J Solution Chem (2011) 40:1965–1986DOI 10.1007/s10953-011-9768-1

Complex Formation Between Ferric(III), Chromium(III),and Cupric(II) Metal Ions and (O,N) and (O,O) DonorLigands with Biological Relevance in Aqueous Solution

Ahmed E. Fazary · Erzalina Hernowo ·Artik Elisa Angkawijaya · Tse-Chuan Chou ·Chih Hung Lin · Mohamed Taha · Yi-Hsu Ju

Received: 8 December 2010 / Accepted: 31 March 2011 / Published online: 17 November 2011© Springer Science+Business Media, LLC 2011

Abstract The complexation equilibria of L-norleucine and gallic acid were studied in aque-ous solutions at 25.00 °C and ionic strength 0.15 mol·dm−3 (NaNO3), by means of poten-tiometry and spectrophotometry. The ferric (FeIII), chromium (CrIII), and cupric (CuII) com-plexing abilities of L-norleucine and gallic acid, along with their overall stability constants,were obtained with the HYPERQUAD 2008 program from the potentiometric data. Theconcentration distributions of the various complex species in solution were evaluated anddiscussed. UV–visible spectroscopic measurements were carried out to give qualitative in-formation about the composition of the complexes formed in these solutions. The cytotoxicactivities of the binary and ternary metal complexes of L-norleucine and gallic acid weretested and evaluated against HEp-2 (human laryngeal carcinoma), Daoy (human medul-loblastoma), MCF-7 (human breast adenocarcinoma), and WiDr (human colon adenocar-cinoma) tumor cell lines. Also, their antioxidant activities were examined by free radicalscavenging assay.

Electronic supplementary material The online version of this article(doi:10.1007/s10953-011-9768-1) contains supplementary material, which is available to authorizedusers.

A.E. Fazary · E. Hernowo · A.E. Angkawijaya · M. Taha · Y.-H. Ju (�)Department of Chemical Engineering, National Taiwan University of Science and Technology,43 Keelung Road, Section 4, Taipei 106-07, Taiwane-mail: yhju@mail.ntust.edu.tw

A.E. Fazarye-mail: aefazary@mail.ntust.edu.tw

Present address:A.E. FazaryChemistry Department, Faculty of Science, King Khalid University, Abha 9044, Kingdom of SaudiArabiae-mail: aefazary@gmail.com

T.-C. Chou · C.H. LinSchool of Pharmacy, College of Medicine, National Taiwan University, Jen-Ai Rd. Sec. 1, Taipei 100,Taiwan

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Scheme 1 Structure of L-methionine, L-norleucine, and L-lysine

Keywords Stability constants · L-Norleucine · Gallic acid · Potentiometry · Spectroscopy ·Cytotoxic · Antioxidant

1 Introduction

L-Norleucine (L-2-aminohexanoic acid; (S)-2-aminocaproic acid, C6H13NO2, Nle) is a non-natural amino acid that is experimentally used to study protein structures and functions[1, 2]. It is structurally similar to methionine; however, it does not contain sulfur and is aderivative of the amino acid lysine (Scheme 1) [3, 4]. It has been the subject of severalstructural [5], spectroscopic [6], and thermoanalytical studies [7]. In recent thermoanalyti-cal investigations, Nle was used as a reference system for the thermal behavior of organiccompounds such as pharmaceuticals [8]. The biological studies showed that Nle works asan antiviral agent for the human parainfluenza virus [9], has antitumor [10] and antifibri-nolytic [11] properties, and prevents brain injury [12].

In addition, much biochemical research has been focused on the role of the antioxidantgallic acid in several biological processes [13–15]. In view of the important roles of non-protein amino acids and phenolic acids in various biological systems, obtaining knowledgeof simultaneous metal ion interaction with such bioactive compounds is one of the most ex-citing subject areas in inorganic biochemistry. Therefore, the study of metal–Nle–gallic acid(GA) complexes may lead to better understanding of the behavior of such compounds in bio-logical systems, and metal–Nle–GA interactions might be of therapeutic interest. A search ofthe literature on interaction between metal ions and Nle showed only a few studies [16–27].

Our recent publications describe the protonation equilibrium processes of essential aminoacids [28], hydroxamic acids [29], phenolic compounds [30], and non-protein Nle [31] andl-canavanine [32] amino acids in aqueous and in aqueous–organic mixed solvent solutions.The present work reports a set of potentiometric and spectrophotometric results on the binaryand mixed-ligand complexes involving the transition metal ions iron(III), chromium(III),and copper(II) with Nle and GA. Our contribution is to gain insight into the complexationproperties of Nle and GA. Quantification of metal-chelating activity was accomplished bya potentiometric technique with a glass electrode, and the experimental data were analyzedusing the TINET [33], GLEE [34], and HYPERQUAD 2008 [35] computer programs. A de-tailed quantitative examination of the complexed species formed in the metal ion–Nle–GAsystems is presented. Graphic representation of the concentration curves of complex speciesis given as distribution diagrams produced by means of HySS modeling program [36], whichfurnishes a variety of data presentations including tables of concentrations of all speciespresent in solution in the selected pH range. Spectroscopic analyses of different complexspecies were performed by means of UV–visible spectroscopy. The biological relevance ofsome complex species in aqueous solutions was investigated using cytotoxic and free radicalscavenging assays.

