rm H)\_r \right] / \left[ \rm M \right] \left[ \rm L\_1 \right] \rm \left[ \rm L\_2 \right] \rm \rm \left[ \rm H \right] \rm \tag{2}
$$

(charges are omitted for simplicity)

where M, L1, L2 and H stand for [Pd(diamine)(H2O)2]2+ ion, ligand(1), ligand(2) and proton, respectively. For OH- the coefficient (r) for H = -1.

Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 83

membrane filter. Great care was taken to ensure that the resulting solution was free of Ag+ ions and that the chloro-complex had been converted into the aqua species, the filtrate made up to the desired volume in a standard volumetric flask. Also, the ligands in the form of hydrochlorides were converted to the corresponding hydronitrate in the same way as

Speciation (based on concentrations of metal ions and complexing species) refers to a program (Pettit) which calculates and plots the species distribution of a series of complexes over a specified pH range. In this program, the input data of total concentrations of metal and ligand, pH range and the best fit set of β values are used to compute equilibrium concentrations of all the available complex species over the given pH range. All types of complexes can be calculated, including mixed complexes, protonated, hydroxo and polynuclear species. The graphical output can thus provide a visual record of the most predominant complex species at any pH especially within the physiological pH range.

The hydrolysis reactions of Pt(II) complexes are among the most important issues which should be considered under physiological conditions. As a consequence, hydrolysis of cisplatin and its derivatives has been thoroughly studied in both solution and solid state (Martin, 1983; Martin, 1999; Faggiani et al., 1977; Faggiani et al., 1977). It is clear from these studies that hydrolysis of cisplatin and other *cis*-diamine platinum(II) species can not be described by the formation of simple monomeric dihydroxo complexes, but various dinuclear (Faggiani et al., 1978) and trinuclear species are also formed (Faggiani et al., 1977; Faggiani et al., 1977). The very slow formation kinetics, however, hampers the determination of stability constants of platinum(II), but the corresponding palladium(II) complexes can be used as appropriate model compounds (Tercero-Moreno et al., 1996).

The main species formed during the hydrolysis of [Pd(diamine)(H2O)2]2+ ion are 10-1, 10-2, 20-1 and 20-2. The first two species are due to deprotonation of the two coordinated water

[Pd(N-N)(H2O)2] [Pd(N-N)(H2O)(OH)] + H

The third species, (20-1), is the hydroxo bridged-dimer formed as result of the combination of the monoaqua hydroxo species (10-1) with the diaqua species (100) [Pd(diamine)(H2O)2]2+

100 10-1

pKa2

pKa1

[Pd(N-N)(H2O)2(OH)] [Pd(N-N)(OH)2] <sup>+</sup>

10-1 10-2

2+ <sup>+</sup> <sup>+</sup>

**2+ complex** 

+ H

+

(3)

(4)

**2.5 Determination of the acid-base equilibria of [Pd(diamine)(H2O)2]**

described above.

**2.4 Speciation distribution as a function of pH** 

molecules, as given by Eqs. 3 and 4.

as given by Eq. 5.


aAdditional data types used in calculations: E, ESR and N, NMR

Table 1. The most commonly used programs for calculating equilibrium constants from potentiometric (V) and spectrophotometric (A) data.

### **2.2 Determination of stability constants of Pd(II) complexes**

As mentioned earlier, determination of formation constants of the Pd(II) complexes is made more difficult than in the case of other metals as a result of the unstable nature of [Pd(H2O)4]2+ in aqueous solution. Some authors have used [PdCl4]2-, as the metal ion source, but in this case the inclusion of the Cl- as a ligand as well as other ligands in the calculation of formation constants becomes necessary (Bóka, et al., 2001). Elding (Elding, 1972) calculated log β14 for [PdCl4]2- to be ~ 10. Therefore, [Pd(NH3)2(H2O)2]2+ and [Pd(diamine)(H2O)2]2+ were considered as the starting metal ions for Pd(II) and the acidbase equilibria of the diaquo complexes were first determined. Secondary ligands were then introduced and the formation constant of the mixed ligand complex calculated using one of the above mentioned programs. Hydrolytic reactions of Pt(II) and Pd(II) complexes are important issues because they are related to the action of the *cis*-platinum(II) anticancer drugs. The very high thermodynamic stability constants of the chelated diamine complexes of palladium(II) result in the complete formation of the species [Pd(diamine)(H2O)2]2+ even under very acidic conditions (pH <2), while the relatively high ratios of the stepwise stability constants suppress the bis(bidentatediamine) complex formation in equimolar solution (Nagy and Sóvágó, 2001). As a consequence, all the Pd(II) species are present in the form of [Pd(bidentatediamine)]2+, and therefore, the ternary complex, Pd(bidentate diamine)-ligand, can be treated as a binary complex.

### **2.3 Preparation of [Pd(diamine)(H2O)2] 2+ complex**

[Pd(diamine)Cl2] complexes were prepared, by reaction of [PdCl4]2- with diamine in the molar ratio 1:1. For equilibrium studies, [Pd(diamine)Cl2] was converted into the diaqua complex [Pd(diamine)(H2O)2](NO3)2 by stirring the chloro-complex with two equivalents of AgNO3 overnight, and removing the AgCl precipitate by filtration through a 0.1 μm pore membrane filter. Great care was taken to ensure that the resulting solution was free of Ag+ ions and that the chloro-complex had been converted into the aqua species, the filtrate made up to the desired volume in a standard volumetric flask. Also, the ligands in the form of hydrochlorides were converted to the corresponding hydronitrate in the same way as described above.

### **2.4 Speciation distribution as a function of pH**

82 Stoichiometry and Research – The Importance of Quantity in Biomedicine

PKAS V (Motekaitis and Martell, 1988)

PSEQUAD V, A (Zekany and Nagypal, 1985)

Table 1. The most commonly used programs for calculating equilibrium constants from

As mentioned earlier, determination of formation constants of the Pd(II) complexes is made more difficult than in the case of other metals as a result of the unstable nature of [Pd(H2O)4]2+ in aqueous solution. Some authors have used [PdCl4]2-, as the metal ion source, but in this case the inclusion of the Cl- as a ligand as well as other ligands in the calculation of formation constants becomes necessary (Bóka, et al., 2001). Elding (Elding, 1972) calculated log β14 for [PdCl4]2- to be ~ 10. Therefore, [Pd(NH3)2(H2O)2]2+ and [Pd(diamine)(H2O)2]2+ were considered as the starting metal ions for Pd(II) and the acidbase equilibria of the diaquo complexes were first determined. Secondary ligands were then introduced and the formation constant of the mixed ligand complex calculated using one of the above mentioned programs. Hydrolytic reactions of Pt(II) and Pd(II) complexes are important issues because they are related to the action of the *cis*-platinum(II) anticancer drugs. The very high thermodynamic stability constants of the chelated diamine complexes of palladium(II) result in the complete formation of the species [Pd(diamine)(H2O)2]2+ even under very acidic conditions (pH <2), while the relatively high ratios of the stepwise stability constants suppress the bis(bidentatediamine) complex formation in equimolar solution (Nagy and Sóvágó, 2001). As a consequence, all the Pd(II) species are present in the form of [Pd(bidentatediamine)]2+, and therefore, the ternary

MINIQUAD V (Sabatini et al., 1974) MINIQUAD75 V (Gans et al.,1976) SUPERQUAD V (Gans et al.,1985)

HYPERQUAD V, A (Sabatini et al., 1992) TITAN V (Gordon, 1982) SCOGS2a V (Chandler et al., 1984) SCOGS2b V (Perrin and Stunzi, 1985) STAR A (Beltrán et al., 1993) HYPNMR N (Frassineti et al., 1995) SPECFIT A(E) (Gampp et al.,1985) SPFAC A(E) (Tauler, et al., 1991)

Program Data typea Reference

aAdditional data types used in calculations: E, ESR and N, NMR

**2.2 Determination of stability constants of Pd(II) complexes** 

complex, Pd(bidentate diamine)-ligand, can be treated as a binary complex.

**2+ complex**  [Pd(diamine)Cl2] complexes were prepared, by reaction of [PdCl4]2- with diamine in the molar ratio 1:1. For equilibrium studies, [Pd(diamine)Cl2] was converted into the diaqua complex [Pd(diamine)(H2O)2](NO3)2 by stirring the chloro-complex with two equivalents of AgNO3 overnight, and removing the AgCl precipitate by filtration through a 0.1 μm pore

potentiometric (V) and spectrophotometric (A) data.

**2.3 Preparation of [Pd(diamine)(H2O)2]**

Speciation (based on concentrations of metal ions and complexing species) refers to a program (Pettit) which calculates and plots the species distribution of a series of complexes over a specified pH range. In this program, the input data of total concentrations of metal and ligand, pH range and the best fit set of β values are used to compute equilibrium concentrations of all the available complex species over the given pH range. All types of complexes can be calculated, including mixed complexes, protonated, hydroxo and polynuclear species. The graphical output can thus provide a visual record of the most predominant complex species at any pH especially within the physiological pH range.

### **2.5 Determination of the acid-base equilibria of [Pd(diamine)(H2O)2] 2+ complex**

The hydrolysis reactions of Pt(II) complexes are among the most important issues which should be considered under physiological conditions. As a consequence, hydrolysis of cisplatin and its derivatives has been thoroughly studied in both solution and solid state (Martin, 1983; Martin, 1999; Faggiani et al., 1977; Faggiani et al., 1977). It is clear from these studies that hydrolysis of cisplatin and other *cis*-diamine platinum(II) species can not be described by the formation of simple monomeric dihydroxo complexes, but various dinuclear (Faggiani et al., 1978) and trinuclear species are also formed (Faggiani et al., 1977; Faggiani et al., 1977). The very slow formation kinetics, however, hampers the determination of stability constants of platinum(II), but the corresponding palladium(II) complexes can be used as appropriate model compounds (Tercero-Moreno et al., 1996).

The main species formed during the hydrolysis of [Pd(diamine)(H2O)2]2+ ion are 10-1, 10-2, 20-1 and 20-2. The first two species are due to deprotonation of the two coordinated water molecules, as given by Eqs. 3 and 4.

$$\begin{array}{ccccc} \text{[Pd(N-N)(H\_2O)\_2]} & \text{2+} & \text{pK}\_{\text{a1}} & + & + & + \\ \text{100} & \text{100} & & \text{10-1} & & \\ \text{100} & & \text{pK}\_{\text{a2}} & & \text{10-1} & \\ \text{[Pd(N-N)(H\_2O)\_2(OH)]} & \text{+} & \text{pK}\_{\text{a2}} & \text{[Pd(N-N)(OH)\_2]} & + & + \\ \text{10-1} & & & \text{10-2} & & \\ \end{array}$$

The third species, (20-1), is the hydroxo bridged-dimer formed as result of the combination of the monoaqua hydroxo species (10-1) with the diaqua species (100) [Pd(diamine)(H2O)2]2+ as given by Eq. 5.

Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 85

Complexa Pka1 pKa2 Reference

*Cis*-[Pt(NH3)2(H2O)2] 2+ 5.6 7.3 (Britten et al., 1982) [Pt(en)(H2O)2] 2+ 5.8 7.6 (Lim et al., 1976)

[Pd(BPY)(H2O)2] 2+ 3.91 8.39 (Shehata, 2001)

Table 2. Comparison of acid dissociation constants of some Pt and Pd-diaquo complexes.

2 3 4 5 6 7 8 9 10 11 pH

Fig. 1. Concentration distribution of various species as a function of pH in the

Pd(AMBI)(H2O)2 system at concentration of 1.25x10-3 mol-dm-3, I = 0.1 mol-dm-3 (NaNO3)

20-2

10-2

10-1

aen, 1,2-DAP, Me2en, Pic and BPY represent ethylenediamine, 1,2-diaminopropane, N,N′-dimethylethylenediamine, picolylamine and 2,2′-bipyridyl, respectively.

100

0

and T = 25 ± 0.1 0C).

10

20

30

40

50

% Species

60

70

80

90

100

[Pd(en)(H2O)2] 2+ 5.6 7.3 (Hohmann, et al., 1991) [Pd(1,2-DAP)(H2O)2] 2+ 5.62 9.35 (Shoukry, et al., 1999) [Pd(Pic)(H2O)2] 2+ 4.81 8.46 (El-Sherif, et al., 2003)

The fourth species, 20-2, is the dimeric di-μ-hydroxo complex of two 10-1 species according to Eq.6.

According to the data in Table 2 (Britten et al., 1982; Lim et al., 1976; Hohmann, et al., 1991; Shoukry, et al., 1999; El-Sherif, et al., 2003 ; Shehata, 2001), the pKa1 and pKa2 values in the bipyridine as a non-leaving group were found to be 3.91 and 8.39, respectively and are lower than the corresponding values of all the PdII-diamine complexes. The [Pd(Pic)(H2O)2]2+ values are intermediate because Pic has one pyridine ring. This can be attributed to the increased positive charge on Pd atom due to the π-acceptor properties of the aromatic moiety of Pyridine ring, leading to an increase in the electrophilicity of the Pd ion and consequently to a decrease in the pKa of the coordinated water molecule. The equilibrium constant for the dimerization reactions (5) and (6) can be calculated with Eqs. 7 and 8 respectively.

$$
\log\_{10} \mathbb{K}\_{\text{dimer}} = \log \| \mathbf{\hat{p}}\_{20-1} - \log \| \mathbf{\hat{p}}\_{10-1} \tag{7}
$$

$$
\log\_{10} \mathbf{K}\_{\text{dirmer}} = \log \beta\_{20\cdot 2} - 2 \log \beta\_{10\cdot 1} \tag{8}
$$

The concentration distribution diagram for [Pd(AMBI)(H2O)2]2+ and its hydrolysed species as a representative example of hydrolysis of [Pd(diamine)(H2O)2] is shown in Fig.1. The concentration of the monohydroxo species, 10-1 and the dimeric species, 20-2 increase with increasing pH, predominating in the pH range 4.8 to 7.8 with formation percentages of ca. 44% and 54% for the monohydroxo (10-1) and dimeric species (20-2), respectively, i.e., they are the main species present in solution in the physiological pH range. A further increase in pH is accompanied by an increase in the dihydroxo species, which is the main species above a pH of ca. 11. In the high pH range the inert dihydroxo complex would be the predominant species, so that the reactivity of DNA to bind the Pd(amine) complex will considerably decrease.

**+**

**logKdimer**

The fourth species, 20-2, is the dimeric di-μ-hydroxo complex of two 10-1 species according

According to the data in Table 2 (Britten et al., 1982; Lim et al., 1976; Hohmann, et al., 1991; Shoukry, et al., 1999; El-Sherif, et al., 2003 ; Shehata, 2001), the pKa1 and pKa2 values in the bipyridine as a non-leaving group were found to be 3.91 and 8.39, respectively and are lower than the corresponding values of all the PdII-diamine complexes. The [Pd(Pic)(H2O)2]2+ values are intermediate because Pic has one pyridine ring. This can be attributed to the increased positive charge on Pd atom due to the π-acceptor properties of the aromatic moiety of Pyridine ring, leading to an increase in the electrophilicity of the Pd ion and consequently to a decrease in the pKa of the coordinated water molecule. The equilibrium constant for the dimerization reactions (5) and (6) can be calculated with Eqs. 7

log10 Kdimer = log β20-1 − log β10-1 (7)

log10 Kdimer = log β20-2 − 2 log β10-1 (8)

The concentration distribution diagram for [Pd(AMBI)(H2O)2]2+ and its hydrolysed species as a representative example of hydrolysis of [Pd(diamine)(H2O)2] is shown in Fig.1. The concentration of the monohydroxo species, 10-1 and the dimeric species, 20-2 increase with increasing pH, predominating in the pH range 4.8 to 7.8 with formation percentages of ca. 44% and 54% for the monohydroxo (10-1) and dimeric species (20-2), respectively, i.e., they are the main species present in solution in the physiological pH range. A further increase in pH is accompanied by an increase in the dihydroxo species, which is the main species above a pH of ca. 11. In the high pH range the inert dihydroxo complex would be the predominant species, so that the reactivity of DNA to bind the Pd(amine) complex will considerably

**+ + 2 3**

**N**

**N**

**10 -1 100 20 -1**

**logKdimer**

**10 -1 20 -2**

**Pd**

**Pd N OH**

**OH2**

**Pd**

**OH2**

**N N**

**+ H2O**

(5)

(6)

**N**

**Pd OH <sup>N</sup>**

**Pd**

**OH**

**N**

**N**

**+**

2H2O

**N**

**+ 3+**

**Pd**

**N**

**N**

to Eq.6.

2

and 8 respectively.

decrease.

**OH2 OH**

**Pd**

**N**

**N**

**OH2**

**OH**

**OH2**

**+**

**OH2**


aen, 1,2-DAP, Me2en, Pic and BPY represent ethylenediamine, 1,2-diaminopropane, N,N′-dimethylethylenediamine, picolylamine and 2,2′-bipyridyl, respectively.

Table 2. Comparison of acid dissociation constants of some Pt and Pd-diaquo complexes.

Fig. 1. Concentration distribution of various species as a function of pH in the Pd(AMBI)(H2O)2 system at concentration of 1.25x10-3 mol-dm-3, I = 0.1 mol-dm-3 (NaNO3) and T = 25 ± 0.1 0C).

Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 87

O

O H

O

O H

O H

O

**<sup>+</sup> <sup>+</sup> <sup>+</sup>**

N H <sup>2</sup>

**-H <sup>2</sup>O**

**-H <sup>3</sup>O <sup>+</sup> pH > 4**

The complexes of general fomula [Pd(diamine)(AA)], where AA = glycine, alanine, valine, proline, phenylalanine, γ-aminobutyric acid, β-alanine and proline are investigated. The potentiometric titration curves of the [Pd(diamine)] with amino acids, lie significantly below the amino acid alone ones. This reveals that the formation of complex species occurs

The stability constants of amino acids with [Pd(amine)(H2O)2] are showed in Tables 3 and 4 (Shoukry et al., 1999; Lim, (1978); Mohamed. and Shoukry, 2001; El-Sherif et al., 2010; Shehata et al., 2008; Shehata et al., 2009; El-Sherif, 2006). The amino acids with no functional groups in the side chains generally form 1:1 complexes with [Pd(diamine)(H2O)2]2+. Their complexes are very stable, with stability constants of log β110 ~ 10-12. They coordinate through both the

The stability constant of 1:1 complex with imidazole have a smaller value than those of amino acids, further supporting that amino acids are coordinating as bidentate ligands.

amino group and the carboxylate oxygen, forming stable five-membered chelate ring.

O

N H O H <sup>2</sup>

N H <sup>2</sup>

N H

**O**

**H <sup>2</sup> N**

**O H <sup>2</sup>**

**N O H <sup>2</sup>**

**P d**

**(m etastable)**

**2 +**

**P d**

O

O

**O**

**H <sup>3</sup> N**

**OH**

**(CH <sup>2</sup> ) n O**

O H

O H

**(CH <sup>2</sup> ) n**

C H <sup>3</sup>

<sup>3</sup>CH

<sup>3</sup>CH

OH

iso-Leucine Hydroxy proline

**N**

**N**

**N**

N H <sup>2</sup>

G l Alanine ycine β -alanine

C H <sup>3</sup>

γ − -aminobutyric acid

Valine Proline

<sup>3</sup>CH

<sup>2</sup>NH

N H <sup>2</sup>

O

N H

β -phenylalanine

**O H <sup>2</sup>**

**P d**

2 **+**

**<sup>N</sup> O H <sup>2</sup>**

**N**

**N**

**N**

O H

O

O H

O

O H

<sup>3</sup>CH

**(CH <sup>2</sup> ) n**

**(CH <sup>2</sup> ) n**

**O**

**- O O**

**H <sup>3</sup> N**

**O**

Scheme 2. Coordination mode of amino acids

**P d**

**+**

through release of hydrogen ions.

**H <sup>2</sup> N**

N H <sup>2</sup>

**+**

## **3. Interactions of [Pd(diamine)(H2O)2] with bio-relevant ligands**

### **3.1 Interactions of [Pd(diamine)(H2O)2] with amino acids**

The potential donor atoms in the amino acids are (the amino-N, the carboxylato- O, as well as the other donor atoms which may be present in the side chains). Pd(II) and Pt(II) form stable complexes with the N-, O-, S-donor atoms present in amino acids, with thermodynamic preference for S- and N- donors over O-donors. Though the thermodynamic preference of the metal ion for a particular donor atom is a very important parameter in determining the choice of donor atoms, at the pH value used for the experiment, these donor atoms may be protonated. Additionally, the effect of chelate ring size may also be a factor in determining the adopted coordination mode.

### **3.1.1 Acid-base equilibria of amino acids**

All amino acids undergo two reversible proton dissociation steps in fairly well separated pH ranges, proceeding according to equilibrium reaction (9).

$$\begin{aligned} \text{pH=2-3} & \quad & \quad \text{pH=8-10} \\ \text{^\*NH\_3CH(R)COOH} & \xrightarrow{\text{^\*NH\_3CH(R)COO}} & \quad \text{NH\_2CH(R)COO}^\* & \xrightarrow{\text{^\*}N=2-} \quad \text{(9)} \\ \text{(H}\text{Li}^+) & \quad \text{(l}\text{Li}^+) & \quad \text{(l}\text{-)} \end{aligned}$$

Besides these two functional groups, most of the essential amino acids contain further functional groups in the side chains of amino acids.

### **3.1.2 Interactions of [Pd(diamine)(H2O)2] with amino acids containing no functional group in the side chain**

### **3.1.2.1 Complexes formed when only one metal coordination site is available**

Multi-NMR studies (Appleton et al., 1986; Appleton, 1997) of complexes as [M(NH3)3(H2O)]2+, M = Pt(II) or Pd(II) with amino acids (AA) showed the initial formation of metastable isomer e.g. [Pd(NH3)3(HGly-O)]2+ is formed at low pH~3, which coordinate through the carboxylate oxygen. Since glycine nitrogen (pKa ~ 9.6) is protonated under these conditions and the carboxyl group (pKa ~ 2.3) partially deprotonated, carboxylate oxygen is more available for reactions than the amine nitrogen. This complex was slowly converted to [Pd(NH3)3(HGly-N)]2+ isomer and the conversion can be slowed at lower pH. β-alanine has an additional methylene group. Its corresponding complex, Hβala-O, did not isomerise at pH = 4.5, but slowly isomerise at higher pH to the complex with β-ala-N. Appleton et al. (Appleton et al., 1986) using multinuclear NMR showed that γ–aminobutyric acid complex, [Pt(NH3)3(γaba-O)]2+, standing at pH = 10 caused only slow displacement of the carboxylatebound ligand by hydroxide. Generally, the O-bound isomer is thermodynamically more stable relative to N-bound form for Pd(II) relative to Pt(II), reflecting a kind of hardness Pd(II) compared to Pt(II) (Appleton, 1997).

### **3.1.2.2 Complexes formed when two metal coordination sites are available**

It has been well established that N,O-chelation is a characteristic coordination mode for glycine bound to palladium(II) (Freeman and Colomb, 1964). The complex-formation equilibria for amino acids may be represented as shown in scheme 2.

The potential donor atoms in the amino acids are (the amino-N, the carboxylato- O, as well as the other donor atoms which may be present in the side chains). Pd(II) and Pt(II) form stable complexes with the N-, O-, S-donor atoms present in amino acids, with thermodynamic preference for S- and N- donors over O-donors. Though the thermodynamic preference of the metal ion for a particular donor atom is a very important parameter in determining the choice of donor atoms, at the pH value used for the experiment, these donor atoms may be protonated. Additionally, the effect of chelate ring

All amino acids undergo two reversible proton dissociation steps in fairly well separated pH

Besides these two functional groups, most of the essential amino acids contain further

Multi-NMR studies (Appleton et al., 1986; Appleton, 1997) of complexes as [M(NH3)3(H2O)]2+, M = Pt(II) or Pd(II) with amino acids (AA) showed the initial formation of metastable isomer e.g. [Pd(NH3)3(HGly-O)]2+ is formed at low pH~3, which coordinate through the carboxylate oxygen. Since glycine nitrogen (pKa ~ 9.6) is protonated under these conditions and the carboxyl group (pKa ~ 2.3) partially deprotonated, carboxylate oxygen is more available for reactions than the amine nitrogen. This complex was slowly converted to [Pd(NH3)3(HGly-N)]2+ isomer and the conversion can be slowed at lower pH. β-alanine has an additional methylene group. Its corresponding complex, Hβala-O, did not isomerise at pH = 4.5, but slowly isomerise at higher pH to the complex with β-ala-N. Appleton et al. (Appleton et al., 1986) using multinuclear NMR showed that γ–aminobutyric acid complex, [Pt(NH3)3(γaba-O)]2+, standing at pH = 10 caused only slow displacement of the carboxylatebound ligand by hydroxide. Generally, the O-bound isomer is thermodynamically more stable relative to N-bound form for Pd(II) relative to Pt(II), reflecting a kind of hardness

**3.1.2 Interactions of [Pd(diamine)(H2O)2] with amino acids containing no functional** 


+NH3CH(R)COOH +NH3CH(R)COO- NH2CH(R)COO-

**3.1.2.1 Complexes formed when only one metal coordination site is available** 

**3.1.2.2 Complexes formed when two metal coordination sites are available** 

equilibria for amino acids may be represented as shown in scheme 2.

It has been well established that N,O-chelation is a characteristic coordination mode for glycine bound to palladium(II) (Freeman and Colomb, 1964). The complex-formation

( H2L+ ) ( HL<sup>±</sup> ) (L- )

**3. Interactions of [Pd(diamine)(H2O)2] with bio-relevant ligands** 

size may also be a factor in determining the adopted coordination mode.

**3.1 Interactions of [Pd(diamine)(H2O)2] with amino acids** 

**3.1.1 Acid-base equilibria of amino acids** 

ranges, proceeding according to equilibrium reaction (9).

functional groups in the side chains of amino acids.

Pd(II) compared to Pt(II) (Appleton, 1997).

**group in the side chain** 

pH=2-3 pH=8-10

Scheme 2. Coordination mode of amino acids

The complexes of general fomula [Pd(diamine)(AA)], where AA = glycine, alanine, valine, proline, phenylalanine, γ-aminobutyric acid, β-alanine and proline are investigated. The potentiometric titration curves of the [Pd(diamine)] with amino acids, lie significantly below the amino acid alone ones. This reveals that the formation of complex species occurs through release of hydrogen ions.

The stability constants of amino acids with [Pd(amine)(H2O)2] are showed in Tables 3 and 4 (Shoukry et al., 1999; Lim, (1978); Mohamed. and Shoukry, 2001; El-Sherif et al., 2010; Shehata et al., 2008; Shehata et al., 2009; El-Sherif, 2006). The amino acids with no functional groups in the side chains generally form 1:1 complexes with [Pd(diamine)(H2O)2]2+. Their complexes are very stable, with stability constants of log β110 ~ 10-12. They coordinate through both the amino group and the carboxylate oxygen, forming stable five-membered chelate ring.

The stability constant of 1:1 complex with imidazole have a smaller value than those of amino acids, further supporting that amino acids are coordinating as bidentate ligands.

Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 89


H2O


H

**N**

**Pd**

**in the side chain** 

**OH2**

**<sup>N</sup> OH2** (CH2)n

+

**N**

**N**

NH2

ionization can be calculated using Eq.10

O

OH OH

**Pd**

<sup>2</sup>NH

S

CH3

S

CH3

**+**

H3N COO

(CH2)n

Scheme 3. Interaction of [Pd(diamine)(H2O)2]2+ with S-containing amino acids.

number of proteolytic enzymes, e.g. chymotrypsin and subtilisin (Bernhard, 1986).

COO


**3.1.4 Interactions of [Pd(diamine)(H2O)2] with amino acids containing hydroxy group** 

Serine and threonine are α-amino acids with β-OH group in the side-chain. They contain only two dissociable protons in the measurable pH range (-NH3+ and -COOH), as the alcoholic hydoxy group is so weakly acidic (pKa > 14), that it does not undergo dissociation in the measurable pH range. The β-alcoholate group in the side chain of the amino acids serine and threonine have been found to play an essential role in the action mechanism of a

Serine Threonine

[Pd(diamine)(H2O)2]2+ promotes the ionization of the alcohol group of serine and threonine, the presence of the species 11-1 indicates the ionization of the OH side-chain. The pKa of the

 pKa =log β110 - log β11-1 (10) The pKa of ionization for serine and threonine are 8.26 and 7.53 with Pd(1,2-DAP), respectively (Shoukry et al.,1999). Values of 8.51 and 8.05 were obtained with Pd(en), Table 3 (Shoukry, et al., 1999; Lim, 1978; Mohamed and Shoukry, 2001; El-Sherif et al., 2010; Shehata,

**N**

**N**

**N**

**N**

<sup>3</sup>CH

**Pd**

<sup>2</sup>NH

**Pd**

OH2

S

CH3

+

S

NH2

OH OH

O

CH3

(CH2)n

(CH2)n


H3N COO

COOH

The stability constants log β110 of [Pd(AEPY)alanine]2+ (10.46) > [Pd(AEPY)-β-alanine]2+ (9.81) > [Pd(AEPY)-γ-aminobutyric acid] (7.81). This trend is attributed to extra stability of five-membered chelate rings for alanine complexes compared to six and seven-membered rings for β-alanine and γ-aminobutyric acid, respectively (Shehata et al., 2009).

### **3.1.3 Interactions of [Pd(diamine)(H2O)2] with amino acids containing sulphur atom in the side chain**

Sulphur containing amino acids (e.g. cysteine, methionine and S-methylcysteine) easily react with Pd(II) because of the great tendency of sulphur (a soft Lewis base) to form bonds with these metals (soft Lewis acids).

The interaction of Pt(II) and Pd(II) with methionine in aqueous solution primarily occurs through the sulphur atom and chelates only in a further step, binding through the amino group (Norman et al., 1992).

The stability constants of S-methyl cysteine (SMC) and methionine with Pd(II) (tables 3 and 4) are lower than those of ordinary amino acids, suggesting that they are not coordinated as glycine complexes. The stability constants with SMC are generally higher than those with methionine due to the formation of more stable 5-membered ring. The reaction between [Pd(diamine)(H2O)2]2+ and S-containing amino acids is showed in scheme 3. At low pH values the coordination site is through S-atom, slowly forming bidentate ligand followed by deprotonation of the carboxylic group.

The stability constants log β110 of [Pd(AEPY)alanine]2+ (10.46) > [Pd(AEPY)-β-alanine]2+ (9.81) > [Pd(AEPY)-γ-aminobutyric acid] (7.81). This trend is attributed to extra stability of five-membered chelate rings for alanine complexes compared to six and seven-membered

**3.1.3 Interactions of [Pd(diamine)(H2O)2] with amino acids containing sulphur atom in** 

Sulphur containing amino acids (e.g. cysteine, methionine and S-methylcysteine) easily react with Pd(II) because of the great tendency of sulphur (a soft Lewis base) to form bonds with

**NH2**

**S-Methyl cysteine Cysteine Methionine**

The interaction of Pt(II) and Pd(II) with methionine in aqueous solution primarily occurs through the sulphur atom and chelates only in a further step, binding through the amino

The stability constants of S-methyl cysteine (SMC) and methionine with Pd(II) (tables 3 and 4) are lower than those of ordinary amino acids, suggesting that they are not coordinated as glycine complexes. The stability constants with SMC are generally higher than those with methionine due to the formation of more stable 5-membered ring. The reaction between [Pd(diamine)(H2O)2]2+ and S-containing amino acids is showed in scheme 3. At low pH values the coordination site is through S-atom, slowly forming bidentate ligand followed by

**O**

**OH**

rings for β-alanine and γ-aminobutyric acid, respectively (Shehata et al., 2009).

**N**

**Pd <sup>N</sup> <sup>N</sup>**

> **N H**

> > **NH2**

**<sup>3</sup>CH OH**

**S**

**O**

**[Pd(N-N)(imidazole)2]+2 (120)**

**NH**

**N**

**N**

**the side chain** 

**S**

**<sup>3</sup>CH**

**Pd**

**<sup>N</sup> <sup>N</sup>**

**[Pd(N-N)(imidazole)]+2**

 **(110)**

these metals (soft Lewis acids).

**NH2**

group (Norman et al., 1992).

deprotonation of the carboxylic group.

**O**

**OH S**

**H**

**OH2**

**N H**

Scheme 3. Interaction of [Pd(diamine)(H2O)2]2+ with S-containing amino acids.

### **3.1.4 Interactions of [Pd(diamine)(H2O)2] with amino acids containing hydroxy group in the side chain**

Serine and threonine are α-amino acids with β-OH group in the side-chain. They contain only two dissociable protons in the measurable pH range (-NH3+ and -COOH), as the alcoholic hydoxy group is so weakly acidic (pKa > 14), that it does not undergo dissociation in the measurable pH range. The β-alcoholate group in the side chain of the amino acids serine and threonine have been found to play an essential role in the action mechanism of a number of proteolytic enzymes, e.g. chymotrypsin and subtilisin (Bernhard, 1986).

[Pd(diamine)(H2O)2]2+ promotes the ionization of the alcohol group of serine and threonine, the presence of the species 11-1 indicates the ionization of the OH side-chain. The pKa of the ionization can be calculated using Eq.10

$$\text{p} \text{K}\_{\text{a}} = \log \| \theta\_{110} \text{ -} \log \theta\_{11\text{-1}} \text{ } \tag{10}$$

The pKa of ionization for serine and threonine are 8.26 and 7.53 with Pd(1,2-DAP), respectively (Shoukry et al.,1999). Values of 8.51 and 8.05 were obtained with Pd(en), Table 3 (Shoukry, et al., 1999; Lim, 1978; Mohamed and Shoukry, 2001; El-Sherif et al., 2010; Shehata,

Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 91




13.36 13.37


13.37

19.20

10.34 2.04

10.29 2.19

13.27 20.53

19.49

16.32

10.02 - -

13.27 15.79

13.19 12.85 10.34



10.61 2.40


10.21 18.68


13.14 19.15


System p q rb Picc AEPYd AMBIe Glycine 1 1 0 9.95 10.33 9.71 Alanine 1 1 0 10.89 10.46 9.98 β-Phenylalanine 1 1 0 11.05 9.86 11.01

Valine 1 1 0 10.33 10.22 9.91 Proline 1 1 0 11.16 10.81 10.85 Iso-leucine 1 1 0 11.76 10.56 11.10 Methionine 1 1 0 9.49 9.08 9.12 S-Methylcysteine 1 1 0 10.52 9.16 10.15

Tyrosine 1 1 0 14.61 - - Tryptophan 1 1 0 10.95 - -

> 11.35 3.05

> 10.40 -

> 13.13 20.54

β-Alanine 1

Cysteine 1

Serine 1

Threonine 1

Ornithine 1

Lysine 1

Histidine 1

Histamine 1

Aspartic acid 1

Glutamic acid 1

0.1 mol dm-3 NaNO3.

