Interaction between δ-‎aminolevulinic Acid Drug (ALA) and Peptides (L)

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Potentiometric investigation on the mixed Cu(II) complexes with δ-Aminolevulinic acid (ALA)  and some selected peptides (L)

Abstract

The interaction between δ-‎aminolevulinic acid drug (ALA) ‎ and peptides (L) with Cu(II) was investigated in aqueous medium at 25 ± 0.1 °C with I = 0.10 mol· L−1 NaCl ‎using ‎the potentiometric technique. The stability constants of the binary and ternary complexes were calculated and discussed.‎ The concentration distribution of the complexes in solution‎ was graphically presented using the ‎HySS program. ‎ In addition, The values of Δ logK, log X and % R. S for the mixed-ligand complexes studied have been evaluated and discussed.

Key words:  Solution equilibrium; Potentiometric methods; Distribution diagrams‎; ‎complexes‎;‎ Cu(II); Drug; Peptides. ‎

1. Introduction

δ-Aminolevulinic acid (ALA), Scheme 1, an endogenous non-proteinogenic amino acid, is the first compound in the porphyrin synthesis pathway, the pathway that leads to heme [1] in mammals and chlorophyll [2] in plants. 5ALA is used in photo-dynamic detection [3-6] and photodynamic surgery of cancer. It is employed in neurosurgical procedures to visualize tumorous tissue. [4] Research from 2006 onwards have determined that the use of this guiding technique intraoperatively might prolong progression-free survival in patients suffering from malignant gliomas, and decrease the residual volume of the tumor [5,6]. Moreover, It was stated that an ALA complex and iron ion could induce murine hair growth in ‎vivo separated from mesenchymal and epithelial cells. Therefore, this complex may potentially ‎become a new beneficial remedy for alopecia [7].‎

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The tripeptide-copper complex has been known to work as a growth factor for the hair. The complex increases vascular endothelial growth factor production and stimulates the proliferation of dermal fibroblasts. One of the tripeptide-‎copper complexes, which is L-alanyl-L-histidyl-L-lysine-Cu2+ encourages human hair follicles growth that is caused as a result of stimulating the ‎preclusion and the proliferation of the apoptosis of dermal papilla cells [8]. Most physiological activities ‎concerning the interactions of nucleic acid are promoted by metal ions by ternary (mixed- ‎ligand) complexes formation [9,10].

When a metal ion is found in a solution that contains two or more different ‎ligands, it is possible for ternary (mixed-ligand) complexes as well as several simple ones to be formed, ‎this is determined by the pH of the system. The actual formation of the complex is dependent on the relative concentration and the affinity of the metal ‎ion towards the different ligands present. ‎Amongst other transition metals, Cu(II) delivers vigorous center in various ‎enzymes. Consequently, It seems to be of substantial recognition to demeanor many ‎studies investigating binary and ternary complexes of Cu(II), connecting peptides and the aminolevulinic acid (ALA), Scheme 1‎, glycyl-glycine (Gly-Gly), glycyl-L-alanine (Gly-Ala), glycyl-L-valine (Gly-Val) and glycyl-L-leucine (Gly-Leu). Furthermore, recent research explains the equilibria linked with the Cu(II) ion with ALA ‎interactions and peptides in aqueous media at 25 °C and ionic strength 0.10 mol· L−1 NaCl using the potentiometric technique. The values of log X, Δ log K and relative stabilization percentages (% R.S.) for mixed-ligand complexes examined have been estimated and reviewed. Ultimately, the distribution of species in solution over a wide range of pHs was also evaluated.

2. Experimental

2.1. Materials

‎ All the chemicals and ligands (Scheme 1) used in this present work were analytical grade purity of Sigma Aldrich products. Stock solutions of CuCl2. 2H2O, HCl and NaCl were prepared in deionized water. A solution of ALA was prepared with two equivalents of HCl. The ionic strength of each solution was adjusted to 0.10 mol· L−1 by addition of NaCl as supporting electrolyte. CO2 free sodium hydroxide, (titrant) solution was standardized potentiometrically with potassium hydrogen phthalate (Merck Chem. Co.).