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2 Experimental

2.1 Materials and Solutions

Analytical grade Nle (Merck, Germany) was used without further purification. This Nlewas assayed in triplicate by titration with a carbonate-free solution of standard sodiumhydroxide (Acros Organics, USA). This assay showed that the purity (%) of the Nle was(99.00 ± 0.05%). The carbonate-free sodium hydroxide solution was prepared by dissolv-ing Analar pellets in ultra pure water, and the solution was standardized potentiometri-cally with potassium hydrogen phthalate (Sigma-Aldrich, USA). A nitric acid (Pancreac,Spain) solution (≈ 0.04 mol·dm−3) was prepared and used after standardization. Ferric ni-trate nonahydrate (Fe(NO3)3·9H2O, 99% purity), sodium hydroxide (NaOH, purity > 99%),and sodium nitrate (NaNO3, 99% purity) were supplied by Acros Organics, USA. Gallicacid, copper chloride dihydrate (CuCl2·2H2O, 99% purity), and chromium chloride hex-ahydrate (CrCl3·6H2O, 99% purity) were obtained from Sigma-Aldrich, USA. Analyticalgrade color-coded buffer solutions of pH = 4.0 (red) and 7.0 (green), were purchased fromcommercial sources. All solutions used throughout the experiments were freshly preparedin ultra pure water obtained from a NANO Pure-Ultrapure water system, providing distilledand deionized water with a resistance of 18.3 M�·cm−1.

2.2 Apparatus and Equilibrium Titration Procedures

The pH-potentiometric titrations were performed using a Metrohm 702 SM titroprocessorwith a 664 dosimate, a 728 magnetic stirrer, coupled with a dosino burette model 683. Theelectrode response can be read to the third decimal place in terms of pH units with a pre-cision of ±0.001 and the potential with a precision of ±0.1 mV. The titroprocessor wascoupled to a personal computer and the titration software TINET version 2.4 was used tocontrol the titration and data acquisition. The pH-titrations were carried out in a 150 cm3

commercial glass vessel. The ionic strength of the solutions was maintained at a constantlevel by using the desired concentration of NaNO3 as the supporting electrolyte. The pH me-ter was calibrated with standard buffer solutions. The standard buffers used for calibratingthe pH meter at 25 °C were potassium hydrogen phthalate, pH = 4.004, and potassium di-hydrogen phosphate/dipotassium hydrogen phosphate, pH = 6.968. Calibrations were per-formed both before and after each series of pH measurements. The strong acid versus al-kali titrations were analyzed using the computer program (GLEE, glass electrode evalua-tion), and the results were used for calibration of the glass electrode by means of a strongacid–strong base titration [34–36]. The program GLEE has been developed as part of theHYPERQUAD suite of programs for stability constant determination [34–37]. This pro-gram provides an estimate of the carbonate concentration of the base, the pseudo-Nernstianstandard potential, and the slope of the electrode response, and (optionally) the concentra-tion of the base and the values of pKw (pKw = 13.77 at ∼ 25 °C). For estimation of theprotonation constants of Nle and GA, the following solutions were prepared (total volume50 cm3) and then titrated potentiometrically against a standard carbonate-free NaOH solu-tion (0.1 mol·dm−3):

(a) 0.003 mol·dm−3 HNO3 + 0.1 mol·dm−3 NaNO3,(b) solution (a) + 0.001 mol·dm−3 Nle,(c) solution (a) + 0.001 mol·dm−3 GA.

1968 J Solution Chem (2011) 40:1965–1986

Nowadays, most equilibrium studies are carried out over large ranges of reactant concen-trations and concentration ratios, but using these ranges always suffers inevitable limitationsdue to factors such as variations of activity coefficients for weak complexes, solubility prob-lems, etc., and some potential applications are to composition regions outside those exper-imentally investigated. In our experiments, for the determination of binary metal complexconstants, solutions containing Nle or GA and metal ions were titrated at 1:1, 1:2 and 1:3metal ion-to-ligand mole ratios to reach the maximum coordination number of the metalion, whereas for ternary systems the 1:1:1 metal ion/Nle/GA ratio was used. The concen-tration of metal ion and ligand solutions in the titrated samples were always the same andwere varied in the range of 0.00004 to 0.0001 mol·dm−3. The pH titrations were carriedout in an 80 cm3 commercial double-walled glass vessel. Each solution was thermostattedat 25.00 °C and left to stand for several minutes before titration, and the cell temperaturewas maintained at the desired value of 25.00 °C by circulating thermostatted water using anoil-bath setup. A magnetic stirrer was used during all titrations. Each titration was repeatedat least 3 times under carefully controlled experimental conditions. Typically, more than 80pH readings (data points of potentiometric measurements) were collected and taken intoaccount for each titration.

2.3 Calculations

For computing the protonation constants of the ligands (Nle and GA) and their metal com-plexes from pH titration data, the best equilibrium model was sought by systematically test-ing various pqr conditions using the advanced software program HYPERQUAD 2008 [35].This software facilitates visual interpretation of refinements, which in turn helps greatly inobtaining the best fit. To compute stability constants from potentiometric and spectropho-tometric data, many software programs such as BEST, LETAGROP, MINIQUAD, PKAS,SUPERQUAD, and HYPERQUAD have been used. All of these programs use the least-squares approach. Among them, HYPERQUAD 2008 is one of the most recent versions ofthe HYPERQUAD program to determine stability constants from potentiometric and spec-trometric data [35].

2.4 Spectrophotometric Measurements

Absorption spectra were obtained using UV–vis spectra (Perkin-Elmer, model Lambda 25).