1

1

1

1

1

1

1

1

1 1

1 1 1 1

1 1

1 1

1 1

1 1

1 1

1 1

1 1

1 1 1

1 1 1 0 1

0 -1

0 -1

0 -1

> 0 1

> 0 1

> 0 1

> 0 1

> 0 1 2

> 0 1 2

aN-N = aromatic diamine, bp, q and r are stoichiometric coefficients corresponding to

eAMBI=2-aminomethylbenzimidazole, data taken from reference (El-Sherif, 2006).

[Pd(diamine)(H2O)2], ligand and H+ respectively, cPic = picolylamine, data taken from reference (El-Sherif et al., 2003), dAEPY = 2-aminoethylpyridine, data taken from reference (Shehata, et al., 2009),

Table 4. Formation constants (log β110) of [Pd(diamine)(H2O)2]a with amino acid at 25 0C and

et al., 2008). This large acidification of ~ 6 log units indicates a large contribution of the OH group in the coordination process at higher pH and participation of the OH group in complex formation is not contributing significantly in the physiological pH range. The pKa value of the alcoholate group incorporated in the Pd(II)-AMBI-serine complex is 8.21 (El-Sherif, 2006). This value is lower than that of the Pd(N,N´-dimethylethylenediamine)-serine complex (8.43) (Mohamed and Shoukry, 2001). This may be due the π-acceptor property of the pyridine ring, which increases the electrophilicity of the Pd(II) ion and consequently decreases the pKa value of the coordinated alcoholate group.


aN-N = aliphatic diamine, bp, q and r are stoichiometric coefficients corresponding to [Pd(diamine)(H2O)2], ligand and H+ respectively, cen = ethylenediamine, data taken from reference (Lim, 1978**)**, dMe2en = N,N′-dimethylethylenediamine, data taken from reference (Mohamed and Shoukry, 2001), e1,2-DAP=1,2-diaminopropane, data taken from reference (Shoukry, et al., 1999); f 1,3- DAP=1,3-diaminopropane, data taken from reference (El-Sherif et al., 2010); gSMC=S-Methyl-L-cysteine, data taken from reference (Shehata, et al., 2008).

Table 3. Formation constants (log β110) of [Pd(diamine)(H2O)2]a with amino acid at 25 0C and 0.1 mol dm-3 NaNO3.



et al., 2008). This large acidification of ~ 6 log units indicates a large contribution of the OH group in the coordination process at higher pH and participation of the OH group in complex formation is not contributing significantly in the physiological pH range. The pKa value of the alcoholate group incorporated in the Pd(II)-AMBI-serine complex is 8.21 (El-Sherif, 2006). This value is lower than that of the Pd(N,N´-dimethylethylenediamine)-serine complex (8.43) (Mohamed and Shoukry, 2001). This may be due the π-acceptor property of the pyridine ring, which increases the electrophilicity of the Pd(II) ion and consequently

System p q rb enc (Me)2end 1,2-DAPe 1,3-DAPf SMCg Glycine 1 1 0 11.21 11.79 11.01 11.12 10.13 Alanine 1 1 0 11.22 10.89 11.42 11.22 10.21 β-Phenylalanine 1 1 0 - 10.09 11.06 - 9.97

Valine 1 1 0 - 11.59 11.36 - 9.82 Proline 1 1 0 12.16 11.14 11.55 - 10.62 Iso-leucine 1 1 0 - - - 10.44 Methionine 1 1 0 9.14 11.27 10.37 10.31 8.75 S-Methylcysteine 1 1 0 9.38 - 10.83 10.64 8.94

> 10.92 2.49

> 20.85

21.19

17.03

13.34

11.19

12.61

[Pd(diamine)(H2O)2], ligand and H+ respectively, cen = ethylenediamine, data taken from reference (Lim, 1978**)**, dMe2en = N,N′-dimethylethylenediamine, data taken from reference (Mohamed and Shoukry, 2001), e1,2-DAP=1,2-diaminopropane, data taken from reference (Shoukry, et al., 1999); f

DAP=1,3-diaminopropane, data taken from reference (El-Sherif et al., 2010); gSMC=S-Methyl-L-cysteine,

Table 3. Formation constants (log β110) of [Pd(diamine)(H2O)2]a with amino acid at 25 0C and



12.00 3.74

3.83

13.65 19.86

11.49 20.44

14.45 14.75 - 11.50

10.70 - - -

10.56 - 9.72


13.37

22.67

0.67

9.76 0.38

20.21

20.69

10.61 13.99

1,3-




10.57 2.11

13.22 - 10.92

13.60

decreases the pKa value of the coordinated alcoholate group.

1

1

1

1

1

1

1

1

1

1

data taken from reference (Shehata, et al., 2008).

1 1

1 1

1 1

1 1

1 1

1 1

1 1

1 1

1 1

1 1 0 1

0 1

0 -1

0 -1

> 0 1

> 0 1

> 0 1

> 0 1

> 0 1

> 0 1

aN-N = aliphatic diamine, bp, q and r are stoichiometric coefficients corresponding to

11.01 2.50

10.96 2.91

β-Alanine 1

Cysteine 1

Serine 1

Threonine 1

Ornithine 1

Lysine 1

Histidine 1

Histamine 1

Aspartic acid 1

Glutamic acid 1

0.1 mol dm-3 NaNO3.

aN-N = aromatic diamine, bp, q and r are stoichiometric coefficients corresponding to [Pd(diamine)(H2O)2], ligand and H+ respectively, cPic = picolylamine, data taken from reference (El-Sherif et al., 2003), dAEPY = 2-aminoethylpyridine, data taken from reference (Shehata, et al., 2009), eAMBI=2-aminomethylbenzimidazole, data taken from reference (El-Sherif, 2006).

Table 4. Formation constants (log β110) of [Pd(diamine)(H2O)2]a with amino acid at 25 0C and 0.1 mol dm-3 NaNO3.

Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 93

111

10-1

110

10-2

20-2

Fig. 2. Concentration distribution of various species as a function of pH in the Pd(AMBI)- Ornithine system at concemtrations of 1.25x10-3 mol-dm-3 for Pd(AMBI)2+ and ornithine,

**3.1.6 Interactions of [Pd(diamine)(H2O)2] with amino acids containing carboxylic acid** 

Aspartic and glutamic are α- amino acids having two carboxylic and one amino group as potential chelating sites. They coordinate with [Pd(diamine)(H2O)2]2+ as bidentate either by the two carboxylate groups or by the amino and one carboxylate group. The stability constant of the aspartic and glutamic acid complexes is in the range of those for amino acids. This may reveal that both amino acids coordinate by the amino and one carboxylate

G lutam ic acid Aspartic acid

HOOC

O

O H

N H <sup>2</sup>

2 3 4 5 6 7 8 9 10 11 **pH**

0

group.

COOH

10

20

30

40

50

**% Species**

60

70

80

90

100

I = 0.1 mol-dm-3 (NaNO3) and T = 25 ± 0.1 0C).

O

O H

N H <sup>2</sup>

**group in the side chain** 

100

The pKa value (9.20) for the Pd(SMC)-serine complex as reported in Table 3 is higher than the corresponding values of the Pd(N, N´ -dimethylethylenediamine)-serine (8.43) [52] and Pd(Picolylamine)-serine complexes (8.30) (El-Sherif et al., 2003). This is due to the strong *trans* labilization effect of sulfur on the coordinated alcoholate group, and in turn hinders the induced proton ionization. This will increase the pKa value of the alcoholate group in the case of the Pd(SMC)-serine complex.

### **3.1.5 Interactions of [Pd(diamine)(H2O)2] with amino acids containing amino group in the side chain**

Ornithine and lysine are α- amino acids having an extra terminal- amino group. They coordinate with [Pd(diamine)(H2O)2]2+ as bidentate either by the two amino groups (N,Ndonor set) or glycine-like, through the α-amino and carboxylate groups (N,O-donor set). The way of coordination is depending on three factors:


The stability constant of the Pd(DAP)-Ornithine complex (logβ110 = 13.65) is higher than those of α- amino acids. This may indicate that ornithine most likely chelates by the two amino groups at higher pH, this is being supported by the great affinity of palladium to nitrogen donor centres. Unlike ornithine, The stability constant of the Pd(DAP)-lysine complex (logβ110 = 11.49) is extremely fair with those of α- amino acids. This may indicate that lysine most likely chelates by the amino and carboxylate groups (glycine-like), because chelates formed through binding with the two amino groups will form unstable eightmembered ring. The concentration distribution diagram of [Pd(AMBI)(ornithine)] complex is given in Fig. 2. It clearly shows that lysine starts to form the protonated species (111) at low pH and predominates between pH (4-9) and attains maximum concentration of ~ 98%, i.e it is the main complex species in physiological pH range. The complex species (110) predominates after pH ~ 9. The (10-1) hydrolysed species is present in very low concentration and the (10-2) species starts to form at higher pH, ca. ~ 10. Therefore, in the physiological pH range the OH- ion doesn't compete with ornithine in the reaction with the palladium (II) complexes.

The pKa value (9.20) for the Pd(SMC)-serine complex as reported in Table 3 is higher than

Pd(Picolylamine)-serine complexes (8.30) (El-Sherif et al., 2003). This is due to the strong *trans* labilization effect of sulfur on the coordinated alcoholate group, and in turn hinders the induced proton ionization. This will increase the pKa value of the alcoholate group in the

**3.1.5 Interactions of [Pd(diamine)(H2O)2] with amino acids containing amino group in** 

Ornithine and lysine are α- amino acids having an extra terminal- amino group. They coordinate with [Pd(diamine)(H2O)2]2+ as bidentate either by the two amino groups (N,Ndonor set) or glycine-like, through the α-amino and carboxylate groups (N,O-donor set).

**Ornithine Lysine**

The stability constant of the Pd(DAP)-Ornithine complex (logβ110 = 13.65) is higher than those of α- amino acids. This may indicate that ornithine most likely chelates by the two amino groups at higher pH, this is being supported by the great affinity of palladium to nitrogen donor centres. Unlike ornithine, The stability constant of the Pd(DAP)-lysine complex (logβ110 = 11.49) is extremely fair with those of α- amino acids. This may indicate that lysine most likely chelates by the amino and carboxylate groups (glycine-like), because chelates formed through binding with the two amino groups will form unstable eightmembered ring. The concentration distribution diagram of [Pd(AMBI)(ornithine)] complex is given in Fig. 2. It clearly shows that lysine starts to form the protonated species (111) at low pH and predominates between pH (4-9) and attains maximum concentration of ~ 98%, i.e it is the main complex species in physiological pH range. The complex species (110) predominates after pH ~ 9. The (10-1) hydrolysed species is present in very low concentration and the (10-2) species starts to form at higher pH, ca. ~ 10. Therefore, in the physiological pH range the OH- ion doesn't compete with ornithine in the reaction with the

**<sup>2</sup>NH**


**NH2**

**O**

**OH**

the corresponding values of the Pd(N, N´

The way of coordination is depending on three factors:

**O**

**NH2**

**OH**

case of the Pd(SMC)-serine complex.

**the side chain** 

**<sup>2</sup>NH**

1. The chelate ring size. 2. The steric effects. 3. The pH of the solution.

palladium (II) complexes.

Fig. 2. Concentration distribution of various species as a function of pH in the Pd(AMBI)- Ornithine system at concemtrations of 1.25x10-3 mol-dm-3 for Pd(AMBI)2+ and ornithine, I = 0.1 mol-dm-3 (NaNO3) and T = 25 ± 0.1 0C).

### **3.1.6 Interactions of [Pd(diamine)(H2O)2] with amino acids containing carboxylic acid group in the side chain**

Aspartic and glutamic are α- amino acids having two carboxylic and one amino group as potential chelating sites. They coordinate with [Pd(diamine)(H2O)2]2+ as bidentate either by the two carboxylate groups or by the amino and one carboxylate group. The stability constant of the aspartic and glutamic acid complexes is in the range of those for amino acids. This may reveal that both amino acids coordinate by the amino and one carboxylate group.

G lutam ic acid Aspartic acid

Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 95

Amide bonds or groups provide the linkage between adjacent amino acids. A protein is composed of a chain of (n) amino acids contains (n-1) peptide (amide) bonds in the backbone. The tetrahedral amino nitrogen in an amino acid with pKa ~ 9.7 loses its basicity upon reaction to give trigonal nitrogen in an amide bond. Amide groups are planar due to 40% double-bond character in the C-N bond, and the *trans* form is strongly favoured (Sigel

**N**

**+**

**R**

**COOH**

**O NH2**

**2 NH**

**H**

**R**

**O**

**\_**

**. ..**

**3.2 Interactions of [Pd(diamine)(H2O)2] with peptides** 

**O**

**.. .**

**R**

**N**

**..**

**R**

**. .. .**

**60% 40%** The presence of the peptide linkage decreases the basicity of the amino group and the

An amide group offers two potential binding atoms, the oxygen and nitrogen atoms. Throughout most of pH range, in the absence of metal ions, the amide group is neutral. It is being a very weak acid for proton loss from the trigonal nitrogen to give a negatively charged species. This very weak acidity makes quantitative equilibrium measurements very difficult. For acetamide it was reported that pKa = 15.1, and the pKa for glycylglycinate is

There are at least four donor groups in the dipeptide (Sigel and Martin, 1982) (amino-N, carboxylate-O, amide-N and carbonyl-O), all are capable of metal ion coordination. Because of the neutrality of the amide group, the terminal amino and carboxylate groups are the most effective binding sites for metal ions in peptides. The coordination of amide group can occur after deprotonation. Other groups may additionally be present in the side chains R1

**H**

**C**

**R**

**2**

**H COO-**

**N H**

**glycinamide glycylglycine glutamine**

Peptides form complexes with stochiometric coefficients 110 and 11-1 according to Scheme (4). Peptides with Pd(II) are known for promoting ionization of the peptide linkage with pKa

**O <sup>2</sup>**

**NH COOH**

**C**

**R**

**1**

**NH3 +** **<sup>H</sup> <sup>N</sup>**

**O**

**NH**

**2**

**H**

and Martin, 1982).

14.1.

and R2.

**NH**

**O <sup>2</sup>**

value calculated by Eq.10.

acidity of the carboxylic group.

### **3.1.7 Interactions of [Pd(diamine)(H2O)2] with amino acids containing imidazole group in the side chain**

The protonated histidine contains three dissociable protons, which can dissociate in the following sequence: carboxylic acid, imidazolium N(3)-H and side chain NH3+. The imidazole N(1)-H is very weekly acidic (pKa = 14.4), and thus it does not dissociate in the measurable pH range (Burger, 1990).

Histidine has three binding sites, provided imidazole, amino and carboxylate groups. It coordinates with [Pd(diamine)(H2O)2]2+ as bidentate either by the α-amino group and imidazole groups (N,N-donor set) or glycine-like, through the α-amino and carboxylate groups (N,O-donor set).

The formation constant value of the (110) species is in fair agreement with that of histamine complex, but higher than those of α-amino acids. This indicates that histidine interacts with [Pd(diamine)(H2O)2] in the same way as histamine does i.e. through the amino and imidazole.

Histidine has shown to form both protonated (111) and deprotonated (110) complex species. The acid dissociation constant of the protonated species is given by the following Eq. (11).

$$\mathbf{p}\mathbf{K}^{\text{H}} = \log \| \boldsymbol{\beta}\_{111} \text{ - } \log \boldsymbol{\beta}\_{110} \tag{11}$$

In the reaction with [Pd(AEPY)(H2O)2]2+ species only two of the above three binding sites are involved in complex formation. The stability constants of the histidine complexes (logβ110 = 13.37) is higher than that of histamine (logβ110 = 12.85) by 0.52 log β units. Moreover, it is higher than those of amino acids (e.g. logβ110 of glycine = 10.33) by 3.04 log units, indicating that both kinds of chelation are involved glycine-like (N,O) at lower pH and histamine-like (N,N) at higher pH. In general, histidine forms more stable complex than histamine due to the negative charge of histidine compared to neutral histamine. Furthermore, palladium may form back bonding to the π–system of imidazole ring, which brings more stable complexes.