 Gly-Gly ‎ Gly-Ala ‎ALA ‎ ‎ Gly-Leu Gly-Val

Scheme 1: The structural formula of ALA and peptides(L).

2.2. Equilibrium measurements

The pH titrations were done at 25 ± 0.1 °C and an ionic strength of 0.10 mol· L−1 NaCl in a double-walled glass container using a digital pH meter Griffin p-J-300-010 G. The electrode system was calibrated in terms of hydrogen-ion ‎concentrations instead of activities. Oxygen and carbonate free, nitrogen gas was bubbled through the solution before and during the titrations. Titrations of the mixed ligand systems were carried out on aliquots (50 ml) of solution containing low concentration (0.005 mol· L−1) of corresponding Cu(II), ALA and peptide ligands (L) in 1:1:1 ratio with known volume of standard CO2 free NaOH.

Quantitatively, the formation of mixed ligand complex equilibrium can be given as (charges omitted for simplicity).

mM + aALA + lL + h H   M m ALAa Ll Hh                                                      (1)

(2)

‎

Where [M], [ALA], [L] and [H] are the concentrations of Cu(II) ion, aminolevulinic acid, ‎ peptides and  ‎proton respectively and the stoichiometric figures are m, a, l and h. The overall stability constant (βmalh ) defined may be used to calculate the species distribution curves that provides the clues for the formation equilibria of the complexes.

All calculations involved in potentiometric studies within the pH range 2.5–12.0 (105-110 data; volume of base/pH) were computed with the aid of HYPERQUAD computer program [11] and the results are given in Table 1. Speciation was calculated via HYSS program [12], which plotted the distribution of species at series of complexes on quantified pH range. Thus, the visual output may provide a graphical record of the major complex species at any specific pH especially in the physiological range of pH.

3. RESULTS AND DISCUSSIONS

3.1. Protonation constants of ligands

Many biological molecules activity depends on the presence of charged groups. Thus, the dissociation constant can be a key parameter for understanding and measuring chemical phenomena or biological activity. Consequently, the passage of many drugs to cells and across other membranes is the pH function in the internal environment and pK of the drug [13].  The acid dissociation constants of the ligands‎ under study were determined and used for determining the stability constants of the binary and ternary complexes formed in the aqueous medium under the experimental conditions and are given in Table 1. The peptides studied here usually have two important functional groups.

ALA exists in solution as zwitterion forming compound. They contain an amine group (basic) and a carboxyl group (acidic).‎ The -NH2 group is the stronger base, and so it picks up H+ from the -COOH group to leave a zwitterion. The analysis of ‎ potentiometric data using a computer program ‎ gave best fit ‎for two protonation constants (Table 1).

Only two deprotonation can be determined for ALAin the range of pH titratable degree at the protonated imino group and carboxylic proton‎ with pKa values of 9.83 and 2.81, respectively. ‎The ionization of ALA in the solution take place according to the following equilibrium:

${{\mathrm{H}}{2}{\mathrm{AlA}}}^{{+}}\mathit{HALA}+{H}^{+}$

(3)

where H2ALA+ is the protonated form and

HALA the zwitterionic form of ALA. This is also illustrated in the species distribution of the ALA ligand in Fig. 1.

Table 1: The protonation constants of the ligands in aqueous media at 25.0 °C and  I = 0.10  mol· L−1 (NaCl) .