2.5 Cytotoxic Activities

The tumor cell lines HEp-2 (human laryngeal carcinoma), Daoy (human medulloblastoma),MCF-7 (human breast adenocarcinoma), and WiDr (human colon adenocarcinoma), usedfor cytotoxic assays, were cultured in the RPMI-1640 medium supplemented with 5% CO2

in an incubator at 37 °C. The cytotoxicity assay depends on the binding of methylene blueto fixed monolayers of cells at pH = 8.5, washing the monolayer, and releasing the dyeby lowering the pH value. The control standard drug was prepared at a concentration of150 µg·mL−1. All complex species samples were prepared in saline water at a concentrationof 1 × 10−6 mol·dm−3 for Nle, GA, Fe(III), Cr(III), and Cu(III), at pH ∼ 7. After seeding2880 cells per well in a 96-well microplate at about 3 µL, 20 µL of sample or standard agentwas placed in each well and incubated at 37 °C for 3 days. After removing the mediumfrom the microplates, the cells were fixed with 10% formaldehyde in 0.9% saline solutionfor 30 min, and then dyed with 1% (w/v) methylene blue in 0.01 mol·dm−3 borate buffer

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(100 µL per well) for 30 min. The 96-well plate was then dipped into a 0.01 mol·dm−3 boratebuffer solution four times in order to remove the dye. Then, 100 µL of EtOH–0.1 mol·dm−3

HCl (1:1) was added to each well as a dye eluting solvent, and the absorbance was measuredon a microtiter plate reader (Dynatech, MR 7000) at 650 nm. The IC50 value was definedby a comparison with the untreated cells when the concentration of test sample resulting in50% reduction of the absorbance. Each experimental variable was run three times, and theresults were expressed via their mean value and standard deviation. A two-tailed P value ofless than 0.05 was considered to be statistically significant.

2.6 Antioxidant Activities

Antiradical activities of the tested complex species were measured using the stable radi-cal 2,2-diphenyl-1-picrylhydrazyl-hydrate (DPPH) [38]. All complex species samples wereprepared in methanol at a concentration of 1 × 10−6 mol·dm−3 for Nle, GA, Fe(III), Cr(III),and Cu(III). About 50 µL of each complex species sample was placed in 1 cm cuvettes,and 2 mL of a methanolic solution of DPPH (6 × 10−5 mol·dm−3) were added to each. Ab-sorbance measurements commenced immediately. The decrease in absorbance at 515 nmwas determined continuously with data acquisition at 2 s intervals, using a spectrophotome-ter, until the absorbance stabilized (16 min). The absorbance of the DPPH radical withoutantioxidant, i.e. the control, was measured daily. Special care, as recommended [38], wastaken to minimize the loss of free radical activity of the DPPH radical stock solution. Alldeterminations were performed in triplicate. The percent inhibition of the DPPH radical bygallic acid and tested compounds was calculated according to the formula:

Percent inhibition = ([AC(0) − AC(t)]/[AC(0)]) × 100

where AC(0) is the absorbance of the control at t = 0 min and AA(t) is the absorbance ofthe reaction solution at t = 16 min. Each experimental variable was run three times, and theresults were expressed via their mean value and standard deviation. A two-tailed P value ofless than 0.05 was considered to be statistically significant.

3 Results and Discussion

In our present work, a pH-potentiometric measurement technique was selected to evalu-ate the stability constants data due to its high degree of accuracy and the reliability of theproton change data obtained with a pH electrode. Before analyzing the experimental datausing the HYPERQUAD program, two important issues must be resolved concerning theirmode and scale of representation. Stability constants may a priori be expressed in terms ofactivity (true thermodynamic parameter) or concentration (stoichiometric constants) quo-tients. Because logarithmic quantities are non-dimensional, it is often forgotten that stabilityconstant’s numerical value depend on the units in which activity terms are expressed, themolar concentration scale being the most commonly used. This remark may seem of minorimportance, but it nonetheless implies that comparisons between logarithmic constants arepermissible for complexes of equivalent stoichiometries only. Therefore, when simulationmodels are used to predict the distribution of such complexes, all these considerations areimplicitly taken into account in the calculations. Basically, the quality of the complexationmodel that can be obtained from the calculations is limited by the experimental data (poten-tiometric accuracy, kinetic effects, precipitation, etc.).

Each titration curve (Figs. 1, 3, 5(a)) of Nle and GA exhibits an inflection point cor-responding to the protonation constants. The protonation constants (log10 β1 and log10 β2)

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Fig. 1 (a) Potentiometrictitration and (b) speciesdistribution curves for the Fe(III)+ L-norleucine (Nle) + gallicacid (GA) system atT = 298.15 K andI = 0.15 mol·dm−3 NaNO3.Percentages are calculated withrespect to the analyticalconcentration of metal ion

evaluated for Nle and GA are presented in Table 1. Certain functional groups found in bi-ological molecules, in particular carboxylic acids or amino groups, can gain or lose a H+

depending on the concentration of hydrogen ions (protons) in the solution. In computer pro-grams such as HYPERQUAD, the protonation equilibira of the ligand are defined as overallassociation constants. This means that for Nle or GA, the two equilibrium constants areexpressed as

H+ + A2− � HA−; [HA−] = β1[H+][A2−]2H+ + A2− � H2A; [H2A] = β2[H+]2[A2−].

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Fig. 2 Species distributiondiagrams for the (a) Fe(III) +L-norleucine (Nle), (b) Fe(III) +gallic acid (GA) binary systemsat T = 298.15 K andI = 0.15 mol·dm−3 NaNO3.Percentages are calculated withrespect to the analyticalconcentration of metal ion

The stepwise acid dissociation constants of Nva or FA are defined as

H2A � H+ + HA−; [H2A] = Ka1[H+][HA−]HA− � H+ + A2−; [HA−] = Ka2[H+][A2−].