### **3.2 Interactions of [Pd(diamine)(H2O)2] with peptides**

94 Stoichiometry and Research – The Importance of Quantity in Biomedicine

**3.1.7 Interactions of [Pd(diamine)(H2O)2] with amino acids containing imidazole group** 

The protonated histidine contains three dissociable protons, which can dissociate in the following sequence: carboxylic acid, imidazolium N(3)-H and side chain NH3+. The imidazole N(1)-H is very weekly acidic (pKa = 14.4), and thus it does not dissociate in the

Histidine has three binding sites, provided imidazole, amino and carboxylate groups. It coordinates with [Pd(diamine)(H2O)2]2+ as bidentate either by the α-amino group and imidazole groups (N,N-donor set) or glycine-like, through the α-amino and carboxylate

NH

(Glycine-like) (Histamine-like) The formation constant value of the (110) species is in fair agreement with that of histamine complex, but higher than those of α-amino acids. This indicates that histidine interacts with [Pd(diamine)(H2O)2] in the same way as histamine does i.e. through the amino and

Histidine has shown to form both protonated (111) and deprotonated (110) complex species. The acid dissociation constant of the protonated species is given by the following Eq. (11).

 pKH = log β111 - log β110 (11) In the reaction with [Pd(AEPY)(H2O)2]2+ species only two of the above three binding sites are involved in complex formation. The stability constants of the histidine complexes (logβ110 = 13.37) is higher than that of histamine (logβ110 = 12.85) by 0.52 log β units. Moreover, it is higher than those of amino acids (e.g. logβ110 of glycine = 10.33) by 3.04 log units, indicating that both kinds of chelation are involved glycine-like (N,O) at lower pH and histamine-like (N,N) at higher pH. In general, histidine forms more stable complex than histamine due to the negative charge of histidine compared to neutral histamine. Furthermore, palladium may form back bonding to the π–system of imidazole ring, which

2 3

Histidine Histamine

N N H2

**N** N **Pd**

**N**

O

NH2

N H O H

**in the side chain** 

groups (N,O-donor set).

NH

**N**

**N**

brings more stable complexes.

imidazole.

measurable pH range (Burger, 1990).

N

**O**

**Pd**

**NH2**

N H2

**O**

**CH2**

<sup>1</sup> <sup>3</sup> <sup>2</sup> <sup>1</sup>

Amide bonds or groups provide the linkage between adjacent amino acids. A protein is composed of a chain of (n) amino acids contains (n-1) peptide (amide) bonds in the backbone. The tetrahedral amino nitrogen in an amino acid with pKa ~ 9.7 loses its basicity upon reaction to give trigonal nitrogen in an amide bond. Amide groups are planar due to 40% double-bond character in the C-N bond, and the *trans* form is strongly favoured (Sigel and Martin, 1982).

The presence of the peptide linkage decreases the basicity of the amino group and the acidity of the carboxylic group.

An amide group offers two potential binding atoms, the oxygen and nitrogen atoms. Throughout most of pH range, in the absence of metal ions, the amide group is neutral. It is being a very weak acid for proton loss from the trigonal nitrogen to give a negatively charged species. This very weak acidity makes quantitative equilibrium measurements very difficult. For acetamide it was reported that pKa = 15.1, and the pKa for glycylglycinate is 14.1.

There are at least four donor groups in the dipeptide (Sigel and Martin, 1982) (amino-N, carboxylate-O, amide-N and carbonyl-O), all are capable of metal ion coordination. Because of the neutrality of the amide group, the terminal amino and carboxylate groups are the most effective binding sites for metal ions in peptides. The coordination of amide group can occur after deprotonation. Other groups may additionally be present in the side chains R1 and R2.

Peptides form complexes with stochiometric coefficients 110 and 11-1 according to Scheme (4). Peptides with Pd(II) are known for promoting ionization of the peptide linkage with pKa value calculated by Eq.10.

Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 97

7.40 3.03

10.73 5.82

0.84

7.75 2.69

12.31 4.71

0.01



[Pd(diamine)(H2O)2], ligand and H+ respectively, cen = ethylenediamine, data taken from reference (Lim, 1978), dMe2en = N,N′-dimethylethylenediamine, data taken from reference (Mohamed and Shoukry 2001), e1,2-DAP=1,2-diaminopropane, data taken from reference (Shoukry et al., 1999**)**; f

DAP=1,3-diaminopropane, data taken from reference (El-Sherif et al., 2010); gSMC=S-Methyl-L-cysteine,

Table 5. Formation constants (log β110) of [Pd(diamine)(H2O)2]a with peptides at 25 0C and

9.30 5.73

10.02 0.36

> 8.31 3.08

> 8.29 4.37

10.06 2.65

> 8.22 3.06

> 7.73 2.39

> 7.73 2.39

[Pd(diamine)(H2O)2], ligand and H+ respectively, cPic = picolylamine, data taken from reference (El-Sherif, 2003), dAEPY = 2-aminoethylpyridine, data taken from reference (Shehata et al., 2009), eAMBI=2-

Table 6. Formation constants (log β110) of [Pd(diamine)(H2O)2]a with peptides at 25 0C and

System p q rb Picc AEPYd AMBIe

8.58 5.35

11.02 2.12

> 9.41 6.02

12.79 6.38

> 7.73 3.30


8.01 4.16

9.11 0.47

8.20 3.56

9.42 1.90

7.75 2.18 SMCg

7.56 -3.38

8.73 1.08

7.73 2.61

2.08

1.98

2.00

1,3-



8.63 5.23

9.24 -0.32

8.01 4.24

8.81 -0.42





8.63 5.23

9.24 -0.32


8.01 4.24

System p q rb enc (Me)2end 1,2-DAPe 1,3-DAPf

8.64 2.47

10.76 9.03

> 9.60 3.76

10.46 6.46

aN-N = aliphatic diamine bp, q and r are stoichiometric coefficients corresponding to

Glycinamide 1

Glutamine 1

Glycylalanine 1

Glycylglycine 1

Asparagine 1

Glycylleucine 1

Glycylvaline 1

0.1 mol dm-3 NaNO3.

Glycinamide 1

Glutamine 1

Glycylalanine 1

Glycylglycine 1

Asparagine 1

Glycylleucine 1

Glycylvaline 1

Leucylalanine 1

0.1 mol dm-3 NaNO3.

1

1

1

1

1

1

1

data taken from reference (Shehata et al., 2008).

1

1

1

1

1

1

1

1

1 1

1 1

1 1

1 1

1 1

1 1

1 1

1 1

0 -1

0 -1

0 -1

0 -1

0 -1

0 -1

0 -1

0 -1

aminomethylbenzimidazole, data taken from reference (El-Sherif, 2006). .

aN-N = aromatic diamine, bp, q and r are stoichiometric coefficients corresponding to

1 1

1 1

1 1

1 1

1 1

1 1

1 1

0 -1

0 -1

0 -1

0 -1

0 -1

0 -1

0 -1

It is found that, most metal ions form complexes with peptides by coordinating with carbonyl oxygen of the peptide group. Only certain specific metal ions are able to promote the deprotonation of the peptide nitrogen and become coordinated with it. Among these metals are Co(II), Ni(II), Cu(II) and Pd(II). The affinity for nitrogen bonding sites over oxygen bonding sites increases from cobalt to palladium corresponding to the stability of their deprotonated complexes. This is consistent with the idea that the deprotonated amide nitrogen is a "soft" base.

It is clear that, the stability of the complex with glycylglycine (log β110) is generally higher than glycinamide as indicated in Tables 5 and 6 (Shoukry et al., 1999 ; El-Sherif, 2003 ; Lim, 1978 ; Mohamed and Shoukry 2001 ; El-Sherif et al., 2010 ; Shehata et al., 2008 ; Shehata et al., 2009; El-Sherif, 2006) due to the negative charge of glycylglycinate compared to neutral glycinamide. The electrostatic interaction between dipositively charged palladium complex and the negatively charged glycylglycine would result in a general free formation energy lowering.

Scheme 4. Mode of coordination of [Pd(diamine)(H2O)2] with peptides

The pKa value for glycinamide complex is lower than those for other peptides. This is due to the bulky substituent group on the other peptides that may hinder the structural changes when going from species 110 to 11-1 (peptide ionization). Glutamine complex has the highest stability, probably due to the presence of α-NH2 that can coordinate first (glycinelike). The α-NH2 of glutamine is more basic than those of other peptides resulting in more stable complexes compared to other peptides. The concentration distribution diagrams of Pd(diamine)-peptide complexes indicate that all peptides form the complex species (110) at low pH with the species (11-1) as the main product at higher pH.


It is found that, most metal ions form complexes with peptides by coordinating with carbonyl oxygen of the peptide group. Only certain specific metal ions are able to promote the deprotonation of the peptide nitrogen and become coordinated with it. Among these metals are Co(II), Ni(II), Cu(II) and Pd(II). The affinity for nitrogen bonding sites over oxygen bonding sites increases from cobalt to palladium corresponding to the stability of their deprotonated complexes. This is consistent with the idea that the deprotonated amide

It is clear that, the stability of the complex with glycylglycine (log β110) is generally higher than glycinamide as indicated in Tables 5 and 6 (Shoukry et al., 1999 ; El-Sherif, 2003 ; Lim, 1978 ; Mohamed and Shoukry 2001 ; El-Sherif et al., 2010 ; Shehata et al., 2008 ; Shehata et al., 2009; El-Sherif, 2006) due to the negative charge of glycylglycinate compared to neutral glycinamide. The electrostatic interaction between dipositively charged palladium complex and the negatively charged glycylglycine would result in a general free formation energy lowering.

K


H + +

The pKa value for glycinamide complex is lower than those for other peptides. This is due to the bulky substituent group on the other peptides that may hinder the structural changes when going from species 110 to 11-1 (peptide ionization). Glutamine complex has the highest stability, probably due to the presence of α-NH2 that can coordinate first (glycinelike). The α-NH2 of glutamine is more basic than those of other peptides resulting in more stable complexes compared to other peptides. The concentration distribution diagrams of Pd(diamine)-peptide complexes indicate that all peptides form the complex species (110) at

O

N H2

(110)

CH2

COO -

pKa

N

N H2

(11-1)

**Pd**

CH2

O

**Pd**

**N**

**N**

**N**

**N**

CH2

N H

CH2

CO2 - +

NH

Glycylglycinate

Scheme 4. Mode of coordination of [Pd(diamine)(H2O)2] with peptides

low pH with the species (11-1) as the main product at higher pH.

CH2 COO-

O

+

2+

CH2 NH2

nitrogen is a "soft" base.

**N**

**N**

**Pd**

(100)

OH2

OH2


aN-N = aliphatic diamine bp, q and r are stoichiometric coefficients corresponding to [Pd(diamine)(H2O)2], ligand and H+ respectively, cen = ethylenediamine, data taken from reference (Lim, 1978), dMe2en = N,N′-dimethylethylenediamine, data taken from reference (Mohamed and Shoukry 2001), e1,2-DAP=1,2-diaminopropane, data taken from reference (Shoukry et al., 1999**)**; f 1,3- DAP=1,3-diaminopropane, data taken from reference (El-Sherif et al., 2010); gSMC=S-Methyl-L-cysteine, data taken from reference (Shehata et al., 2008).

Table 5. Formation constants (log β110) of [Pd(diamine)(H2O)2]a with peptides at 25 0C and 0.1 mol dm-3 NaNO3.


aN-N = aromatic diamine, bp, q and r are stoichiometric coefficients corresponding to

[Pd(diamine)(H2O)2], ligand and H+ respectively, cPic = picolylamine, data taken from reference (El-Sherif, 2003), dAEPY = 2-aminoethylpyridine, data taken from reference (Shehata et al., 2009), eAMBI=2 aminomethylbenzimidazole, data taken from reference (El-Sherif, 2006). .

Table 6. Formation constants (log β110) of [Pd(diamine)(H2O)2]a with peptides at 25 0C and 0.1 mol dm-3 NaNO3.

Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 99

**N**

**9**

**A denosine m onophosphate**

The nucleic acids are polymers built up from nucleotides via phosphodiester bond formation

The sequence of the nucleotides is extremely important as it constitutes the genetic code in DNA. Different nucleotides vary in the nature of the purine and pyrimidine bases (Hay, 1985).

The accepted models for DNA complex formation with [Pd(diamine)(H2O)2]2+ are consistent with the formation of 1:1 and 1:2 complexes, as shown in Tables 7 and 8 (Shoukry et al.,1999; El-Sherif et al., 2003; Lim, 1978; Mohamed and Shoukry, 2001; El-Sherif, et al., 2010; Shehata, et al., 2008); Shehata, et al., 2009; El-Sherif, 2006). Generally, from Table 8, the complexes with DNA constituents are more stable (higher log β values) with bipyridine than with all other amine ligands. They are probably stabilized by intramolecular stacking between the bipyridine aromatic ring and the purine rings (Fisher and Sigel, 1980). The stability of the corresponding complexes with picolylamine has intermediate log β values, probably

The pyrimidines, uracil and thymine have only basic nitrogen donor atoms (N3-C4O group).

O

4

N H

1 3 2

Uracil

Thymine Inosine

<sup>O</sup> <sup>O</sup>

NH

**3.3.2 Ternary complexes involving DNA constituents and [Pd(diamine)(H2O)2]** 

**2 ´**

**N**

**3**

**Sugar**


because in this case there is only one pyridine ring.

**N H <sup>2</sup>**

**N**

**1**

**B ase**

**N U C L E O S I D E**

between the 3´

NH

O

O

4

CH3

N H

1 3 2

**N**

**7**

**O**

**O H O H**

**5 ´ 1 ´**

**4 ´**

**3 ´**

**N U C L E O T ID E**

**O P O O**

**O**


OH OH

OH N

N

9

N

1 7 3

O

6

NH

Fitting the potentiometric data for the Pd(DAP)-glutathione system indicated the formation of complex species with the stoichiometric coefficients 110 and 111. Glutathione has various binding sites, namely oxygen atom of carboxylic group, nitrogen atom of amino group and sulphur atom of sulfhydryl group. The stability constant of the (110) complex (log β = 15.92) is higher than the ones of α-amino acids (log β[Pd(DAP)(glycine)] = 11.12). This indicates that glutathione interacts with Pd(II) ion by the amino and deprotonated SH groups and not by the amino and caboxylate group like simple α-amino acids. This is in good agreement with the fact that Pd(II) has a high affinity for S-donor ligands. The concentration distribution diagram of [Pd(DAP)(glutathione)] given in Fig. 3, shows the formation of the protonated complex 111 with a formation degree of 81% at pH 3.1. At pH 6, the complex species (110) predominates with a concentration of 99 % i.e. the reaction of [Pd(DAP)]2+ goes to completion in the physiological pH range. This may suggest that GSH will compete with DNA for the reaction with the Pd(II) complex.

Fig. 3. Concentration distribution of various species as a function of pH in the Pd(DAP)- Glutathione system at concentrations of 1.25x10-3 mol-dm-3 for Pd(DAP)2+ and glutathione, I = 0.1 mol-dm-3 (NaNO3) and T = 25 ± 0.1 0C).

### **3.3 Interactions of [Pd(diamine)(H2O)2] with DNA constituents**

### **3.3.1 Nucleosides, nucleotides and nucleic acid**

Nucleosides are composed of a purine or pyrimidine base attached to the sugar ribose via the N-9 and C-1 atoms, respectively. In nucleotides, the sugar is linked to a phosphate group.

Fitting the potentiometric data for the Pd(DAP)-glutathione system indicated the formation of complex species with the stoichiometric coefficients 110 and 111. Glutathione has various binding sites, namely oxygen atom of carboxylic group, nitrogen atom of amino group and sulphur atom of sulfhydryl group. The stability constant of the (110) complex (log β = 15.92) is higher than the ones of α-amino acids (log β[Pd(DAP)(glycine)] = 11.12). This indicates that glutathione interacts with Pd(II) ion by the amino and deprotonated SH groups and not by the amino and caboxylate group like simple α-amino acids. This is in good agreement with the fact that Pd(II) has a high affinity for S-donor ligands. The concentration distribution diagram of [Pd(DAP)(glutathione)] given in Fig. 3, shows the formation of the protonated complex 111 with a formation degree of 81% at pH 3.1. At pH 6, the complex species (110) predominates with a concentration of 99 % i.e. the reaction of [Pd(DAP)]2+ goes to completion in the physiological pH range. This may suggest that GSH will compete with

**12345678 pH**

Fig. 3. Concentration distribution of various species as a function of pH in the Pd(DAP)- Glutathione system at concentrations of 1.25x10-3 mol-dm-3 for Pd(DAP)2+ and glutathione,

Nucleosides are composed of a purine or pyrimidine base attached to the sugar ribose via the N-9 and C-1 atoms, respectively. In nucleotides, the sugar is linked to a phosphate

**110**

DNA for the reaction with the Pd(II) complex.

I = 0.1 mol-dm-3 (NaNO3) and T = 25 ± 0.1 0C).

**3.3.1 Nucleosides, nucleotides and nucleic acid** 

**3.3 Interactions of [Pd(diamine)(H2O)2] with DNA constituents** 

**111**

**100**

group.

**% Species**

**A denosine m onophosphate**

The nucleic acids are polymers built up from nucleotides via phosphodiester bond formation between the 3´ -OH group of one nucleotide and the 5´ -OH group of the adjacent nucleotide. The sequence of the nucleotides is extremely important as it constitutes the genetic code in DNA. Different nucleotides vary in the nature of the purine and pyrimidine bases (Hay, 1985).