 Ligands pK’s of Ligands pKa1 pKa2 Aminolevulinic‎ (ALA) 2.81(0.01) 9.83(0.02) Glycyl-glycine (Gly-Gly) 3.21(0.05) 8.04(0.03) Glycyl-L-alanine (Gly-Ala)‎ 2.70(0.006) 7.56(0.01) ‎ Glycyl-L-valine (Gly-Val) ‎ 3.27 (0.03) 7.96 (0.02) Glycyl-L-leucine (Gly-Leu) 4.32(0.02) 9.13(0.002) Glutamine (Glu) 2.17(0.02) 9.13(0.01) Glycinamide (Gly) 1.78 (0.01) ‎8.06 (0.01)‎

Figure 1.  Representative concentration distribution curves as a function of pH calculated for ALA system at 25.0 °C and I = 0.10  mol· L−1 (NaCl) .

Binary copper (II) complex formation equilibria with ligands

Analysis of the binary complexes curves  indicates that the free ligand solutions shift the buffer region of the ligand to lower pH values. This reflects that complexation reaction proceeds by releasing of protons from such ligands (ALA or peptides). The formation constants were determined by fitting potentiometric data on the basis of possible composition models. The selected model with the best statistical fit was found to consist Cu(II)-ALA (1100), Cu(II)-(ALA)2 (1200), Cu(II)-(ALA)2 H (1201), Cu(II)-(ALA)2 H2 (1202),Cu(II)-ALA (OH) 110-1, Cu(II)-L (1010), Cu(II)-(L)2 (1020), Cu(II)-(L) H-2 (101-1) and Cu(II)-(ALA)2 H-1 ‎‎(102-1) ‎  complexes. In the complexation between peptides and copper (II), the chelation starts at the amino end of the molecule, with the assistance of ‎carbonyl oxygen, and continues with the sequential deprotonation and ‎coordination of the amide groups [14, 15‎].

ALA  acts as an ON bidentate ligand, where it chelates to the copper ion through the carboxylic oxygen and imino nitrogen. Evaluation of the concentration distribution of the various species as a function of pH provides a useful description of metal ion binding in biological system. In all species distribution curves the concentration of the formed complex increases with increasing pH, thus making the complex formation more favored in the physiological pH rang.‎Species distribution diagram of Cu(II)-ALA system is given in Fig. (2) and the respective obtained overall stability constants of the species which are presented in Table (2).

Figure 2.  Representative concentration distribution curves as a function of pH calculated for Cu(II)-ALA system at 25C and 0.10 mol· L−1 ionic strength.

Table 2: Stability constants of the binary species in the Cu(II)-ALA or peptides systems at 25°C and ‎‎0.1 mol/L ionic strength.‎

 Systems m a l hn log βb Cu(II) – ALA 1 1 0 0 8.67 (0.003) 1 2 0 0 15.78(0.01) 1 2 0 1 22.90(0.01) 1 2 0 2 28.01(0.02) 1 1 0 -1 1.56(0.01) Cu(II) – Gly-Gly ‎ 1 0 1 0 7.60 (0.02) 1 0 2 0 13.04 (0.01) 1 0 1 -1 1.34 (0.01) 1 0 1 -2 -7.76 (0.01) 1 0 2 -1 4.41 (0.03) Cu(II) – Gly-Ala 1 0 1 0 8.21 (0.01) 1 0 2 0 15.15(0.02) 1 0 1 -1 1.98 (0.01) 1 0 1 -2 -6.78 (0.01) 1 0 2 -1 3.72 (0.01) Cu(II) – Gly-Val 1 0 1 0 8.38 (0.01) 1 0 2 0 16.05(0.001) 1 0 1 -1 0.43 (0.01) 1 0 1 -2 -6. 98 (0.001) 1 0 2 -1 3.67 (0.01) Cu(II) – ‎Gly-Leu 1 0 1 0 8.21 (0.01) 1 0 2 0 16.10(0.01) 1 0 1 -1 1.56 (0.002) 1 0 1 -2 -7. 04 (0.001) 1 0 2 -1 4.55 (0.01) Cu(II) – ‎Glu 1 0 1 0 7.81 (0.01) 1 0 2 0 13.63(0.01) 1 0 1 -1 1.06 (0.01) 1 0 2 -1 3.92 (0.01) Cu(II) ‎- Gly 1 0 1 0 4.27 (0.01) 1 0 2 0 9.33(0.01) 1 0 1 -1 0.48 (0.03) 1 0 1 -2 -5. 61 (0.001)

n m, a, l and h represents stoichiometric constants corresponding to Cu(II), ALA, L and H+, respectively. bStandard deviations are given in parentheses and coefficient −1 designate proton loss.