It is noteworthy that the measured pKa1 (2.31) and pKa2 (9.35) values of Nle can beassociated with the carboxylic acid and amino functions, respectively, since log10 β = pKa2

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Fig. 3 (a) Potentiometrictitration and (b) speciesdistribution curves for the Cr(III)+ L-norleucine (Nle) + gallicacid (GA) system atT = 298.15 K andI = 0.15 mol·dm−3 NaNO3.Percentages are calculated withrespect to the analyticalconcentration of metal ion

and log10 β2 = pKa1 + pKa2. While for GA, the protonation constants pKa1 (4.33) and pKa2

(8.71) were obtained for the carboxylic acid and the hydroxyl group at C(4) in the meta-position, respectively. From the chemical structures shown in Scheme 2 and the protona-tion equilibria of Nle and GA, one can conclude that protonation equilibrium constants arecontrolled by the electronic effects of substituent groups, aromatic nucleus, and the spacebetween the functional groups.

From the experimental data shown in Table 1, it was observed that the protonation con-stants of Nle and GA obtained in this work are consistent with those reported in the literaturewithin a very reasonable range, after allowing for the variation in experimental conditionssuch as ionic strength and background medium as well as the methods of calculation [16,22, 25, 26].

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Fig. 4 Species distributiondiagrams for the (a) Cr(III) +L-norleucine (Nle), (b) Cr(III) +gallic acid (GA) binary systemsat T = 298.15 K andI = 0.15 mol·dm−3 NaNO3.Percentages are calculated withrespect to the analyticalconcentration of metal ion

It was known that the use of protonation constants as references for determining metalcomplex stability constants is preferred. The formation constants (log10 βpqrs) for the binaryand mixed-ligand systems involving the transition metal ions (Fe2+, Cr3+, and Cu2+) withNle and GA, as well as the pH intervals in which the data were collected, are presented inTables 2–6.

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Fig. 5 (a) Potentiometric titration and (b) species distribution curves for the Cu(II) + L-norleucine (Nle) +gallic acid (GA) system at T = 298.15 K and I = 0.15 mol·dm−3 NaNO3. Percentages are calculated withrespect to the analytical concentration of metal ion

The complexation between metal ion (M) and Nle and GA can be described by the gen-eral equilibrium reaction:

pM + qNle + rGA + sH � MpNleqGArHs

βpqrs = [MpNleqGArHs]/[M]p[Nle]q[GA]r [H]s

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Table 1 Logarithms of the protonation constants (βqqrH) of L-norleucine (Nle) and gallic acid (GA) in

water at the ionic strength I = 0.15 mol·dm−3 and temperature t = 25.00 °Ca

Species p q r s log10 βpqrs pH range S.D.

L-Norleucine (Nle)

NleH− 0 1 0 1 9.35 (9.67)b 2.59–11.10 0.0272

NleH2 0 1 0 2 11.66 (12.01)b 2.59–11.10 0.1161

Gallic acid (GA)

GAH+ 0 1 1 1 8.71 (8.70)c 2.53–11.10 0.0240

GAH2 0 1 1 2 13.04 (12.96)c 2.53–11.10 0.0374

aAll the values were calculated with the program Hyperquad 2008 from potentiometric investigations at

25.00 °C and I = 0.15 mol·dm−3 NaNO3. The symbols p, q , r , and s are used in the programs to indicatethe stoichiometric coefficients associated with the possible equilibria in solutionsbData between parentheses were extracted from Refs. [16, 22]cData between parentheses were extracted from Ref. [25]

Scheme 2 Protonation equilibria of L-norleucine and gallic acid

where p, q , r , s are the stoichiometric coefficients associated with the possible equilibria insolutions for metal ion, ligand Nle, ligand GA and H-atoms, respectively. The stoichiome-tries and stability constants of the complexes formed were determined by trying variouspossible composition models. The selected model was the one that gave the best statisticalfit, seemed chemically sensible, and was consistent with the titration data without giving anysystematic drifts in the magnitudes of the various residuals.

Figures 1, 3 and 5(a) show the pH-metric titration curves of the metal ion–Nle–GA binaryand ternary systems. Experimental and calculated titration curves are represented by sym-bols and dashed lines, respectively. Good fitting between these curves and the experimentalresults confirms the validity of the obtained complexation models. In those figures, we cansee a good overlap of experimental and calculated values of pH. Except for the [Fe(III)–GA]complexes, refinements using the entire pH range were successful for the other systems,which were carried out over the pH range ∼2.5 to 5.7 and ∼2.5 to 9.6 for [Cr(III)–Nle]and [Cr(III)–GA] complexes, respectively. In all titrations, the experimental pH titrationcurves recorded for the binary and ternary metal ion–Nle–GA systems, when comparedwith the pH titration curves of Nle and GA ligands alone as shown in Figs. 1, 3, 5(a),

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Table 2 Logarithms of the overall stability constants (βqqrH) of binary and ternary ferric (Fe(III)) complexes

with L-norleucine (Nle) and gallic acid (GA) in water at the ionic strength I = 0.15 mol·dm−3 NaNO3 andtemperature t = 25.00 °Ca

Complex series p q r s log10 βpqrs pH range S.D.

L-Norleucine (Nle) complexesFeNle+ 1 1 0 0 9.92 2.53–11.14 0.1021Fe(Nle)−2 1 2 0 0 20.11 2.53–11.14 0.0318

Fe(Nle)3−3 1 3 0 0 29.83 2.53–11.14 0.1388

FeNleH2+ 1 1 0 1 13.08 2.53–11.14 0.0888FeNle(H)−1 1 1 0 −1 7.40 2.53–11.14 0.0893FeNle(H)−−2 1 1 0 −2 1.37 2.53–11.14 0.1215