### **3.3.2 Ternary complexes involving DNA constituents and [Pd(diamine)(H2O)2]**

The accepted models for DNA complex formation with [Pd(diamine)(H2O)2]2+ are consistent with the formation of 1:1 and 1:2 complexes, as shown in Tables 7 and 8 (Shoukry et al.,1999; El-Sherif et al., 2003; Lim, 1978; Mohamed and Shoukry, 2001; El-Sherif, et al., 2010; Shehata, et al., 2008); Shehata, et al., 2009; El-Sherif, 2006). Generally, from Table 8, the complexes with DNA constituents are more stable (higher log β values) with bipyridine than with all other amine ligands. They are probably stabilized by intramolecular stacking between the bipyridine aromatic ring and the purine rings (Fisher and Sigel, 1980). The stability of the corresponding complexes with picolylamine has intermediate log β values, probably because in this case there is only one pyridine ring.

The pyrimidines, uracil and thymine have only basic nitrogen donor atoms (N3-C4O group).


Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 101

8.03 14.47 - -

8.25 13.72

8.91 10.86

5.25

19.03

14.05 18.56 - -

11.64 11.89 - - -

9.18 16.35 13.93 - - -

9.23 13.44 15.16 - - -

8.57 10.86 - -







10.96 17.17 13.50 22.15

16.88 13.29 22.65


11.95 16.59 15.97 25.76 30.25

9.71 14.89 12.55 20.11 25.37 -

10.17 14.80 16.65 21.49 28.50 20.98

11.95 16.59 15.97 25.76 30.25


9.08 12.98

9.43 13.11

12.94

12.93


10.02 - 12.06 - - 14.40

10.05 - 15.81 - - 17.79






System p q rb Picc AEPYd BPYe AMBIf

9.17 15.98

9.00 14.96

8.96 15.62

9.17 15.21

10.42 - 16.46 - - 18.73

10.83 - 17.35 - - 21.01

Uracil 1

Uridine 1

Thymine 1

Thymidine 1

Cytosine 1

Adenosine 1

Guanosine 1

Adenine 1

Inosine 1

IMP 1

GMP 1

CMP 1

1 1 1

1 1 1

1

1

1 1

1

1

data taken from reference (El-Sherif, 2006)

25 0C and 0.1 mol dm-3 NaNO3.

1 2

1 2

1 2 1

1 2

1 2

0 0

0 0

0 0 1

0 0

0 0

aN-N = aromatic diamine, bp, q and r are stoichiometric coefficients corresponding to

[Pd(diamine)(H2O)2], ligand and H+ respectively, cPic = picolylamine, data taken from reference

(El-Sherif, 2003), dAEPY = 2-aminoethylpyridine, data taken from reference (Shehata et al., 2009), eBPY = 2,2′-bipyridyl, data taken from reference (Shehata, 2001), eAMBI=2-aminomethylbenzimidazole,

Table 8. Formation constants (log β110) of [Pd(diamine)(H2O)2]a with DNA constituents at


aN-N = aliphatic diamine bp, q and r are stoichiometric coefficients corresponding to [Pd(diamine)(H2O)2], ligand and H+ respectively, cen = ethylenediamine, data taken from reference (Lim, 1978), dMe2en = N,N′-dimethylethylenediamine, data taken from reference (Mohamed and Shoukry 2001), e1,2-DAP=1,2-diaminopropane, data taken from reference (Shoukry et al., 1999); f 1,3-DAP=1,3-diaminopropane, data taken from reference (El-Sherif, et al., 2010); gSMC=S-Methyl-Lcysteine, data taken from reference (Shehata et al., 2008).

Table 7. Formation constants (log β110) of [Pd(diamine)(H2O)2]a with DNA constituents at 25 0C and 0.1 mol dm-3 NaNO3.


8.35 14.88

8.70 14.37

15.14

8.75 14.53

8.03 12.40 12.74

8.76 15.26 18.50 12.31 21.69 28.45


[Pd(diamine)(H2O)2], ligand and H+ respectively, cen = ethylenediamine, data taken from reference (Lim, 1978), dMe2en = N,N′-dimethylethylenediamine, data taken from reference (Mohamed and Shoukry 2001), e1,2-DAP=1,2-diaminopropane, data taken from reference (Shoukry et al., 1999); f 1,3-DAP=1,3-diaminopropane, data taken from reference (El-Sherif, et al., 2010); gSMC=S-Methyl-L-

Table 7. Formation constants (log β110) of [Pd(diamine)(H2O)2]a with DNA constituents at



DAPe

8.74 15.43

8.90 15.80

8.92 14.84








1,3- DAPf

8.61 14.76

9.02 15.65

9.69


11.14 - 9.14


SMCg

8.18 12.40

12.21

8.63 13.28

14.10

8.54

8.44

6.81 10.16 10.84

13.62 14.17

10.60 - 10.51 - -

11.35 14.52

11.96 12.57

7.67 11.59

13.53 13.84


System p q rb enc (Me)2end 1,2-

8.35 14.88

8.70 14.37

8.84 14.69

6.83 - 11.26

8.76 15.26 18.50 12.31

0 0

0 0

0 0

0 0

0 0

0 0

0 1 0

0 1 0

0 1 0

0 1 0

0 1 0

0 1 0

aN-N = aliphatic diamine bp, q and r are stoichiometric coefficients corresponding to

Uracil 1

Uridine 1

Thymine 1

Thymidine 1

Cytosine 1

Cytidine 1

Inosine 1

UMP 1

IMP 1

GMP 1

Adenine 1

CMP 1

TMP 1

25 0C and 0.1 mol dm-3 NaNO3.

1

1

1

1

1

1

1 1

1 1

1 1

1 1

1 1

1 1 1 2

1 2

1 2

1 2

1 2

1 2

1 1 2

1 1 2

1 1 2

1 1 2

1 1 2

1 1 2

cysteine, data taken from reference (Shehata et al., 2008).


aN-N = aromatic diamine, bp, q and r are stoichiometric coefficients corresponding to [Pd(diamine)(H2O)2], ligand and H+ respectively, cPic = picolylamine, data taken from reference (El-Sherif, 2003), dAEPY = 2-aminoethylpyridine, data taken from reference (Shehata et al., 2009), eBPY = 2,2′-bipyridyl, data taken from reference (Shehata, 2001), eAMBI=2-aminomethylbenzimidazole, data taken from reference (El-Sherif, 2006)

Table 8. Formation constants (log β110) of [Pd(diamine)(H2O)2]a with DNA constituents at 25 0C and 0.1 mol dm-3 NaNO3.

Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 103

complexes. For Pd(AMBI)-complexes, the pKa values of the protonated species of the IMP complex (112) are 1.98 (log β112 - log β111) and 5.76 (log β111 - log β110). The corresponding values for GMP are 3.21 and 6.17. The former pKa value for IMP and GMP corresponds to the N1H group while the second is assignable to the -PO2(OH) group. The N1H groups were acidified upon complex formation by 7.23 (9.21-1.98) and 6.07 (9.28-3.21) pK units for IMP and GMP, respectively. Acidification of the N1H group upon complex formation is consistent with previous reports for IMP and GMP complexes (Sigel et al. 1994). In the fully protonated species (112), the two protons bound to N1 and the phosphate groups, exist at pH ~ 3 or lower. In the (111) complex species, which reaches its maximum concentration of 88 % at pH ~ 4.8 (Fig. 4), the single proton binds to phosphate. Therefore, monoprotonated species (111) is an N1-coordinated. The stability constant difference between the (112) and (111) complexes is 3.21, due to the pKa of the N1 deprotonation process. The phosphate group was not acidified upon complex formation since it is too far from the coordination center. A proposed coordination process of Pd(AMBI)2+ with 5′

**2 3 4 5 6 7 8 9 10 11 12 pH**

Fig. 4. Concentration distribution of various species as a function of pH in the Pd(AMBI)-5´-

**110**

**1-01**

GMP system at concentrations of 1.25x10-3 mol-dm-3 for Pd(AMBI)2+ and 5´

GMP is reported in Scheme 5.

**111**

**0**

**10**

**20**

**30**

**40**

**50**

**% Species**

**60**

**112**

**100**

mol-dm-3 (NaNO3) and T = 25 ± 0.1 0C).

**70**

**80**

**90**

**100**


**10-2**


The thymine complex is more stable than the uracil one, most probably owing to the high basicity of the N3 group of thymine resulting from the extra electron donating methyl group. As a result of the high pKa values of pyrimidines (pKa ≈ 9) and the fact that they are monodentates, the complexes are formed only above pH 6, supporting the view that the negatively charged nitrogen donors of pyrimidine bases are important binding sites in the neutral and slightly basic pH ranges. The purines like inosine have two metal ion binding centres N1 and N7 nitrogens. Inosine can be protonated at N7 forming a (N1H-N7H) monocation. The pKa of N1H is 8.43 (El-Sherif, 2006) and the pKa of N7H is 1.2 (Martin, 1985**)**. It was reported that, in the acidic pH range, N1 remained protonated, while the metal ion is coordinated to N7 i.e. these N-donors are pH dependent binding sites and there is a gradual change from N7- binding to N1-binding with increase of pH (Maskos, 1985). The results showed that inosine form the complexes 110 and 111. The speciation diagrams of Pd(diamine)-inosine complexes indicated that the species 111 is formed in acidic pH range and it corresponds to the N7 coordinated complex, while N1 nitrogen is in protonated form. Inosine is slightly more acidic than the pyrimidine bases, a property which can be related to the existence of a higher number of resonance forms for the inosine anion. Based on the existing data, uracil and thymine ligate in the deprotonated form through the N3 atom.

Several solid Pd(II) complexes of guanosine and inosine have suggested to exhibit N7-O6 chelation (Pneumatikakis, 1984 ; Pneumatikakis et al., 1988). Also, according to *ab initio* SCF calculations, N7-O6 chelation is the energetically favored bonding mode (Del Bene, 1984) ; Anwader et al., 1987).

The IR spectrum of [Pd(BPY)(Inosine)]NO3 showed a shift of the ν(C=O) stretching vibration from 1700 cm-1 and 1676 cm-1 in the free inosine (which corresponds to keto-enol forms) to 1639 in the complex (Shehata, 2001). This clearly indicates the involvement of C=O in coordination. At higher pH, the N1H is deprotonated and the negative charge on N1 resonates with C=O, increasing the negative charge on the oxygen atom. This is in consistent with the large acidification of the N1H.

Inosine-5′ -monophosphate (5′ -IMP) forms a stronger complex with [Pd(diamine)(H2O)2] than does inosine. The extra stabilization can be attributed to the triply negatively charged 5′ -IMP3- ion. The purines, inosine-5´-monophosphate and guanosine-5´-monophosphate form 110, 111 and 112 complexes. The protonated species are easily detected with aromatic amine (BPY, Pic and AMBI) rather than with aliphatic amines (en, Me2en, 1,2-DAP and 1,3- DAP). This may be interpreted on the basis that the protonated species are stabilized through the back bonding to the π–system of aromatic rings, which makes more stable

The thymine complex is more stable than the uracil one, most probably owing to the high basicity of the N3 group of thymine resulting from the extra electron donating methyl group. As a result of the high pKa values of pyrimidines (pKa ≈ 9) and the fact that they are monodentates, the complexes are formed only above pH 6, supporting the view that the negatively charged nitrogen donors of pyrimidine bases are important binding sites in the neutral and slightly basic pH ranges. The purines like inosine have two metal ion binding centres N1 and N7 nitrogens. Inosine can be protonated at N7 forming a (N1H-N7H) monocation. The pKa of N1H is 8.43 (El-Sherif, 2006) and the pKa of N7H is 1.2 (Martin, 1985**)**. It was reported that, in the acidic pH range, N1 remained protonated, while the metal ion is coordinated to N7 i.e. these N-donors are pH dependent binding sites and there is a gradual change from N7- binding to N1-binding with increase of pH (Maskos, 1985). The results showed that inosine form the complexes 110 and 111. The speciation diagrams of Pd(diamine)-inosine complexes indicated that the species 111 is formed in acidic pH range and it corresponds to the N7 coordinated complex, while N1 nitrogen is in protonated form. Inosine is slightly more acidic than the pyrimidine bases, a property which can be related to the existence of a higher number of resonance forms for the inosine anion. Based on the existing data, uracil and thymine ligate in the

Several solid Pd(II) complexes of guanosine and inosine have suggested to exhibit N7-O6 chelation (Pneumatikakis, 1984 ; Pneumatikakis et al., 1988). Also, according to *ab initio* SCF calculations, N7-O6 chelation is the energetically favored bonding mode (Del Bene, 1984) ;

N N

7 9

N

The IR spectrum of [Pd(BPY)(Inosine)]NO3 showed a shift of the ν(C=O) stretching vibration from 1700 cm-1 and 1676 cm-1 in the free inosine (which corresponds to keto-enol forms) to 1639 in the complex (Shehata, 2001). This clearly indicates the involvement of C=O in coordination. At higher pH, the N1H is deprotonated and the negative charge on N1 resonates with C=O, increasing the negative charge on the oxygen atom. This is in consistent

than does inosine. The extra stabilization can be attributed to the triply negatively charged


1 6 3

O

Pd

+

N

N

N

O

OH


OH

OH

deprotonated form through the N3 atom.

with the large acidification of the N1H.


Inosine-5′

5′

Anwader et al., 1987).

complexes. For Pd(AMBI)-complexes, the pKa values of the protonated species of the IMP complex (112) are 1.98 (log β112 - log β111) and 5.76 (log β111 - log β110). The corresponding values for GMP are 3.21 and 6.17. The former pKa value for IMP and GMP corresponds to the N1H group while the second is assignable to the -PO2(OH) group. The N1H groups were acidified upon complex formation by 7.23 (9.21-1.98) and 6.07 (9.28-3.21) pK units for IMP and GMP, respectively. Acidification of the N1H group upon complex formation is consistent with previous reports for IMP and GMP complexes (Sigel et al. 1994). In the fully protonated species (112), the two protons bound to N1 and the phosphate groups, exist at pH ~ 3 or lower. In the (111) complex species, which reaches its maximum concentration of 88 % at pH ~ 4.8 (Fig. 4), the single proton binds to phosphate. Therefore, monoprotonated species (111) is an N1-coordinated. The stability constant difference between the (112) and (111) complexes is 3.21, due to the pKa of the N1 deprotonation process. The phosphate group was not acidified upon complex formation since it is too far from the coordination center. A proposed coordination process of Pd(AMBI)2+ with 5′ - GMP is reported in Scheme 5.

Fig. 4. Concentration distribution of various species as a function of pH in the Pd(AMBI)-5´- GMP system at concentrations of 1.25x10-3 mol-dm-3 for Pd(AMBI)2+ and 5´-GMP, I = 0.1 mol-dm-3 (NaNO3) and T = 25 ± 0.1 0C).

Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 105

between the phosphate and the exocyclic amine is also thought to contribute to the higher

It is interesting to compare the the stability constants obtained from earlier kinetic results of Pd(diamine) with DNA and those estimated from potentiometric measurements. Much of the kinetic work was done in an acidic pH range in order to simplify the speciation of the system. Under these conditions, Pd(Pic)2+ for example, binds to IMP through the N7 site, leaving the N1 site and the phosphate groups protonated. The stability constant (K) of the

The log K value was found to be 3.52. This is comparable with the value obtained from the kinetic investigation (log K = 2.09) (Rau et al., 1997). The difference can be related to different experimental conditions (the kinetic study was performed at 10 0C with an ionic strength of 0.5 M), techniques employed and the acidity range selected for the kinetic measurements, where more than one PdII complex and/or IMP acid-base forms may

It was previously shown that N-donor ligands such as DNA constituents have an affinity for [Pd(AEPY)(H2O)2]2+, which may have important biological implications. However, the preference of Pd(II) to coordinate to S-donor ligands was demonstrated as shown in Tables 3 & 4. These results suggest that Pd(II)-N adducts can easily be converted into Pd-S adducts. Consequently, the equilibrium constant for such conversion is of biological significance. If we consider inosine as a typical DNA constituent (presented by HL) and cysteine as a typical thiol ligand (presented by H2B), the equilibria involved in the complex-formation

HL H+ + L-

 [Pd(AEPY)]2+ + L- [Pd(AEPY)L]+ (13a) (100) (110)

H2B 2H+ + B2-

 [Pd(AEPY)]2+ + B2- [Pd(AEPY)B] (14a) (100) (110)

Keq

[Pd(AEPY)L]+ + B2- [Pd(AEPY)B] + L-

β110[Pd(AEPY)L]+=[Pd(DAP)L]+/ [Pd(AEPY)]2+ [L-] (13b)

β110[Pd(AEPY)B] = [Pd(AEPY)B] / [Pd(AEPY)]2+ [B2-] (14b)

(15)

log K = log β112 – log β012 (12)

stability of the nucleotides over that of the nucleosides (Reedik, 1992).

