Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 271–280

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Theoretical spectroscopic studies and identification of metal-citrate (Cd and Pb) complexes by ESI-MS in aqueous solution Alexandre C. Bertoli a,⇑, Ruy Carvalho a, Matheus P. Freitas a, Teodorico C. Ramalho a, Daiana T. Mancini a, Maria C. Oliveira b, Amarílis de Varennes c, Ana Dias b a b c

Departamento de Química, Universidade Federal de Lavras, Caixa Postal 3037, 37200-000 Lavras, MG, Brazil Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisbon, Portugal Departamento de Química Ambiental, Instituto Superior de Agronomia, Universidade de Lisboa, 1399-017 Lisbon, Portugal

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

2+ and Pb2+) were studied by theoretical calculations, ESI MS and FTIR-ATR.  The thermodynamic stability of the metal complex was analyzed.  Theoretical calculations allowed the band assignments.

 Citrate-metal complexes (Cd

a r t i c l e

i n f o

Article history: Received 6 May 2014 Received in revised form 7 August 2014 Accepted 23 August 2014 Available online 1 September 2014 Keywords: Cd/Pb complexes ESI-MS FTIR-ATR DFT PM6

a b s t r a c t The combined use of ESI-MS, FTIR-ATR and theoretical calculations for the determination of metal-citrate (metal = Cd and Pb) structures are reported. Mass spectrometry allowed to determine the stoichiometry 1:1 and 2:1 of the complexes, corroborating the theoretical calculations. The species found in the ratio 2:1 had their molecular structures readjusted, since the deprotonation of citric acid differed from what was simulated. The calculations of thermodynamic stability (DH0(aq.)) for the complexes obtained by B3LYP/ LANL2DZ were more exoenergetic than those found by PM6. However, for both methods, the stability of the complexes follows a trend, that is, the lowest-energy isomers in PM6 are also the most stable in B3LYP/LANL2DZ. The infrared analysis suggested that carboxyl groups are complexation sites and hydrogen bonds can help in the stability of the complexes. The vibrational frequencies in B3LYP/LANL2DZ had a good correlation with the experimental infrared results. Ó 2014 Elsevier B.V. All rights reserved.

Introduction Over the years, heavy metal ions have been established as toxic environmental pollutants that can affect vital processes in the physiology of plants and humans [1]. The influence of these toxic

⇑ Corresponding author. Tel.: +55 35 3829 1276; fax: +55 35 3829 1271. E-mail address: [email protected] (A.C. Bertoli). http://dx.doi.org/10.1016/j.saa.2014.08.053 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

elements has been under extensive investigation, in order to prevent catastrophic events at the microscopic level that may endanger the integrity and survival of living organisms. Anthropic activities have contributed significantly to the increase of these metals in the atmosphere, hydrosphere and lithosphere of the planet, further increasing the risk of their solubility and mobility in the environment. The heavy metals cadmium (Cd2+) and lead (Pb2+) have shown clear toxicity profiles for a number of different biological systems [2,3].