FeNle(H)2−−3 1 1 0 −3 −6.17 2.53–11.14 0.1044

Fe(Nle)2(H)2−−1 1 2 0 −1 15.63 2.53–11.14 0.1420

Fe(Nle)2(H)4−−3 1 2 0 −3 −2.35 2.53–11.14 0.1027

Fe(OH)2+ 1 0 0 −1 −0.84 2.53–11.14 0.1249Fe(OH)+2 1 0 0 −2 −5.34 2.53–11.14 0.1318Gallic acid (GA) complexesFeGA+ 1 0 1 0 11.00 (11.392)b 2.49–5.74 0.1067Fe(GA)−2 1 0 2 0 18.73 (19.61)b 2.49–5.74 0.1350

Fe(Nle)3−3 1 0 3 0 26.08 (24.26)b 2.49–5.74 0.1395

FeGAH2+ 1 0 1 1 14.26 2.49–5.74 0.1169Fe(OH)2+ 1 0 0 −1 −2.42 2.49–5.74 0.1244Fe(OH)+2 1 0 0 −2 −5.04 2.49–5.74 0.1332Fe(OH)3 1 0 0 −3 −10.64 2.49–5.74 0.0909Mixed ligand complexesFeNleGA− 1 1 1 0 21.02 2.41–11.16 0.0921FeNleGAH 1 1 1 1 25.74 2.41–11.16 0.0938FeNleGAH+

2 1 1 1 2 29.21 2.41–11.16 0.1086

FeNleGA(H)2−−1 1 1 1 −1 14.96 2.41–11.16 0.1100

FeNleGA(H)3−−2 1 1 1 −2 6.62 2.41–11.16 0.0934

aAll the values were calculated with the program Hyperquad 2008 from potentiometric investigations at

25.00 °C and I = 0.15 mol·dm−3 NaNO3. The symbols p, q , r , and s are used in the programs to indicatethe stoichiometric coefficients associated with the possible equilibria in solutionsbData between parentheses were extracted from Refs. [16–27]

prove that complex formation did occur between the metal ions studied (Fe3+, Cr3+, andCu2+) and the two ligands (Nle and GA). Ternary complex formation may proceed eitherthrough a stepwise or simultaneous mechanism depending on the chelating potentials of Nleand GA.

The color of the solution was dark brown for the Fe3+–Nle–GA systems at pH < 3,changed to dark purple at pH ∼ 3.6, and then became light purple at pH ∼ 6.3. For theCr3+–Nle–GA systems the solution was initially pinkish blue, changed to dark greenish-blue at 4.3 < pH < 6, then to green at pH ∼ 6.38, then became turbid orange at pH ∼ 7.43,and finally turbid green at pH ∼ 9.8. As for the Cu2+–Nle–GA system, its color changedfrom initially light yellow to turbid orange at pH = 5.5, then to greenish blue at pH = 9.67.No precipitation occurred in any solutions during titrations. However, a slight turbidity wasobserved in the case of Cr3+–Nle–GA systems at pHs ∼ 5.38 to 8.0.

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Table 3 Logarithms of the overall stability constants of (βqqrH) of binary and ternary chromium (Cr(III))

complexes with L-norleucine (Nle) and gallic acid (GA) in water at the ionic strength I = 0.15 mol·dm−3

and temperature t = 25.00 °Ca

Complex series p q r s log10 βpqrs pH range S.D.

L-Norleucine (Nle) complexes

CrNle+ 1 1 0 0 8.41 2.57–9.34 0.0822

Cr(Nle)−2 1 2 0 0 15.71 2.57–9.34 0.0768

Cr(Nle)3−3 1 3 0 0 22.49 2.57–9.34 0.1190

Cr(Nle)2H 1 2 0 1 21.10 2.57–9.34 0.1257

CrNleH2+ 1 1 0 1 12.05 2.57–9.34 0.1233

CrNle(H)−−2 1 1 0 −1 −4.28 2.57–9.34 0.0722

Cr(Nle)2(H)3−−2 1 2 0 −2 0.97 2.57–9.34 0.1388

CrNle2(H)2−−1 1 2 0 −1 9.30 2.57–9.34 0.1281

Cr(OH)2+ 1 0 0 −1 −3.38 2.57–9.34 0.1390

Gallic acid (GA) complexes

CrGA+ 1 0 1 0 8.30 (9.50)b 2.52–9.63 0.0688

Cr(Ga)−2 1 0 2 0 14.25 (18.5)b 2.52–9.63 0.0304

CrGAH2+ 1 0 1 1 12.66 2.52–9.63 0.0546

CrGA(H)−1 1 0 1 −1 1.97 2.52–9.63 0.0964

CrGA(H)−−2 1 0 1 −2 −4.75 2.52–9.63 0.1318

CrGA2H 1 0 2 1 20.20 2.52–9.63 0.1239

Cr(OH)2+ 1 0 0 −1 −3.45 2.52–9.63 0.1419

Cr(OH)3 1 0 0 −3 −16.63 2.52–9.63 0.1432

Mixed ligand complexes

CrNleGA− 1 1 1 0 19.75 2.55–11.17 0.0550

CrNleGA(H)2+3 1 1 1 3 34.03 2.55–11.17 0.0550

CrNleGAH 1 1 1 1 25.67 2.41–11.17 0.0354

CrNleGAH+2 1 1 1 2 30.37 2.41–11.17 0.0448

CrNleGA(H)2−−1 1 1 1 −1 12.67 2.55–11.17 0.4160

CrNleGA(H)3−−2 1 1 1 −2 3.59 2.55–11.17 0.0663

CrNleGA(H)4−−3 1 1 1 −3 −5.90 2.55–11.17 0.0632

aAll the values were calculated with the program Hyperquad 2008 from potentiometric investigations at

25.00 °C and I = 0.15 mol·dm−3 NaNO3. The symbols p, q , r , and s are used in the programs to indicatethe stoichiometric coefficients associated with the possible equilibria in solutionsbData between parentheses were extracted from Refs. [16–27]