**3.3.3 Comparison of thermodynamic and kinetic data** 

species formed under this condition is calculated using Eq. 12.

contribute to the kinetic result.

and displacement reactions are:

Scheme 5. Proposed coordination process of Pd(AMBI)2+ with 5′ -GMP

The IMP and GMP complexes are more stable than those of the pyrimidines. The extra stabilization can be explained on the basis of different columbic forces operating between the ions resulting from the negatively charged phosphate group. Hydrogen bonding between the phosphate and the exocyclic amine is also thought to contribute to the higher stability of the nucleotides over that of the nucleosides (Reedik, 1992).

### **3.3.3 Comparison of thermodynamic and kinetic data**

104 Stoichiometry and Research – The Importance of Quantity in Biomedicine

N

OH2

Pd

H2 N

H2O

O P

N

OH2

6

1 7 3


N1GMPN7H-Pd(AMBI)

O

N

2 NH

The IMP and GMP complexes are more stable than those of the pyrimidines. The extra stabilization can be explained on the basis of different columbic forces operating between the ions resulting from the negatively charged phosphate group. Hydrogen bonding

OH2

Pd

N

N H

+

O

Pd

N

9

N

NH2

pKa = ~ 3.21

OH OH

(112) pH ~ 3

**O**

**O**


OH

O P

O

N

9

N

OH OH

 N1GMPN7-Pd(AMBI) (111) pH ~ 5


**O**

**O**

OH

N H

O P OH **O**

**O**

6

1 7 3

O

NH

NH N

Scheme 5. Proposed coordination process of Pd(AMBI)2+ with 5′

NH2

2 NH

N

O

N

9

N

OH OH

6

1 7 3

O

NH

2 NH

N

It is interesting to compare the the stability constants obtained from earlier kinetic results of Pd(diamine) with DNA and those estimated from potentiometric measurements. Much of the kinetic work was done in an acidic pH range in order to simplify the speciation of the system. Under these conditions, Pd(Pic)2+ for example, binds to IMP through the N7 site, leaving the N1 site and the phosphate groups protonated. The stability constant (K) of the species formed under this condition is calculated using Eq. 12.

$$
\log \mathcal{K} = \log \| \boldsymbol{\beta}\_{112} - \log \boldsymbol{\beta}\_{012} \tag{12}
$$

The log K value was found to be 3.52. This is comparable with the value obtained from the kinetic investigation (log K = 2.09) (Rau et al., 1997). The difference can be related to different experimental conditions (the kinetic study was performed at 10 0C with an ionic strength of 0.5 M), techniques employed and the acidity range selected for the kinetic measurements, where more than one PdII complex and/or IMP acid-base forms may contribute to the kinetic result.

It was previously shown that N-donor ligands such as DNA constituents have an affinity for [Pd(AEPY)(H2O)2]2+, which may have important biological implications. However, the preference of Pd(II) to coordinate to S-donor ligands was demonstrated as shown in Tables 3 & 4. These results suggest that Pd(II)-N adducts can easily be converted into Pd-S adducts. Consequently, the equilibrium constant for such conversion is of biological significance. If we consider inosine as a typical DNA constituent (presented by HL) and cysteine as a typical thiol ligand (presented by H2B), the equilibria involved in the complex-formation and displacement reactions are:


$$[\text{Pd(AEPY)}]^{2\*} + \text{L} \cdot \xleftarrow{} \xleftarrow{} \xleftarrow{} [\text{Pd(AEPY)} \text{L}]^{\*}\tag{13a}$$

$$\beta\_{110} \text{[^{Pd(AEPY)L}]^{\*}} \text{=[^{Pd(DAP)L}]^{\*} / [^{Pd(AEPY)}]^{2\*} [L:]} \text{ [L:]} \tag{13b}$$

$$\mathbf{^{H\_2B}} \quad \begin{array}{c} \implies \mathbf{^{2H^\*} + \mathbf{B^2}} \end{array}$$

$$\begin{array}{cccc}\text{[Pd(AEPY)]}^{2+} + \text{B}^{2-} & \overbrace{\longleftrightarrow}^{\text{}} & \text{[Pd(AEPY)B]} & \text{[14a)}\\\text{(100)} & \text{(110)}\end{array}$$

$$\left[\beta\_{110}\text{[Pd(AEPY)B]} = \left[\text{Pd(AEPY)B}\right] / \left[\text{Pd(AEPY)}\right] \text{?} ^\text{[B2]}\text{[}^\text{[B2]}\text{]}\text{} \tag{14b}$$

$$\begin{array}{cccc} \text{[Pd(AEPY)L]} + \text{B} & \begin{array}{c} \text{K}\_{\text{eq}} \\ \text{[Pd(AEPY)B]} + \text{L} \end{array} & \begin{array}{c} \text{[Pd(AEPY)B]} + \text{L} \\ \text{[The} \end{array} \end{array} \tag{15}$$

Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 107

1,2-DAPa 6.05 - (Shoukry et al., 1999) 1,3-DAPb 7.16 - (El-Sherif et al., 2010) Pica 8.09 10.91 (El-Sherif et al., 2003) AMBIa 7.55 10.53 (El-Sherif, 2006) BPYa 8.47 11.37 (Shehata, 2001)

a1,2-dap,1,3-DAP, Pic, AMBI and BPY are 1,2-diaminopropane, 1,3-diaminopropane Picolylamine,

The stability constants of the CBDCA complex with [Pd(aromaticdiamine)(H2O)2]2+ is higher than those for [Pd(aliphaticdiamine)(H2O)2]2+. Moreover, the protonated species were not observed in similar palladium complexes of CBDCA and aliphatic amines (Shoukry et al., 1999; El-Sherif et al., 2010**)** using potentiometric technique. The higher stability of CBDCA complexes with [Pd(aromaticdiamine)(H2O)2] and the stabilization of the protonated species may be attributed to the π-acceptor properties of the pyridine rings. The pKa of the protonated species of Pd(BPY) with CBDCA is 2.92; lower value than the one measured for HCBDCA-, indicating acidification upon first chelation to Pd through one carboxylate group by 2.56 pK units (5.48-2.92). The pKa value of this protonated species was estimated previously from UV-Vis. measurements to be ca. 2.5 at 25 °C and 0.1 M ionic strength (Shoukry et al., 1998). The involvement of the carboxylic oxygen in coordination is confirmed by the shift of the asymmetric and symmetric stretching frequencies of COO-

lower and higher frequencies, respectively. υas and υs, which can be found at 1706 and 1293 cm-1 in H2CBDCA are shifted to 1645 and 1354 cm-1 in the [Pd(BPY)(CBDCA)] complex. This

**3.4.2 Ring-opening of [Pd(DAP)(CBDCA)] and the formation of [Pd(DAP)(CBDCA-O)** 

The potentiometric data for the system consisting of [Pd(diamine)(H2O)2]2+, CBDCA and DNA constituents were fitted assuming different models. The accepted model for the investigated DNA constituents is consistent with the formation of the 1110, 1111 and 1112 species (Table 10) (El-Sherif et al., 2003; Shehata, 2001; Mohamed and Shoukry, 2001). The results were further verified by comparing the experimental potentiometric data with the theoretically calculated curve. This supports the formation of the quaternary complex. It is interesting to notice that the quaternary complex of inosine is more stable than those of pyrimidines. This may be explained on the premise that the cyclobutane ring forms a close hydrophobic contact with the purine ring of inosine. Such contacts may contribute to the stabilization of the quaternary complexes. These studies bring to the conclusion that CBDCA is attached through one carboxylate oxygen while 5´-GMP is attached through N7 of the purine base. The same finding was obtained from an NMR investigation of the ring opening reaction of carboplatin with phosphate, chloride, and 5´-guanosine monophosphate (5´- GMP) in aqueous solution at 310 K using 1H, 15N and 31P NMR spectroscopy (Frey et al., 1993). In each case a ring-opened species containing monodentate CBDCA was detected during reaction development. A structure of cis-[Pt(NH3)2(CBDCA-O)(5´-GMP)] is proposed, taking into account the equivalence of all six cyclobutane ring protons. There is a

to

Complex log β110 log β111 Reference

2-aminomethylbenzimidazole, and 2,2′-bipyridyl respectively at 25 C and I = 0.1 M.

Table 9. Stability constants of [Pd(diamine)(CBDCA)].

corresponds to a unidentate chelation mode (Nakamoto, 1997).

**(DNA)]** 

The equilibrium constant for the displacement reaction given in equation (15) is given by:

$$\mathbf{K\_{eq}} = \left[ \text{Pd(AEPY)B} \right] \left[ \text{L} \cdot \right] / \left[ \text{Pd(AEPY)L} \right] \text{\*} \left[ \text{B}^{2} \right] \tag{16}$$

Substitution from eq. (13b) and (14b) in eq. (16) results in :

$$\mathbf{K}\_{\rm eq} = \left\langle \mathbf{J}\_{110} \text{[Pd(AEFY)B]} \right\rangle \left\langle \mathbf{J}\_{110} \text{[Pd(AEFY)L]} \right\rangle \tag{17}$$

The 100 species, [Pd(AEPY)(H2O)2]2+, is represented in the above equations as [Pd(AEPY)]2+ for simplicity reasons. logβ110 values for [Pd(AEPY)(L)]+ and [Pd(AEPY)B] complexes taken from Tables 4 and 8 amount to 7.78 and 15.11, respectively, and by substitution in equation (17) it was found that log Keq = 7.33. In the same way the equilibrium constants for the displacement of coordinated inosine by glycine and S-methylcysteine are log Keq = 2.55 and 1.38, respectively. These values clearly indicate how sulfhydryl ligands such as cysteine and by analogy glutathione are effective in displacing the DNA constituent, i.e., the main target in tumour chemotherapy. Chelated cyclobutanedicarboxylate (log Keq = 7.11) may undergo displacement reaction with inosine. Log Keq for such a reaction was calculated as described above and amounts to 0.68. The low value of the equilibrium constant for reaction is of biological significance since it is in line with the finding that carboplatin interacts with DNA through ring opening of chelated CBDCA and not through displacement of CBDCA.

### **3.4 Ternary complexes involving CBDCA**

Cyclobutane-1,1-dicarboxylic acid (H2CBDCA) is a diprotic acid with pKa1 and pKa2 of 2.75 and 5.48, respectively at 25 0C and 0.1M ionic strength (El-Sherif, et al., 2010 ; Shehata, et al., 2009). The acid-base equilibria are schematized as follows:

### **3.4.1 Ternary complexes involving CBDCA and [Pd(diamine)(H2O)2]**

The potentiometric data for H2CBDCA complex-formation with [Pd(diamine)(H2O)2]2+ were fitted considering the formation of 110 and the monoprotonated complex species 111 (Table 9) (Shoukry et al., 1999; El-Sherif et al., 2003 ; Shehata, 2001; El-Sherif et al., 2010; El-Sherif, 2006) according to Scheme (6).

Scheme 6. Coordination mode of CBDCA with [Pd(diamine)(H2O)2]


a1,2-dap,1,3-DAP, Pic, AMBI and BPY are 1,2-diaminopropane, 1,3-diaminopropane Picolylamine, 2-aminomethylbenzimidazole, and 2,2′-bipyridyl respectively at 25 C and I = 0.1 M.

Table 9. Stability constants of [Pd(diamine)(CBDCA)].

106 Stoichiometry and Research – The Importance of Quantity in Biomedicine

 Keq = β110[Pd(AEPY)B]/ β110[Pd(AEPY)L]+ (17) The 100 species, [Pd(AEPY)(H2O)2]2+, is represented in the above equations as [Pd(AEPY)]2+ for simplicity reasons. logβ110 values for [Pd(AEPY)(L)]+ and [Pd(AEPY)B] complexes taken from Tables 4 and 8 amount to 7.78 and 15.11, respectively, and by substitution in equation (17) it was found that log Keq = 7.33. In the same way the equilibrium constants for the displacement of coordinated inosine by glycine and S-methylcysteine are log Keq = 2.55 and 1.38, respectively. These values clearly indicate how sulfhydryl ligands such as cysteine and by analogy glutathione are effective in displacing the DNA constituent, i.e., the main target in tumour chemotherapy. Chelated cyclobutanedicarboxylate (log Keq = 7.11) may undergo displacement reaction with inosine. Log Keq for such a reaction was calculated as described above and amounts to 0.68. The low value of the equilibrium constant for reaction is of biological significance since it is in line with the finding that carboplatin interacts with DNA

through ring opening of chelated CBDCA and not through displacement of CBDCA.

Cyclobutane-1,1-dicarboxylic acid (H2CBDCA) is a diprotic acid with pKa1 and pKa2 of 2.75 and 5.48, respectively at 25 0C and 0.1M ionic strength (El-Sherif, et al., 2010 ; Shehata, et al.,

pKa1=2.75 pKa2=5.48


COO-

COOH

N N

P d OOC

**(110)**

P d

**(111)**

N N

OOC

OOC


α β

β

γ

The potentiometric data for H2CBDCA complex-formation with [Pd(diamine)(H2O)2]2+ were fitted considering the formation of 110 and the monoprotonated complex species 111 (Table 9) (Shoukry et al., 1999; El-Sherif et al., 2003 ; Shehata, 2001; El-Sherif et al., 2010; El-Sherif,

O H <sup>2</sup> COOH <sup>N</sup>

] / [Pd(AEPY)L]+[B2-] (16)

COO-

COO-

The equilibrium constant for the displacement reaction given in equation (15) is given by:

Keq =[Pd(AEPY)B] [L-

**3.4 Ternary complexes involving CBDCA** 

COOH

COOH

2006) according to Scheme (6).

P d

O H <sup>2</sup>

O H <sup>2</sup> HOOC

HOOC

N

**(100)**

2009). The acid-base equilibria are schematized as follows:

**3.4.1 Ternary complexes involving CBDCA and [Pd(diamine)(H2O)2]** 

+ - H <sup>3</sup> <sup>O</sup> <sup>+</sup>

Scheme 6. Coordination mode of CBDCA with [Pd(diamine)(H2O)2]

Substitution from eq. (13b) and (14b) in eq. (16) results in :

The stability constants of the CBDCA complex with [Pd(aromaticdiamine)(H2O)2]2+ is higher than those for [Pd(aliphaticdiamine)(H2O)2]2+. Moreover, the protonated species were not observed in similar palladium complexes of CBDCA and aliphatic amines (Shoukry et al., 1999; El-Sherif et al., 2010**)** using potentiometric technique. The higher stability of CBDCA complexes with [Pd(aromaticdiamine)(H2O)2] and the stabilization of the protonated species may be attributed to the π-acceptor properties of the pyridine rings. The pKa of the protonated species of Pd(BPY) with CBDCA is 2.92; lower value than the one measured for HCBDCA-, indicating acidification upon first chelation to Pd through one carboxylate group by 2.56 pK units (5.48-2.92). The pKa value of this protonated species was estimated previously from UV-Vis. measurements to be ca. 2.5 at 25 °C and 0.1 M ionic strength (Shoukry et al., 1998). The involvement of the carboxylic oxygen in coordination is confirmed by the shift of the asymmetric and symmetric stretching frequencies of COO to lower and higher frequencies, respectively. υas and υs, which can be found at 1706 and 1293 cm-1 in H2CBDCA are shifted to 1645 and 1354 cm-1 in the [Pd(BPY)(CBDCA)] complex. This corresponds to a unidentate chelation mode (Nakamoto, 1997).

### **3.4.2 Ring-opening of [Pd(DAP)(CBDCA)] and the formation of [Pd(DAP)(CBDCA-O) (DNA)]**

The potentiometric data for the system consisting of [Pd(diamine)(H2O)2]2+, CBDCA and DNA constituents were fitted assuming different models. The accepted model for the investigated DNA constituents is consistent with the formation of the 1110, 1111 and 1112 species (Table 10) (El-Sherif et al., 2003; Shehata, 2001; Mohamed and Shoukry, 2001). The results were further verified by comparing the experimental potentiometric data with the theoretically calculated curve. This supports the formation of the quaternary complex. It is interesting to notice that the quaternary complex of inosine is more stable than those of pyrimidines. This may be explained on the premise that the cyclobutane ring forms a close hydrophobic contact with the purine ring of inosine. Such contacts may contribute to the stabilization of the quaternary complexes. These studies bring to the conclusion that CBDCA is attached through one carboxylate oxygen while 5´-GMP is attached through N7 of the purine base. The same finding was obtained from an NMR investigation of the ring opening reaction of carboplatin with phosphate, chloride, and 5´-guanosine monophosphate (5´- GMP) in aqueous solution at 310 K using 1H, 15N and 31P NMR spectroscopy (Frey et al., 1993). In each case a ring-opened species containing monodentate CBDCA was detected during reaction development. A structure of cis-[Pt(NH3)2(CBDCA-O)(5´-GMP)] is proposed, taking into account the equivalence of all six cyclobutane ring protons. There is a

Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 109

<sup>1010</sup> <sup>1011</sup>

1111

1 3 5 7 911

Traditionally, water has been considered as the solvent that best represents biological conditions. Although this is a general assumption, a lower polarity has been detected in some biochemical micro-environments, such as active sites of enzymes and side chains in proteins (Rees, 1980 ; Rogersa et al., 1985; Akerlof and Short, 1953). It was suggested that these properties approximately correspond to those (or can be simulated by those) existing in the water/dioxane mixtures. Consequently, a study of the Pd(Pic)-CBDCA and Pd(1,3-DAP)-CBDCA complex formation, taken as a typical example for [Pd(aliphaticdiamine)CBDCA] and [Pd(aromaticdiamine)CBDCA] respectively, in dioxanewater solutions of different compositions could be of biological significance. In order to characterize the formation equilibria of the Pd(diamine)-CBDCA complex in dioxane-water solutions, all other equilibria involved, namely acid-base equilibria of CBDCA and [Pd(diamine)(H2O)2]2+, have to be studied in the same solvent. The equilibrium constants are reported in Table 11 (El-Sherif et al., 2003, 2010). The hydrolysis of Pd(diamine)2+ complex in dioxane-water solution leads to the formation of mono- and dihydroxy species. The dihydroxo bridged dimer was not detected. The pKa values of CBDCA and those of the coordinated water molecules in [Pd(diamine)(H2O)2]2+ increase linearly with increasing of dioxane concentration. This may be correlated with the ability of a relatively low dielectric solvent to increase the electrostatic attraction between the proton and the ligand anion in

Fig. 5. Concentration distribution of various species as a function of pH in the Pd(Pic)- CBDCA-IMP system at concentrations of 1.25x10-3 mol-dm-3 for Pd(Pic)2+; CBDCA and IMP,

**pH**

100-2

1110

1100

1000

0

20

40

**% Species** 60

80

100

1101

1012

I = 0.1 mol-dm-3 (NaNO3) and T = 25 ± 0.1 0C).