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However, metal mobilization in biological fluids requires the presence of organic ligands, capable of promoting the formation of coordination complexes, which may enable metal absorption or retention, if they produce structures with high steric hindrance [4]. Among the physiological ligands that are able to promote aqueous interactions with metal ions, there are the hydroxycarboxylic acids, of which citric acid is highlighted. Citric acid is a tricarboxylic organic compound, which is present in human plasma at a concentration of 0.1 mM, is part of the citric acid cycle in the Krebs cycle and is involved in metalloenzyme systems, such as Aconitase and NifV nitrogenase in nitrogen fixation [5]. In addition to its important physiological functions, citric acid has also been used as a soil extractor to predict the phytoavailability of heavy metals. Through extractors, it is sought to determine the sufficiency or deficiency degree of soil nutrients, and also to quantify the accumulation of toxic metals [6]. Therefore, the chemistry of formation of metal complexes with citric acid or citrate is delicate, due to the different bonding modes reported for divalent metal ions. Ion binding can occur at four locations: through three carboxylate groups (pKa1 = 3.13; pKa2 = 4.76; pKa3 = 6.40), and the hydroxyl group (pKa4  11), though stability constants are sensitive to the medium [7]. Furthermore, the deprotonation of the hydroxyl group has proved to be a challenge for the determination of stability constants for metal-citrate complexes; thus, most of the papers deal with citrate as an H3L tribasic ligand [8]. In order to conduct studies on these organometallic species, techniques such as mass spectrometry and infrared spectroscopy are certainly some of the powerful techniques for structure elucidation and, when used together, are able to provide important details about the structures of various compounds. Moreover, theoretical calculations can be used as a very useful tool to corroborate experimental data [4]. Given the above, the objective of this study is to put these techniques together, in order to investigate the structures of citrate complexed with Cd2+ and Pb2+, which is of special interest in chemistry, since the weight, size and shape of these complexes can influence on several biological systems. Thus, the study was divided into four sections (’Theoretical calculations: Structures and thermodynamic stability of the metal:citrate system, Identification of the complexes in aqueous solution by ESI-MS, Structural rearrangement and thermodynamic stability of the metal:citrate system by PM6 and B3LYP/LANL2DZ, and Vibrational assignments’). The first is related to the molecular structures suggested for the formation of metal complexes, citrate:Cd2+ and citrate:Pb2+, as well as their thermodynamic and structural properties. The second deals with analyses by electrospray ionization mass spectrometry (ESI-MS), in order to identify the species proposed by theoretical calculations. In the third part, the structural rearrangement of the complexes found by ESI-MS is presented, comparing the thermodynamic properties between the methods of semi-empirical calculation and DFT. Finally, the last part presents the analyses of the complex by infrared spectroscopy (FTIR-ATR) and the vibrational frequencies found by theoretical methods.

Methodology Computational details For the modeling and optimization of possible structures of complexes formed from the metal-citrate ratio, the Gaussian 09W program was used [9]. Initially, estimates of the molecular structures of free citrate and the complexes citrate:Cd and citrate:Pb were performed using the PM6 semi-empirical method [10], which is parameterized for most transition metals. Assays

by electrospray ionization mass spectrometry (ESI-MS) were carried out to try to confirm the structures and ratios metal:citrate proposed, according to Section ‘Electrospray ionization-mass spectrometry of the system metal:citrate in aqueous solution’. Taking into account the species found by ESI-MS, rearrangements of the complexes initially proposed were made. The molecular structures and vibrational frequencies were calculated again in PM6 and taken as an input structure for the calculations using density functional theory (DFT), applying the B3LYP functional and the LANL2DZ basis, which includes the effective core potential (ECP) [11]. All calculations were performed considering the free molecules in vacuum and in solution, implicitly considering the solvent water by the polarizable continuum model (PCM) [12]. Thermodynamic studies The thermodynamic study aims to provide a theoretical discussion on the complexes, in order to obtain the parameters that determine their chemical properties. To do so, the absolute energy values (DH0) of the complexes were obtained at different metalcitrate ratios, using the thermodynamic cycle of Fig. 1. The DH(aq.) of a complex in the thermodynamic cycle was calculated by Eq. (1) [13].

DHðaq:Þ ¼ DHðgÞ þ ½DHðsolv:Þ ðM  CitrateÞ

n

 ðDHðsolv:Þ M2þ

þ DHðsolv:Þ CitrateÞ

ð1Þ (DDH0(aqueous))

The calculation of relative energy was performed to identify the most stable stereoisomer in relation to the same metal and same stoichiometry. The DDH0(aqueous) was determined by the difference between the energy variation of a higher-energy isomer (DH02) and the lowest-energy isomer (DH01), according to Eq. (2).