Ternary copper (II) complex formation equilibria involving ALA and some peptides

The formation ‎constants of the Cu (II) complexes with ALA and ‎those of ‎peptides, ‎cited in Table 3, are of the ‎same order. Consequently, the ligation of ‎ALA ‎and ‎ peptides will ‎proceed simultaneously. Evidence for formation of mixed ligand ‎complex by simultaneous mechanism ‎was further verified by the good agreement ‎observed between the observed  and ‎calculated data, as shown in Fig. 3 for the ‎glycyl-glycine system, taken as a ‎representative example.‎ The formation constants were determined by fitting potentiometric data on the basis of possible ‎composition ‎models. ‎The most acceptable model was found to be consistent with the formation of the complexes with stoichiometric coefficients 1110 [Cu(ALA)(L)] and 111-1 [Cu(ALA)(LH−1)].  In the 1110 case, the peptide is bound through the amino and carbonyl oxygen groups. On increasing the pH, the coordination sites should switch from the carbonyl oxygen to the amide nitrogen. The amide groups undergo deprotonation and the[Cu (ALA) LH−1]  complexes are formed. The pKa values are calculated by the following equation:

pKa=logβ1110logβ111−1                                                              (3)

The pKa’s of the amide groups for Gly-Gly, ‎ Gly-Ala, Gly-Val, -‎Gly-Leu, Glu and Gly are 9.18, 9.16, 9.01, 9.59, 10.80 and 5.66 respectively. Itis clear that, the pKa value for glycinamide complex is lower than those for other peptides. This may be due to the bulky substituent group on the other peptides that may hinder the structural changes when going from species 1110 to 111-1.

Fig. 3 Potentiometric titration curves for the Cu(II)–ALA- Gly-Gly ‎ system at 25C and 0.10 mol· L−1 ionic strength.

The ∆log K value for deprotonated ternary complexes formed through simultaneous mechanism are given by Eq. (4) ‎ whereas those of the induce deprotonated peptide complex can be calculated using Eq. (5):

∆logK=logβ1110logβ1100logβ1010                                                               (4)

∆logK=logβ111−1logβ1100logβ101−1                                                            (5)

‎ The values of ∆log K for the mixed ligand complexes studied in this paper are listed in Table 2. The theoretical ∆ log K value for a distorted-octahedral Cu(II) complex is -0.9 [16]. The tendency to form ternary complexes was compared with this value, so that if ∆ log K is greater than-0.9, this should be taken as an indication that the ternary complex is favored. The ∆ log K values for the induced deprotonated peptide ternary complexes, [Cu(ALA)(LH−1)] are also considerably more negative than -0.9. This may be taken as an indication that formation of the ternary peptide complexes is less favoured than of binary ones. This may be explained on the premise that the deprotonated peptide is coordinated with the free Cu(II) ion as a tridentate ligand, whereas in the ternary complex two coordination sites are available in the [Cu(ALA)] complex.

Table 3. Stability constants of the ternary species in the Cu(II)-ALA-peptides systems at 25°C and 0.1 mol/L ionic strength.