Estimation of the concentration distributions of the various species in solution providesa useful picture of metal ion binding. The equilibrium distributions of various complexspecies formed in water solutions are shown as functions of pH in Figs. 1–4 and 5(b).The graphic distribution diagrams in Figs. 1–4 and 5(b) show that the protonated bi-nary and mixed-ligand complex species, which include FeNleH2+, FeGAH2+, FeNleGAH,FeNleGAH+

2 , CrNleH2+, CrNle2H, CrGAH2+, CrNleFAH, CrNleGAH+2 , CrNleGAH2+

3 ,CuNleH+, CuGAH+, CuNleGAH2, and CuNleGAH3, started forming at very low pH.The deprotonated binary and mixed-ligand complexes such as FeNle+, FeNle−

2 , FeNle3−3 ,

1978 J Solution Chem (2011) 40:1965–1986

Table 4 Logarithms of the overall stability constants (βqqrH) of binary and ternary cupric (Cu(II)) com-

plexes with L-norleucine (Nle) and gallic acid (GA) in water at ionic strength I = 0.15 mol·dm−3 and tem-perature t = 25.00 °Ca

Complex series p q r s log10 βpqrs pH range S.D.

L-Norleucine (Nle) complexes

CuNle 1 1 0 0 8.11 (8.18)b 2.58–11.06 0.0686

Cu(Nle)2−2 1 2 0 0 14.97 (14.88)b 2.58–11.06 0.0893

CuNleH+ 1 1 0 1 12.16 2.58–11.06 0.0598

CuNle(H)−−1 1 1 0 −1 2.09 2.58–11.06 0.0860

Cu(Nle)2(H)3−−1 1 2 0 −1 7.97 2.58–11.06 0.0903

Gallic acid (GA) complexes

CuGA 1 0 1 0 10.82 (9.75)b 2.49–11.06 0.1419

Cu(GA)2−2 1 0 2 0 18.43 (19.50)b 2.49–11.06 0.0624

CuGAH+ 1 0 1 1 16.70 2.49–11.06 0.0425

CuGA(H)−−1 1 0 1 −1 6.23 2.49–11.06 0.1055

CuGA(H)3−−3 1 0 1 −3 −8.75 2.49–11.06 0.1259

Cu(GA)2(H)3−−1 1 0 2 −1 13.01 2.49–11.06 0.0542

Cu(GA)2(H)4−−2 1 0 2 −2 5.26 2.49–11.06 0.0724

CuGA(H)3−2 1 0 1 2 21.29 2.49–11.06 0.0863

Mixed ligand complexes

CuNleGA2− 1 1 1 0 19.70 2.54–11.14 0.0498

CuNleGA(H)2 1 1 1 2 30.02 2.54–11.14 0.0385

CuNleGa(H)+3 1 1 1 3 33.14 2.54–11.14 0.0938

CuNleGA(H)3−−1 1 1 1 −1 14.36 2.54–11.14 0.0975

CuNleGA(H)4−−2 1 1 1 −2 7.40 2.54–11.14 0.0582

aAll the values were calculated with the program Hyperquad 2008 from potentiometric investigations at

25.00 °C and I = 0.15 mol·dm−3 NaNO3. The symbols p, q , r , and s are used in the programs to indicatethe stoichiometric coefficients associated with the possible equilibria in solutionsbData between parentheses were extracted from Refs. [16–27]

FeGA+, FeGA−2 , FeGA3−

3 F, FeNleGA2−−1, CrNle+, CrNle−

2 , CrNle3−3 , CrGA+, CrGA−

2 ,CrNleGA+, CuNle, CuNle2−

2 , CuGA, CuGA2−2 , CuNleGA2− were formed under acidic con-

ditions. On the other hand, the systems FeNleH−1, FeNle2H−1, FeNle2H4−−3, FeNleH1−

−2,FeNleH2−

−3, FeNleGAH2−−1, FeNleGAH3−

−2, CrNleH1−−2, CrNle2H3−

−2, CrNle2H2−−1, CrGAH−1,

CrGAH1−−2, CrNleGAH2−

−1, CrNleGAH3−−2, CrNleGAH4−

−3, CuNleH−−1, CuNle2H3−

−1, CuGAH−−1,

CuGAH3−−3, CuGA2H3−

−1, CuGA2H4−−2, CuNleGAH3−

−1, and CuNleGAH4−−2 started to form

at neutral pH. The metal hydroxy complex species were formed under alkaline condi-tions.

In coordination chemistry, the HSAB theory classifies acceptors (metal ions, proton) anddonors (ligands: Nle, GA) as being hard or soft, based on their thermodynamic behaviors(or coordination affinities) in aqueous solutions. The simple rule of this useful theory is that‘hard acceptors prefer hard donors and soft acceptors prefer soft donors’ [27]. Both iron(III)and chromium(III) are considered to be hard acceptors, while copper(II) is considered to

J Solution Chem (2011) 40:1965–1986 1979

Table 5 Cytotoxic activities of L-norleucine, gallic acid and their metal complexes against human tumorcells