**4. Effect of solvent on the stability constants** 

close hydrophobic contact between the cyclobutane ring of monodentate CBDCA and the purine ring of coordinated 5´-GMP. The reaction of carboplatin with 5´-GMP (kobs 4.1 × 10-6 s-1) was faster than that with phosphate (kobs 4.3 × 10-7 s-1) and chloride (kobs 1.2 × 10-6 s-1), or water alone (< 5 × 10-9 s-1), suggesting that direct attack of nucleotides on carboplatin may be importance crucial step in the mechanism of action for this drug. Estimation of the concentration distribution of the various species in solution provides a useful picture of metal ion binding. To illustrate the main features observed in the species distribution plots in these systems, the speciation diagram obtained for the Pd(Pic)-CBDCA-IMP system as a representative example of [Pd(Pic)(CBDCA-O)(DNA)] is shown in Fig. 5. The Pd(Pic)- CBDCA species (1100) predominates at pH = 4.3 with maximum concentration of 73%. The Pd(Pic)-IMP species (1010) reaches the maximum concentration of 16 % at pH = 7.4. The quaternary species Pd(Pic)-CBDCA-IMP (1110) attains a maximum of 80 % in the pH range 7.6-9. This reveals that in the physiological pH range the ring opening of chelated CBDCA by DNA is quite feasible.


a l, p, q and r are the stoichiometric coefficients corresponding to Pd(diamine)(H2O)2], cyclobutane-1,1′ dicarboxylate, DNA and H+, respectively. bMe2en = N,N′-dimethylethylenediamine, data taken from reference (Mohamed and Shoukry, 2001), cPic = picolylamine, data taken from reference (El-Sherif et al., 2003), dBPY = 2,2′-bipyridyl, data taken from reference (Shehata, 2001).

Table 10. Formation constants (log β1110) for mixed ligand complexes of [Pd(diamine)(H2O)2] with cyclobutanedicarboxylic acid and some DNA units at 25 0C and 0.1M ionic strength.

close hydrophobic contact between the cyclobutane ring of monodentate CBDCA and the purine ring of coordinated 5´-GMP. The reaction of carboplatin with 5´-GMP (kobs 4.1 × 10-6 s-1) was faster than that with phosphate (kobs 4.3 × 10-7 s-1) and chloride (kobs 1.2 × 10-6 s-1), or water alone (< 5 × 10-9 s-1), suggesting that direct attack of nucleotides on carboplatin may be importance crucial step in the mechanism of action for this drug. Estimation of the concentration distribution of the various species in solution provides a useful picture of metal ion binding. To illustrate the main features observed in the species distribution plots in these systems, the speciation diagram obtained for the Pd(Pic)-CBDCA-IMP system as a representative example of [Pd(Pic)(CBDCA-O)(DNA)] is shown in Fig. 5. The Pd(Pic)- CBDCA species (1100) predominates at pH = 4.3 with maximum concentration of 73%. The Pd(Pic)-IMP species (1010) reaches the maximum concentration of 16 % at pH = 7.4. The quaternary species Pd(Pic)-CBDCA-IMP (1110) attains a maximum of 80 % in the pH range 7.6-9. This reveals that in the physiological pH range the ring opening of chelated CBDCA

System l P q ra (Me)2en b Picc BPYd

16.18 - -

15.17 - -

15.71 - -

12.29 17.72 -

a l, p, q and r are the stoichiometric coefficients corresponding to Pd(diamine)(H2O)2], cyclobutane-1,1′ dicarboxylate, DNA and H+, respectively. bMe2en = N,N′-dimethylethylenediamine, data taken from reference (Mohamed and Shoukry, 2001), cPic = picolylamine, data taken from reference (El-Sherif et al.,

Table 10. Formation constants (log β1110) for mixed ligand complexes of [Pd(diamine)(H2O)2] with cyclobutanedicarboxylic acid and some DNA units at 25 0C and 0.1M ionic strength.



14.18 - -

14.18 - -

14.34 - -

16.26 - -

21.58 -

20.86 - -



18.31 24.76 27.05

20.14 26.74 28.62


23.24 27.08

22.77 25.58


16.00 22.42 27.92 31.49

by DNA is quite feasible.

1 1

1 1

1 1

1 1

1 1

1 1

1 1

1 1 1

1 1 1

1 1 1

1 1 1

1 1 1

1 1 1

1 1 1

1 1 1

1 1 1

1 1 1

1 1 1

1 1 1

1 1 1

1 1 1

1 1 1

2003), dBPY = 2,2′-bipyridyl, data taken from reference (Shehata, 2001).

0 1 2

0 1 2

0 1 2

0 1 2

0 1 2

0 1 2

0 1 2

Uracil 1

Uridine 1

Thymine 1

Thymidine 1

Adenine 1

Inosine 1

GMP 1

IMP 1

Fig. 5. Concentration distribution of various species as a function of pH in the Pd(Pic)- CBDCA-IMP system at concentrations of 1.25x10-3 mol-dm-3 for Pd(Pic)2+; CBDCA and IMP, I = 0.1 mol-dm-3 (NaNO3) and T = 25 ± 0.1 0C).

### **4. Effect of solvent on the stability constants**

Traditionally, water has been considered as the solvent that best represents biological conditions. Although this is a general assumption, a lower polarity has been detected in some biochemical micro-environments, such as active sites of enzymes and side chains in proteins (Rees, 1980 ; Rogersa et al., 1985; Akerlof and Short, 1953). It was suggested that these properties approximately correspond to those (or can be simulated by those) existing in the water/dioxane mixtures. Consequently, a study of the Pd(Pic)-CBDCA and Pd(1,3-DAP)-CBDCA complex formation, taken as a typical example for [Pd(aliphaticdiamine)CBDCA] and [Pd(aromaticdiamine)CBDCA] respectively, in dioxanewater solutions of different compositions could be of biological significance. In order to characterize the formation equilibria of the Pd(diamine)-CBDCA complex in dioxane-water solutions, all other equilibria involved, namely acid-base equilibria of CBDCA and [Pd(diamine)(H2O)2]2+, have to be studied in the same solvent. The equilibrium constants are reported in Table 11 (El-Sherif et al., 2003, 2010). The hydrolysis of Pd(diamine)2+ complex in dioxane-water solution leads to the formation of mono- and dihydroxy species. The dihydroxo bridged dimer was not detected. The pKa values of CBDCA and those of the coordinated water molecules in [Pd(diamine)(H2O)2]2+ increase linearly with increasing of dioxane concentration. This may be correlated with the ability of a relatively low dielectric solvent to increase the electrostatic attraction between the proton and the ligand anion in

Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 111

Carboplatin or *Cis*-diamine(1,1-cyclobutanedicarboxylate)platinum(II), is a clinically successful second generation platinum complex. When Pt antitumor drugs are injected into the blood, it is crucial that they reach their final target, the intracellular DNA, without reacting with other nucleophiles within the cytoplasm. In human blood plasma the chloride content is quite high (~ 100 mM), so that hydrolysis of *cis*-[PtCl2(NH3)2] is less likely to occur than within the cell where the chloride concentration is as low as 4 mM (Rosenberg, 1980). The high chloride concentration allows the neutral complex, *cis*-[PtCl2(NH3)2], to flow almost unchanged through the blood. It then diffuses through the cell membrane and finally hydrolyzed to give more reactive aquated species as shown in scheme 7. Due to the lower

**Cl Pt NH3**

**NH3**

A realistic extrapolation of the present study to biologically relevant conditions will require information on the effect of the chloride concentration on the reported stability constants. The reactivity of CBDCA toward the different Pd(II) species increases markedly when chloride ions of Pd(AMBI)Cl2 are replaced successively by one and two water molecules. A similar qualitative conclusion has been reached in the case of Pt(en)Cl2 by Lim and Martin (Lim and Martin, 1976), based on equilibrium distribution of Pt(en) and on rates of reactions

The equilibrium constants for Pd(AMBI)-CBDCA complex obtained for different chloride ion concentrations, keeping the ionic strength constant at 0.30 mol-dm-3, are summarized in

Passive diffusion **[Cl-**

**Cl**

**RNA**

**] = 4mM**

**Cl Pt NH3**

**NH3**

Hydrolysis

**Cl Pt NH3**

**NH3**

**Cl**

**OH2**

**5. Effect of chloride on the stability constants** 

chloride concentration the aquated complexes can react with DNA.

**[Cl-**

**Cell Nucleus DNA**

**] = 100mM**

of pyridine with Pt(dien) complexes.

Scheme 7. The cellular uptake of cisplatin and its targets.

case of CBDCA and between a proton and the hydrolysed form of Pd(II) species. The variation in the stability constant of the [Pd(diamine)(H2O)2]2+ complex with CBDCA as a function of solvent composition, is shown in Fig. 6. The stability constant for the Pd(diamine)-CBDCA complex increases linearly with increasing dioxane concentration. This is explained in terms of complex formation involving oppositely charged ions as in the Pd(diamine)-CBDCA complex, which is favoured by the low dielectric constant of the medium, i.e. with increasing dioxane concentration. The results show that the CBDCA complex with Pd(diamine)2+ will be more favoured in biological environments of lower dielectric constant.


ap, q and r are stoichiometric coefficients corresponding to [Pd(diamine)(H2O)2], ligand and H+ respectively, bPic = picolylamine, data taken from reference (El-Sherif et al., 2003), c1,3-DAP = 1,3 diaminopropane, data taken from reference (El-Sherif et al., 2010).

Table 11. Effect of dioxane on the formation constant (log β110) of [Pd(diamine)(H2O)2] with CBDCA at 25 0C and 0.1 mol dm-3 NaNO3.

Fig. 6. Effect of dioxane concentration on the stability of the Pd(diamine)-CBDCA system and logK refers to the 110 species.

### **5. Effect of chloride on the stability constants**

110 Stoichiometry and Research – The Importance of Quantity in Biomedicine

case of CBDCA and between a proton and the hydrolysed form of Pd(II) species. The variation in the stability constant of the [Pd(diamine)(H2O)2]2+ complex with CBDCA as a function of solvent composition, is shown in Fig. 6. The stability constant for the Pd(diamine)-CBDCA complex increases linearly with increasing dioxane concentration. This is explained in terms of complex formation involving oppositely charged ions as in the Pd(diamine)-CBDCA complex, which is favoured by the low dielectric constant of the medium, i.e. with increasing dioxane concentration. The results show that the CBDCA complex with Pd(diamine)2+ will be more favoured in biological environments of lower

% Dioxane p q ra Picb 1,3-DAPc 12.5 1 1 0 8.93 8.13 25 1 1 0 9.36 8.57 37.5 1 1 0 9.89 8.98 50 1 1 0 10.58 9.48 62.5 1 1 0 10.99 10.13

ap, q and r are stoichiometric coefficients corresponding to [Pd(diamine)(H2O)2], ligand and H+ respectively, bPic = picolylamine, data taken from reference (El-Sherif et al., 2003), c1,3-DAP = 1,3-

Table 11. Effect of dioxane on the formation constant (log β110) of [Pd(diamine)(H2O)2] with

**0 10 20 30 40 50 60 70 Dioxane %**

Fig. 6. Effect of dioxane concentration on the stability of the Pd(diamine)-CBDCA system

**Pd(Pic)-CBDCA Pd(1,3-DAP)-CBDCA**

diaminopropane, data taken from reference (El-Sherif et al., 2010).

CBDCA at 25 0C and 0.1 mol dm-3 NaNO3.

dielectric constant.

**5**

and logK refers to the 110 species.

**6**

**7**

**8**

**9**

**log K**

**10**

**11**

**12**

Carboplatin or *Cis*-diamine(1,1-cyclobutanedicarboxylate)platinum(II), is a clinically successful second generation platinum complex. When Pt antitumor drugs are injected into the blood, it is crucial that they reach their final target, the intracellular DNA, without reacting with other nucleophiles within the cytoplasm. In human blood plasma the chloride content is quite high (~ 100 mM), so that hydrolysis of *cis*-[PtCl2(NH3)2] is less likely to occur than within the cell where the chloride concentration is as low as 4 mM (Rosenberg, 1980). The high chloride concentration allows the neutral complex, *cis*-[PtCl2(NH3)2], to flow almost unchanged through the blood. It then diffuses through the cell membrane and finally hydrolyzed to give more reactive aquated species as shown in scheme 7. Due to the lower chloride concentration the aquated complexes can react with DNA.

Scheme 7. The cellular uptake of cisplatin and its targets.

A realistic extrapolation of the present study to biologically relevant conditions will require information on the effect of the chloride concentration on the reported stability constants. The reactivity of CBDCA toward the different Pd(II) species increases markedly when chloride ions of Pd(AMBI)Cl2 are replaced successively by one and two water molecules. A similar qualitative conclusion has been reached in the case of Pt(en)Cl2 by Lim and Martin (Lim and Martin, 1976), based on equilibrium distribution of Pt(en) and on rates of reactions of pyridine with Pt(dien) complexes.

The equilibrium constants for Pd(AMBI)-CBDCA complex obtained for different chloride ion concentrations, keeping the ionic strength constant at 0.30 mol-dm-3, are summarized in

Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 113

**6. Biological activity of mixed ligand Pd(II) complexes with biologically active** 

Four new palladium(II) and platinum(II) complexes of formula [M(BPY)(AA)]+ (where BPY is 2,2´-bipyridylamine, AA is an anion of glycine or L-alanine, and M is Pd(II) or Pt(II) have been synthesized (Paul et al., 1993) and characterized with amino acids bound as bidentate ligands. These complexes are 1:1 electrolyte in conductivity water. Among the studied complexes, the two L-alanine complexes show ID50 values against lymphocytic leukemia cells lower than cis-diammine dichloroplatinum(II), whereas the two glycine complexes show ID50 values higher than cisplatin. The interaction of calf thymus DNA with the above complexes shows significant spectral changes in the presence of [Pt(BPY)(gly)]Cl, [Pd(BPY)(ala)]Cl, and [Pt(BPY)(ala)]Cl and the binding mode between these complexes and

Nine palladium(II) complexes of the formula [Pd(BPY)(AA)]n+ (where BPY is 2,2´ bipyridylamine, AA is an anion of L-cysteine, L-aspartic acid, L-glutamic acid, L-methionine, L-histidine, L-arginine, L-phenylalanine, L-tyrosine, L-tryptophan, n = 0 or1) have been synthesized by the interaction of [Pd(BPY)Cl2] with an appropriate sodium salt of amino acids in water. The Pd(II)-complexes have shown growth inhibition against L1210 lymphoid leukemic, P388 lymphocytic leukemic sarcoma 180 and Ehrlich ascites tumor cells. Some of these complexes show ID50 values comparable to *cis*-platin (Puthraya et al.,1986).

The amino acids (AA) complexes of [Pt(phen)(AA)]NO3·xH2O and [Pd(phen)(AA)]NO3·x H2O have been prepared by the interaction of palladium and platinum complexes, [Pt(phen)Cl2] and [Pd(phen)Cl2] with salts of amino acids in methanol and characterized by 1H NMR and IR spectroscopy which confirmed the formation of a very large variety of compounds (Jin et al., 2000). All of these new compounds have been isolated and tested for cytotoxicity on Molt-4, a human leukaemia cell line. The IC50 values of [Pt(phen)(Pro)]Cl·2H2O and [Pd(phen)(Asp)]Cl·1.5H2O are 9.8 and 7.31 μM, respectively,

PaIladium(II) complexes of type [Pd(phen)(AA)]+ (where AA is an anion of glycine, L-alanine, L-leucine, L-phenylalanine, L-tyrosine, L-tryptophan, L-valine, L-proline, or L-serine), have been synthesized. These palladium(II) complexes have been characterized by ultraviolet-visible, infrared, and 1H NMR spectroscopy. They have also been screened for cytotoxicity in P388 lymphocytic cells. Only two complexes, [Pd(Phen)(Gly)]+ and [Pd(phen)(Val)]+, showed a comparable cytotoxictty with cisplatin (Mital, et al., 1991).