 Systems m a l hn log βb pKa Δ log K ${{\mathbit{log}}{\mathbit{K}}}_{{\mathbit{Cu}}\left({\mathbit{ALA}}\right){\mathbit{L}}}^{{\mathbit{Cu}}\left({\mathbit{ALA}}\right)}$ % R. Sc. ${\mathbit{logx}}$ Cu(II) – ALA-Gly-Gly ‎ 1 1 1 0 13.33 (0.01) 9.18 -2.94 -5.86 4.66 5.73 -38.68 -2.16 1 1 1 -1 4.15 (0.01) Cu(II) – ALA-Gly-Ala 1 1 1 0 13.92 (0.01) 9.16 -2.96 -5.89 5.25 5.71 -36.05 -3.09 1 1 1 -1 4.76(0.01) Cu(II) – ALA-Gly-Val 1 1 1 0 13.01 (0.02) 9.01 -4.04 -5.10 4.34 4.63 -48.21 -5.81 1 1 1 -1 4.00 (0.01) Cu(II) – ALA-‎Gly-Leu 1 1 1 0 13.34 (0.01) 9.59 -3.54 -6.48 4.67 5.13 -43.12 -5.20 1 1 1 -1 3.75(0.01) Cu(II) – ALA-‎Glu 1 1 1 0 13.83 (0.02) 10.80 -2.65 -6.65 5.16 6.02 -33.93 -1.75 1 1 1 -1 3.03(0.01) Cu(II) – ALA-Gly 1 1 1 0 10.87 (0.01) 5.66 -2.07 -3.94 2.20 6.60 -48.48 -3.37 1 1 1 -1 5.21 (0.001)

Can another parameter definition, which is the percentage of relative stability (% RS) to measure the stability of a ternary complex‎, as follows:

(6)

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For all systems, the parameter % R.S. is negative ‎(Table 3)‎. This may be considered as evidence for the occurrence of enhanced stabilities. Negative values of % R.S. agree with the  ∆ log K values.

log X (nonproportional dissociation constant) can be used to describe the formation tendency of ternary complexes. It measures the tendency of 1 mol each of the binary complexes Cu(ALA)2 and Cu(L)2 to disproportionate forming 2 mol of Cu(ALA)L. This constant equilibrium expression is calculated by equilibrium equations:

(7)

(8)

The log X values were calculated (Tables 3) and the results showed that the values are higher than that expected on a statistical basis (0.6) [17]. This means that the formation of binary complexes is more prevalent than ‎ ternary complexes in these systems. A species distribution diagram obtained for Cu(II) –ALA- glycyl-glycine, considered as representative peptide and is provided in Figure 3. [Cu (ALA)L](1110) starts to format pH at ∼2.8 and, with increasing pH, its ‎concentration increases reaching a maximum of 33% at pH = 5.0.Another increase in pH is accompanied by a decrease in the concentration of 1110 and an increase in the formation of [Cu (ALA) LH-1] (111-1)].

Fig. 4 Representative concentration distribution curves as a function of pH calculated for

Cu(II)–ALA- Gly-Gly system at 25C and 0.10 mol· L−1 ionic strength.

Conclusion

A potentiometric titration technique has been used to determine the formation equilibria of ternary complexes of Cu(II) with ALA as drug ligand and peptides (L). The acidity constants of the ligands selected were determined and used for determining the stability constants of the complexes formed in aqueous solution at 25 °C and ionic strength I = 0.10 mol/L (NaCl). The order of stability of the Cu(II) ion obtained in the mixed ligand complex systems examined in aqueous solution in this study is as follows: Cu(II):ALA: Gly-Val>Cu(II):ALA: Gly-Leu > Cu(II):ALA: Gly-Gly ‎> Cu(II):ALA: ‎ Gly-Val. The fact that the negative log   log X and %RS values were obtained from the mixed ligand complex systems shows that the stability of binary complex systems is more dominant than that of the mixed ligand complex systems. When we examine Figure 4, we can see that CuALA or CuL binary coordination compound is dominant in pH = 3.0–12 range. However, ternary coordination compound is formed after pH=4.0, and it is the only dominant type after pH = 10.0.

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