Compound/cancer cell linea

IC50 (µg·mL−1)b

HEp-2 Daoy MCF-7 WiDr

Nle 9.15 ± 0.21 10.32 ± 0.61 5.89 ± 0.83 4.63 ± 0.51

GA 17.36 ± 0.14 13.41 ± 0.10 11.39 ± 0.12 18.16 ± 0.14

FeNle+ 3.61 ± 0.11 4.72 ± 0.13 5.19 ± 0.13 4.03 ± 0.11

Fe(Nle)−2 4.31 ± 0.14 3.41 ± 0.14 5.26 ± 0.12 4.06 ± 0.14

Fe(Nle)3−3 3.15 ± 0.11 3.71 ± 0.13 5.19 ± 0.21 4.03 ± 0.11

FeGA+ 9.16 ± 0.14 10.41 ± 0.18 11.21 ± 0.17 8.36 ± 0.15

Fe(GA)−2 10.65 ± 0.21 11.76 ± 0.15 12.19 ± 0.13 4.62 ± 0.18

Fe(GA)3−3 9.36 ± 0.13 10.49 ± 0.16 11.36 ± 0.17 8.96 ± 0.14

FeNleGA− 13.25 ± 0.31 14.27 ± 0.27 15.09 ± 0.23 14.28 ± 0.17

CrNle+ 3.36 ± 0.14 4.49 ± 0.11 2.36 ± 0.17 2.96 ± 0.14

Cr(Nle)−2 2.65 ± 0.11 3.76 ± 0.21 1.89 ± 0.10 1.63 ± 0.09

Cr(Nle)3−3 2.36 ± 0.14 2.49 ± 0.11 1.36 ± 0.07 0.96 ± 0.04

CrGA+ 3.65 ± 0.11 4.76 ± 0.13 5.89 ± 0.18 4.63 ± 0.12

Cr(GA)−2 4.36 ± 0.14 5.49 ± 0.14 6.36 ± 0.17 5.96 ± 0.14

CrNleGA− 3.69 ± 0.10 4.96 ± 0.10 5.23 ± 0.83 4.68 ± 0.02

CuNle 11.86 ± 0.24 11.46 ± 0.16 11.86 ± 0.19 10.98 ± 0.21

Cu(Nle)2−2 13.15 ± 0.21 14.16 ± 0.21 12.19 ± 0.13 12.63 ± 0.22

CuGA 19.96 ± 0.64 17.49 ± 0.14 15.36 ± 0.17 18.96 ± 0.14

Cu(GA)2−2 19.65 ± 0.21 17.76 ± 0.11 15.89 ± 0.13 17.63 ± 0.21

CuNleGA2− 19.98 ± 0.17 18.49 ± 0.22 17.36 ± 0.18 18.96 ± 0.21

aThe IC50 value is the molarity at which 50% tumor cell death was observed after 72 h under standard tissueculture conditions. Each experimental variable was run three times, and the results were expressed via theirmean value and standard deviation. A two-tailed P value of less than 0.05 was considered to be statisticallysignificantbCancer cell lines: HEp-2 (human laryngeal carcinoma), Daoy (human medulloblastoma), MCF-7 (humanbreast adenocarcinoma), and WiDr (human colon adenocarcinoma) tumor cell lines

be a borderline ion. On the other hand, Nle possesses two different donor atoms: N (of theamino group) and O (of the carboxyl group), while GA possesses one donor atom O (of thephenolic group) and another O (of the carboxylic group). Hence, during complexation ofFe3+, Cr3+ and Cu2+ with Nle or GA, these donor atoms are involved.

The coordination chemistry of Cu2+ with ligands containing N and O donors has asignificant effect on its fate, transport, and reactivity in biological and environmental sys-tems [39]. The hydrated Cu2+ ion is the predominant species in aqueous solution at pH < 7,while the mono- and polynuclear hydroxide complexes are formed in appreciable amountsat pH > 7 [39]. The hydrolysis reactions of Cu2+ are critical to understanding the speciationof the Cu2+–hydroxide complexes in aqueous and alkaline solutions of biological and in-dustrial importance [39]. Generally, there are two aspects to metal hydrolysis that we needto consider during our modeling of the experimental data: (i) hydrolysis of the metal ionitself and (ii) hydrolysis of a binary or ternary complex. From our experimental results,we conclude that experimental conditions may alter the hydrolytic behavior of iron(III),chromium(III) and copper(II) ions as reported previously by many other researchers. In theconcentration range selected for the metal ions studied, the log10 β values are not consid-

1980 J Solution Chem (2011) 40:1965–1986

Table 6 DPPH radicalscavenging assay ofL-norleucine, gallic acid andtheir metal complexes

aEach experimental variable wasrun three times, and the resultswere expressed via mean valueand standard deviation.A two-tailed P value of less than0.05 was considered to bestatistically significant

Complex species DPPH inhibition (%)a

Nle 33.65 ± 1.18

GA 89.12 ± 2.14

FeNle+ 53.25 ± 1.12

Fe(Nle)−2 49.16 ± 1.09

Fe(Nle)3−3 57.62 ± 1.15

FeGA+ 96.36 ± 2.27

Fe(GA)−2 93.68 ± 2.24

Fe(GA)3−3 97.36 ± 2.12

FeNleGA− 93.85 ± 2.13

CrNle+ 23.12 ± 1.21

Cr(Nle)−2 21.62 ± 1.12

Cr(Nle)3−3 23.15 ± 1.17

CrGA+ 49.86 ± 1.19

Cr(GA)−2 67.92 ± 1.25

CrNleGA− 79.46 ± 1.21

CuNle 53.26 ± 1.24

Cu(Nle)2−2 44.25 ± 1.31

CuGA 94.12 ± 2.22

Cu(GA)2−2 93.11 ± 2.17

CuNleGA2− 92.85 ± 2.27

erably affected in the same ionic medium. However the data obtained previously at varioustemperatures significantly contribute to understanding the hydrolysis behavior of these metalions. With all of these considerations, knowledge of hydrolysis constants of the iron(III),chromium(III) and Cu(II) metal ions appears to be of primary value, and the present workmay also contribute to the data collection.

A summarization of all of the logarithms of overall stability constants (log10 βpqrs ) ofbinary and mixed ligand complexes investigated is reported in Tables 2–4. As can be seenfrom this data, we can concluded that:

(i) The stability constants of Fe3+–GA, Cr3+–GA, Cu2+–Nle, and Cu2+–GA binary com-plexes show non-significant differences from the data reported previously in the litera-ture [16–27].