DDH0ðaq:Þ ¼ DH02  DH01

ð2Þ

Electrospray ionization-mass spectrometry of the system metal:citrate in aqueous solution The reagent solutions C6H8O7, CdCl2H2O and (CH3COO)2Pb3H2O were prepared with a concentration of 1  103 mol L1. For the formation of the possible complexes, a series of metal:citrate solutions was prepared with molar ratios ranging from 1:1 to 6:1 at different pH values: 3.0; 7.0 and 9.0. The pH was adjusted by the addition of an aqueous NH4OH solution, and the solutions were prepared 48 h before the analysis. For injection into the mass spectrometer, 20% methanol was added, and the pH was checked again. ESI mass spectra acquired in the negative mode were obtained on a 500-MS quadrupole ion trap mass spectrometer (Varian Inc., Palo Alto, CA, USA). The samples were introduced into the ESI ion source through a syringe at a flow rate of 20 lL min1. The ion spray voltage was ±5 kV; capillary voltage: 60–80 V and RF load of 80%. Nitrogen was used as a nebulizer and drying gas at pressures of 35 psi and 10 psi, respectively; the temperature of the drying gas was 350 °C. The spectra were recorded in the range of 100–1000 Da., with an average of 20–35 scans. The isotopic distribution patterns were calculated using the program ISOPRO 3.1. Infrared spectroscopy (FTIR-ATR) The FTIR-ATR spectra were collected using a Nicolet Nexus spectrometer, equipped with an attenuated total reflectance accessory (ATR) with a ZnSe crystal. The spectra were acquired with 64 scans and a resolution of 4 cm1.

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273

Fig. 1. Thermodynamic cycle.

Results and discussion Theoretical calculations: Structures and thermodynamic stability of the metal:citrate system A theoretical study on the formation of complexes was conducted, so that the different modes of interaction and stability between citric acid and the metal cations Cd2+ and Pb2+ could be understood. It is important to highlight that, due to the complexity presented by compounds involving transition metals, there are still some difficulties in the study of these chemical systems. This complexity is directly related to the description of transition metals, where certain characteristics inherent to the participation of ‘‘d’’ orbitals of the metal center is observed. At first, the species formed between citric acid and the metals can present a large number of conformations, due to the presence of carboxylic groups. Although several conformers are possible, the structures of citrate (Cit3) are considered completely deprotonated in the calculations, in which the carboxyl groups are always coordinated to the central metal. Different metal:citrate conditions (Figs. 2 and 3) were taken into consideration: 1:1 (one citrate molecule to a metal cation); 2:1 (two citrate molecules to a metal cation) and 3:1 (three citrate molecules to a metal cation). The calculation method used for the optimizations was the semiempirical (PM6), which uses some parameters obtained from experimental data [10]. Figs. 2 and 3 present, respectively, the most stable citrate:cadmium and citrate:lead complexes in different stoichiometric ratios, after optimization. The energy results for the complexes under different conditions, 1:1; 2:1 and 3:1, are given in Table 1. The energies calculated for the formation reactions of the complexes were given in DH0. These values can be approximated to DG0, since DS0 is considered negligible in conformational changes [14]. According to the results shown in Table 1, in the 1:1 ratio, the energy difference between the isomers of Cd2+ complexes is 13.47 kcal mol1. The energy difference is attenuated to 0.07 kcal mol1 for Pb2+ isomers in the same ratio. It was observed that the complexes [Pb2+(Cit3)] in the 1:1 ratio have energy values lower than the species [Cd2+(Cit3)], suggesting a greater stability of Pb2+ complexes.

The stability of the complexes can be explained by the fact that metal atoms sometimes behave as Lewis acids, and citrate molecules behave as a Lewis base [15]. Pb2+ is considered an intermediate Lewis acid regarding hardness and softness, and Cd2+ a soft acid. On the other hand, carboxylate groups, to which metals bond, are considered hard bases and, according to the principle suggested by Pearson, hard acids bind to hard bases and soft acids bind to soft bases, which could possibly make Pb2+ more available than Cd2+, since it is an intermediate and higher acid (more accessible to the ligand) [16]. In the 2:1 stoichiometry, it was observed that the energy difference between the species [Cd2+(Cit3)2]4 was 5.15 kcal mol1, while Pb2+ complexes presented a variation of only 1.67 kcal mol1 in the same ratio. The complex [Cd2+(Cit3)2]4 presented tetrahedral geometry, differing from the species [Pb2+(Cit3)2]4, which showed an octahedral arrangement. Analyzing the preference of the complex for an octahedral or tetrahedral environment, complexes with tetrahedral geometry occur, in general, as a result of ligand–ligand repulsions, which exceed the energy difference in the formation of metal–ligand bonds and are assigned to metals with a small atomic radius and ligands with high steric hindrance [15]. By increasing the number of citrate ligands in the system, several possibilities (stereoisomers) involving metals can be formed. Therefore, for the metal:citrate 3:1 ratio, four stereoisomers were simulated. It is delicate to compare the stoichiometric conditions reported, since they are systems with different numbers of atoms. However, the energy values found for the 3:1 stoichiometry greatly differ from the other ratios. While the isomers of the complex [Cd2+(Cit3)3]7 had negative energy values, species related to [Pb2+(Cit3)3]7 showed positive energy values. These results may indicate restrictions in the formation of these species, once the positive energy related to Pb2+ complexes reveals no spontaneity in the reactions. This conclusion is in good agreement with the experimental results obtained. A justification for this energy variation in the complexes with a 3:1 ratio can be attributed to classical intramolecular interactions – repulsion effects between bulky groups and electrostatic interactions [17]. These isomers showed a trigonal planar geometry, in which only a carboxylate group of each citrate could coordinate