Five new palladium(II) complexes of the formula [Pd(AMBI)(AA)]n+ (where AMBI = 2-aminomethyl benzimidazole, AA is an anion of glycine, alanine, cysteine, methionine and threonine) have been synthesized (El-Sherif, 2011). These palladium(II) complexes have been ascertained by elemental, molar conductance, infrared and 1HNMR spectroscopy. The isolated Pd(II)-complexes were screened for their antibacterial and cytotoxic activities and the results are reported and discussed. The activity of these compounds against Staphylococcus pyogenes decreased in the order Tavanic (standard) > [Pd(AMBI)(Met)]∼ [Pd(AMBI)(Cys)] > [Pd(AMBI)(Ser)] > [Pd(AMBI)(Ala)] > [Pd(AMBI)(Gly)] and against E. coli decreased in the order Tavanic ∼ [Pd(AMBI)(Met)] > [Pd(AMBI)(Cys)] > [Pd(AMBI)(Ser)] > [Pd(AMBI)(Ala)] > [Pd(AMBI)(Gly)] under experimental conditions. Cytotoxic study of the compounds against colon carcinoma (HCT116) and larynx carcinoma

exhibiting a significant activity which is close to *cis*-[PtCl2(NH3)2].

**ligands** 

DNA seems to be noncovalent.

Table 12 (El-Sherif et al., 2003; El-Sherif, 2006). The stability constants of the Pd(AMBI)- CBDCA complex, tend to decrease while increasing the chloride ion concentration. This can be accounted for on the basis that the concentrations of the active species, the mono- and the diaqua complexes, decrease with increasing [Cl- ]; this will in turn affect the stability of the complexes formed. The variation in stability constant of the Pd(diamine)2+ complex with CBDCA as a function of Cl ion concentration, is shown in Fig. 7.


ap, q and r are stoichiometric coefficients corresponding to [Pd(diamine)(H2O)2], ligand and H+ respectively, bPic = picolylamine, data taken from reference (El-Sherif et al., 2003), cAMBI = 2 aminomethylbenzimidazole, data taken from reference (El-Sherif, 2006).

Table 12. Effect of chloride on the formation constant (log β110) of [Pd(diamine)(H2O)2] with CBDCA at 25 0C and 0.3 mol dm-3 (KCl + NaNO3).

Fig. 7. Effect of chloride ion concentration on the stability of the Pd(diamine)-CBDCA system and logK refers to the 110 species.

Table 12 (El-Sherif et al., 2003; El-Sherif, 2006). The stability constants of the Pd(AMBI)- CBDCA complex, tend to decrease while increasing the chloride ion concentration. This can be accounted for on the basis that the concentrations of the active species, the mono- and the diaqua complexes, decrease with increasing [Cl-]; this will in turn affect the stability of the complexes formed. The variation in stability constant of the Pd(diamine)2+ complex with

ion concentration, is shown in Fig. 7.

]/M p q ra Picb AMBIc 0.05 1 1 0 3.95 3.55 0.10 1 1 0 3.56 3.15 0.15 1 1 0 3.30 2.80 0.20 1 1 0 3.00 2.40 0.25 1 1 0 2.62 -

ap, q and r are stoichiometric coefficients corresponding to [Pd(diamine)(H2O)2], ligand and H+ respectively, bPic = picolylamine, data taken from reference (El-Sherif et al., 2003), cAMBI = 2-

Table 12. Effect of chloride on the formation constant (log β110) of [Pd(diamine)(H2O)2] with

**0 0.05 0.1 0.15 0.2 0.25 Chloride ion conc.** 

Fig. 7. Effect of chloride ion concentration on the stability of the Pd(diamine)-CBDCA

**Pd(Pic)-CBDCA Pd(AMBI)-CBDCA**

aminomethylbenzimidazole, data taken from reference (El-Sherif, 2006).

CBDCA at 25 0C and 0.3 mol dm-3 (KCl + NaNO3).

CBDCA as a function of Cl-

[Cl-

**1.5**

system and logK refers to the 110 species.

**2**

**2.5**

**3**

**log K**

**3.5**

**4**

**4.5**

### **6. Biological activity of mixed ligand Pd(II) complexes with biologically active ligands**

Four new palladium(II) and platinum(II) complexes of formula [M(BPY)(AA)]+ (where BPY is 2,2´ -bipyridylamine, AA is an anion of glycine or L-alanine, and M is Pd(II) or Pt(II) have been synthesized (Paul et al., 1993) and characterized with amino acids bound as bidentate ligands. These complexes are 1:1 electrolyte in conductivity water. Among the studied complexes, the two L-alanine complexes show ID50 values against lymphocytic leukemia cells lower than cis-diammine dichloroplatinum(II), whereas the two glycine complexes show ID50 values higher than cisplatin. The interaction of calf thymus DNA with the above complexes shows significant spectral changes in the presence of [Pt(BPY)(gly)]Cl, [Pd(BPY)(ala)]Cl, and [Pt(BPY)(ala)]Cl and the binding mode between these complexes and DNA seems to be noncovalent.

Nine palladium(II) complexes of the formula [Pd(BPY)(AA)]n+ (where BPY is 2,2´ bipyridylamine, AA is an anion of L-cysteine, L-aspartic acid, L-glutamic acid, L-methionine, L-histidine, L-arginine, L-phenylalanine, L-tyrosine, L-tryptophan, n = 0 or1) have been synthesized by the interaction of [Pd(BPY)Cl2] with an appropriate sodium salt of amino acids in water. The Pd(II)-complexes have shown growth inhibition against L1210 lymphoid leukemic, P388 lymphocytic leukemic sarcoma 180 and Ehrlich ascites tumor cells. Some of these complexes show ID50 values comparable to *cis*-platin (Puthraya et al.,1986).

The amino acids (AA) complexes of [Pt(phen)(AA)]NO3·xH2O and [Pd(phen)(AA)]NO3·x H2O have been prepared by the interaction of palladium and platinum complexes, [Pt(phen)Cl2] and [Pd(phen)Cl2] with salts of amino acids in methanol and characterized by 1H NMR and IR spectroscopy which confirmed the formation of a very large variety of compounds (Jin et al., 2000). All of these new compounds have been isolated and tested for cytotoxicity on Molt-4, a human leukaemia cell line. The IC50 values of [Pt(phen)(Pro)]Cl·2H2O and [Pd(phen)(Asp)]Cl·1.5H2O are 9.8 and 7.31 μM, respectively, exhibiting a significant activity which is close to *cis*-[PtCl2(NH3)2].

PaIladium(II) complexes of type [Pd(phen)(AA)]+ (where AA is an anion of glycine, L-alanine, L-leucine, L-phenylalanine, L-tyrosine, L-tryptophan, L-valine, L-proline, or L-serine), have been synthesized. These palladium(II) complexes have been characterized by ultraviolet-visible, infrared, and 1H NMR spectroscopy. They have also been screened for cytotoxicity in P388 lymphocytic cells. Only two complexes, [Pd(Phen)(Gly)]+ and [Pd(phen)(Val)]+, showed a comparable cytotoxictty with cisplatin (Mital, et al., 1991).

Five new palladium(II) complexes of the formula [Pd(AMBI)(AA)]n+ (where AMBI = 2-aminomethyl benzimidazole, AA is an anion of glycine, alanine, cysteine, methionine and threonine) have been synthesized (El-Sherif, 2011). These palladium(II) complexes have been ascertained by elemental, molar conductance, infrared and 1HNMR spectroscopy. The isolated Pd(II)-complexes were screened for their antibacterial and cytotoxic activities and the results are reported and discussed. The activity of these compounds against Staphylococcus pyogenes decreased in the order Tavanic (standard) > [Pd(AMBI)(Met)]∼ [Pd(AMBI)(Cys)] > [Pd(AMBI)(Ser)] > [Pd(AMBI)(Ala)] > [Pd(AMBI)(Gly)] and against E. coli decreased in the order Tavanic ∼ [Pd(AMBI)(Met)] > [Pd(AMBI)(Cys)] > [Pd(AMBI)(Ser)] > [Pd(AMBI)(Ala)] > [Pd(AMBI)(Gly)] under experimental conditions. Cytotoxic study of the compounds against colon carcinoma (HCT116) and larynx carcinoma

Coordination Chemistry of Palladium(II) Ternary Complexes with Relevant Biomolecules 115

7. Pd(diamine)-CBDCA complex formation is more favoured in biological environments

8. Hopefully that these data will bring a significant contribution in carrying out

The author thanks the Chemistry Department, Cairo University, Faculty of Science, Egypt for financial support and Prof. Dr. M.M. Shoukry for his efforts and co-operation. The author is also grateful to the chairman of Chem. Department (Prof. Dr. M. Badway) and the Dean of Faculty of Science (Prof. Dr. Gamal Abd El-Nasar for their constant encouragement

diaminopropane; 1,2-DAP = 1,2-diaminopropane**;** AEPY = 2-aminoethyl pyridine; UMP =

Akerlof, G. Short, O.A. (1953) The dielectric constant of dioxane-water mixtures between 0

Anwader, E.H.S., Probst, M.M. and Rode. B.M. (1987) Investigations of Zn(II) complexes

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the palladium(II) and mixed metal complexes of peptides containing methionyl

platinum(II) chloride and (diethylenetriamine)nitratoplatinum(II) nitrate and some comments on the existence of PtII-OH2 and PtII–OH bonds in the solid state. Acta

with DNA/RNA bases by means of quantum chemical calculations. Inorg Chim

chain-length, n, on the reactions of amino-acids, +NH3(CH2)NCO2 (N = 1, 2, 3), with






of lower dielectric constant.

monophosphate; CMP = Cytidine-5′

Acta. 137, 203-208.

= Phenylalanine; Ala = Alanine.

**8. Aknowledgement** 

for this work.

Uridine-5′

**9. Abbreviations** 

**10. References** 

8.

mechanistic studies in biological media.

Pic = Picolylamine (2-Aminomethylpyridine); BPY = 2,2′

and 80 degrees-correction. J Am Chem Soc. 75, 6357.

acids and related molecules. Coord Chem Rev 166, 313-359.

platinum(II) ammine complexes. Aus J Chem. 39(9), 1347-1362.

biological active systems, Ellis Horwood Ltd., England.

residues. Journal of Inorganic Biochemistry 83, 77-89.

Crystallogr Sect B 38(8), 2148-2155.


(HEP2) cells indicate that, [Pd(AMBI)(Met)]Cl.H2O complex shows significant activity against (HCT116) cells with IC50 value of 0.74 μg/ml, while [Pd(AMBI)(Cys)] complex shows significant activity against (HEP2) with IC50 value of 0.60 μg/ml. These results confirm the chemotherapeutically significance of these compounds (El-Sherif, 2011).

### **7. Conclusions**

In this review, a detailed survey of the formation equilibria of [Pd(diamine)(H2O)2]2+ with ligands of biological significance is presented. The main conclusions may be summarized as follows:


### **8. Aknowledgement**

114 Stoichiometry and Research – The Importance of Quantity in Biomedicine

(HEP2) cells indicate that, [Pd(AMBI)(Met)]Cl.H2O complex shows significant activity against (HCT116) cells with IC50 value of 0.74 μg/ml, while [Pd(AMBI)(Cys)] complex shows significant activity against (HEP2) with IC50 value of 0.60 μg/ml. These results

In this review, a detailed survey of the formation equilibria of [Pd(diamine)(H2O)2]2+ with ligands of biological significance is presented. The main conclusions may be summarized as

1. The stability constants of the hydroxo complexes are higher for [Pd(aromaticdiamine)(H2O)2]2+ than [Pd(aliphaticdiamine) )(H2O)2]2+, suggesting that the presence of aromatic residues increases the affinity of palladium(II) for hydrolysis. 2. Combining of stability constants data of such diaqua-complexes with amino acids, peptides and DNA constitutents, it would be possible to calculate the equilibrium distibution of the metal species in biological fluids where all types of ligands are present simultaneously. This would constitute a powerful starting point for understanding the mode of action of such metal species under physiological conditions. 3. Amino acids form highly stable complexes, with the substituent on the α- carbon atom possessing a significant effect on the stability of the formed complex. The thioether group in S-methylcysteine increase the stability constant of its complex due to the highest coordination potentiality of the sulphur atom. The imidazole group in histidine increases the stability of the complex due to high affinity of PdII to the nitrogen donor group. On the other hand the extra carboxylic group in aspartic acid does not contribute to the stability of the formed complex, as the extra-carboxylic group is not competing

4. The present study clearly shows clearly that [Pd(diamine)(H2O)2]2+ complex can form strong bonds with peptides and promote easy deprotonation of the peptide. The relative magnitudes of the pKa values of the Pd(II) complexes with peptides have interesting biological implications. Under normal physiological condition (pH 6-7), the peptides would coordinate to [Pd(diamine)(H2O)2]2+ in entirely different ways. Glutaminate exists solely in its protonated form, whereas the other peptides are present entirely in the deprotonated form. Furthermore, the slight difference in the side chain of the peptides seem to produce dramatic differences in their behaviour toward the Pd(II) complex. 5. Anti-tumour Pt(II) amines are usually administrated as cisdichloro complexes. This form persists in human blood plasma because of its high chloride content (0.1 M). The net zero charge on the complex facilitates its passage through cell walls. Within many cells the chloride ion concentration is much lower (only ca. 4 mM), giving more reactive aquated species. Due to the lower chloride concentration, the complex diffuses through the cell membrane and is then hydrolyzed to give the more reactive aquated complexes

6. The reactivity of CBDCA toward the different Pd(II) species increases markedly when chloride ions of Pd(diamine)Cl2 are replaced successively by one and two water molecules. A similar qualitative conclusion has been reached in the case of Pt(en)Cl2 by Lim and Martin, based on equilibrium distribution of en Pt(II) and on rates of reactions

of pyridine with dien Pt(II) complexes (Lim, and Martin, 1976).

confirm the chemotherapeutically significance of these compounds (El-Sherif, 2011).

with the amino group in complex formation.

which can then react with DNA

**7. Conclusions** 

follows:

The author thanks the Chemistry Department, Cairo University, Faculty of Science, Egypt for financial support and Prof. Dr. M.M. Shoukry for his efforts and co-operation. The author is also grateful to the chairman of Chem. Department (Prof. Dr. M. Badway) and the Dean of Faculty of Science (Prof. Dr. Gamal Abd El-Nasar for their constant encouragement for this work.

### **9. Abbreviations**

Pic = Picolylamine (2-Aminomethylpyridine); BPY = 2,2′ -bipyridyl; 1,3-DAP = 1,3 diaminopropane; 1,2-DAP = 1,2-diaminopropane**;** AEPY = 2-aminoethyl pyridine; UMP = Uridine-5′ -monophosphate; TMP = Thymidine-5′ -monophosphate; GMP = Guanosine-5′ monophosphate; CMP = Cytidine-5′ -monophosphate; IMP = Inosine-5′ -monophosphate; Phe = Phenylalanine; Ala = Alanine.

### **10. References**


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**5** 

**Heavy Metal Ion Extraction Using Organic Solvents: An Application** 

**of the Equilibrium Slope Method** 

Tjoon Tow Teng, Yusri Yusup and Ling Wei Low

The separation procedure of a chemical species from a matrix is essentially based on the transportation of the solute between the two involved phases, generally an organic and an inorganic one. Specifically, solvent extraction uses the concept of unique solute distribution ratios between two immiscible solvents. However, there are several situations where solutes have been observed to completely move from the inorganic to the organic phase

Organic solvent extraction is the transport of solutes, e.g. heavy metal ions, from an inorganic (or aqueous) phase to an organic phase. Solvents used comprise of an extractant + diluent combination. The roles of each are as follows: 1) the extractant, as a specific metal ion extractant; 2) the diluent, as a solvent condition controller, i.e. hydrophobicity, which can affect the molecules extractability (Watson, 1999; Cox, 2004). Occasionally, a phase modifier can be added to solve the problem of emulsion formation, aside from improving

Solvent extraction is widely applied to processes of metal ions recovery, ranging from aqueous solutions in hydrometallurgical treatment to environmental applications. It is also considered a useful technique to increase the initial concentration of the solute, commonly

In the biomedical field, supported liquid membrane methodology was used for trace analytes determination by facilitating chromatogram differentiation between samples, water and blood plasma (Jonsson and Mathiasson, 1999). It is also used to enrich human wastes (e.g. urine) with heavy metals prior to concentration determination using atomic absorption

Numerous organic solvents have been utilized to remove heavy metals (Leopold et al., 2010; Rafighi et al., 2010; Chang et al., 2011; Fu et al., 2011; Mishra and Devi, 2011). Most of them are, in part, made from petroleum. Recently, solvents such as vegetable oil (Venkateswaran

the phase demarcation process in an aqueous organic system (Cox, 2008).

used in the separation processes of analytical applications (Reddy et al., 2005).

spectroscopy (AAS) (Lindegrade et al., 1992; Djane et al., 1997a; Djane et al., 1997b).

**1. Introduction** 

(Anthemidis and Ioannou, 2009).

**1.1 Organic solvents** 

*Universiti Sains Malaysia* 

*Malaysia* 