(ii) The overall stabilities of ferric(III), chromium(III), cupric(II) binary complexes of Nleare significantly higher than those of GA.

(iii) By comparing the stability constants of binary and mixed-ligand complexes, it wasdemonstrated that ternary complexation is responsible for the stabilization of the mixedcomplexes.

UV–visible absorption spectra are known to be useful for proving the presence of Nleand GA binary and ternary complexes of iron(III), chromium(III), and copper(II) metalions. As shown in Figs. 7–9, Nle shows almost no absorption signals, whereas GA showsa strong broad absorption band at 320 nm. The UV–visible spectroscopic measurementsclearly show an obvious absorption trend for each binary and ternary complex species as

J Solution Chem (2011) 40:1965–1986 1981

Fig. 6 Species distributiondiagrams for the (a) Cu(II) +L-norleucine (Nle), (b) Cu (II) +gallic acid (GA) binary systemsat T = 298.15 K andI = 0.15 mol·dm−3 NaNO3.Percentages are calculated withrespect to the analyticalconcentration of metal ion

shown in Figs. 7–9. The spectra of the ternary systems are quite different from those of thebinary systems, emphasizing their formation in solution.

Acknowledgements Financial Support was provided by National Taiwan University of Science and Tech-nology Research Fund (contract No. 99-0541064-001-0001).

1982 J Solution Chem (2011) 40:1965–1986

Fig. 7 Absorption spectra for (—) L-norleucine, (- - -) gallic acid, (– – –) binary system (0.001 mol·dm−3

FeIII + 0.001 mol·dm−3 L-norleucine), (–·–) binary system (0.001 mol·dm−3 FeIII + 0.001 mol·dm−3 gal-lic acid), (–··–) ternary system (0.001 mol·dm−3 FeIII + 0.001 mol·dm−3 L-norleucine + 0.001 mol·dm−3

gallic acid)

Fig. 8 Absorption spectra for (—) L-norleucine, (- - -) gallic acid, (– – –) binary system (0.001 mol·dm−3

CrIII + 0.001 mol·dm−3 L-norleucine), (–·–) binary system (0.001 mol·dm−3 CrIII + 0.001 mol·dm−3 gal-lic acid), (–··–) ternary system (0.001 mol·dm−3 CrIII + 0.001 mol·dm−3 L-norleucine + 0.001 mol·dm−3

gallic acid)

J Solution Chem (2011) 40:1965–1986 1983

Fig. 9 Absorption spectra for (—) L-norleucine), (- - -) gallic acid, (– – –) binary system (0.001 mol·dm−3

CuII + 0.001 mol·dm−3 L-norleucine), (–·–) binary system (0.001 mol·dm−3 CuII + 0.001 mol·dm−3 gallicacid), (–··–) ternary system (0.001 mol·dm−3 CuII +0.001 mol·dm−3 L-norleucine+0.001 mol·dm−3 gallicacid)

Supplementary Data

Potentiometric titration experimental data points for all systems associated with this article,listed in 3 tables as a supplementary file and can be found in the online version.

Appendix: Cytotoxic Activities and Antioxidant Activities of L-norleucine, GallicAcid and Their Metal Complexes

A.1 Cytotoxic Activities

Some of the nonprotein amino acid and phenolic chelates studied were found to behaveas rather efficient anti-proliferative and cytotoxic agents, with their effect on healthy cellsbeing reversible upon drug removal. From the results now obtained (Table 5), it is possi-ble to conclude that for the complexes studied under physiological conditions, the nature ofthe amine ligand(s) (e.g., the number of N atoms), the characteristics of the leaving groupson the copper, chromium, and iron ions, the number and coordination mode of the metalion(s), and their chemical environment determine their antitumor activity, most probablythrough induction of DNA structural rearrangements. Thus, the design of new, more effec-tive anticancer drugs should be governed by these crucial factors, since slight changes inthe metal coordination are sufficient to significantly change the in vitro anti-proliferativeand/or cytotoxic properties of these compounds. Among the complex species tested againstthe four human cancer cell lines, i.e., HEp-2 (human laryngeal carcinoma), Daoy (humanmedulloblastoma), MCF-7 (human breast adenocarcinoma), and WiDr (human colon ade-nocarcinoma), the chromium complexes of Nle and GA showed weak cytotoxic activities. It

1984 J Solution Chem (2011) 40:1965–1986

was observed that, the copper complexes of Nle and GA exhibited strong cytotoxic activitiesagainst the four cancer cell lines, while iron complex species showed a moderate cytotoxicactivity (Table 5).

A.2 Antioxidant Activities

It is known that copper, chromium, and iron metal ions are essential trace metals for manyliving organisms [40]. These metal ions play a crucial role in various enzymes that cat-alyze oxidation/reduction reactions correlated with the antioxidant system of these organ-isms [41]. It has been reported that certain copper, chromium, iron complexes catalyze rad-ical formation while others seem to have antioxidant efficacy [42]. However, these differentbehaviors depend upon the chemical environment and nature of the chelating agent. In thepresent work, the copper and iron complexes of GA showed a very strong antioxidant activ-ity, while chromium complexes of GA exhibited moderate antioxidant activities (Table 6).Also, the iron, and copper complexes of Nle exhibited a moderate antioxidant activity, whilethe chromium complexes of Nle exhibited weak antioxidant activities. It worth noting thatthe iron and copper ternary complexes of Nle and GA showed a very strong antioxidant ac-tivity, while chromium ternary complexes of Nle and GA showed good antioxidant activity(Table 6).

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