Fig. 2. Most stable complexation forms in the gas phase, obtained by optimization in PM6: (A) [Cd2+(Cit3)], (B) [Cd2+(Cit3)2]4 and (C) [Cd2+(Cit3)3]7.

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Fig. 3. Most stable complexation forms in the gas phase, obtained by optimization in PM6: (A) [Pb2+(Cit3)], (B) [Pb2+(Cit3)2]4 and (C) [Pb2+(Cit3)3]7.

Table 1 Enthalpy values (kcal mol1) for the formation reactions of the complexes in solution (DDH0(aqueous)) between citrate (Cit3) and the metal cations Cd2+ and Pb2+. Structures optimized in the semi-empirical method (PM6).

DDH0(aq.)

Models 2+

3

[Cd (Cit )] Isomer 1 Isomer 2

0.00 13.47

[Cd2+(Cit3)2]4 Isomer 1 Isomer 2 2+

3

0.00 5.15

7

[Cd (Cit )3] Isomer 1 Isomer 2 Isomer 3 Isomer 4

3



[Pb (Cit )] Isomer 1 Isomer 2

0.07 0.00

[Pb2+(Cit3)2]4 Isomer 1 Isomer 2

1.67 0.00

2+

8.51 7.08 6.49 0.00

DDH0(aq.)

Models 2+



3

7

[Pb (Cit )3] Isomer 1 Isomer 2 Isomer 3 Isomer 4

56.33 23.63 0.00 64.36

with the central metal cation, possibly due to steric repulsion between the ligands. Another explanation may be related to the electron pair repulsion theory (VSEPR), in which regions of high electron concentrations repel each other and, due to this repulsion, they are organized so that they are distant from each other [18]. Table 2 shows the bond lengths between the oxygen atoms of the carboxylate groups and the metals studied in the formation of the complexes. The smallest bonding distances for O-Metal, evaluated in Table 2, were obtained for Pb2+ and Cd2+ stereoisomers in the ratio 1:1. These results can help in the interpretation of thermodynamic studies for these complexes, once they showed low DH0(aq.) values, suggesting the formation of stable species. The bond lengths for CdAO ranged from 2.11 to 2.43 Å. These values are similar to the corresponding CdAO bonding distances in other complexes obtained experimentally using X-ray diffraction: [Cd2+(HCit2)(H2O)]n (2.22–2.36 Å) [5] and (NH+4)[Cd2+ (Cit3)(H2O)]H2O (2.27–2.64 Å) [1]. In both studies, the complexes had octahedral geometry. However, when the ligand is less bulky, such as some organic acids, Cd2+ complexes, they can adopt the tetrahedral coordination environment [19,20]. Regarding Pb2+ complexes, PbAO bonds ranged from 2.07 to 2.70 Å. Through X-ray diffraction, PbAO bonding lengths have been reported in other studies and are similar to those found in this study. The complex synthesized [Pb2+(HCit2)]nnH2O showed PbAO distances from 2.39 to 3.27 Å [21], while the compound {Na(H2O)3}[Pb5(C6H5O7)3(C6H6O7)(H2O)3]9.5H2O, PbAO obtained the bonding lengths from 2.39 to 3.32 Å [7]. In Pb:DTPA complexes, was observed PbAO bonding lengths of 2.40, 2.41 and 2.39 Å, values that differ from those found in the present study [22]. However, it is noteworthy that the authors used Density Functional Theory (DFT) calculations as a method

and, although the metal is coordinated to the carboxylate groups of DTPA, it is a bulky ligand, in which repulsion effects may exist. Another factor that must be considered is that, due to its large atomic radius, Pb2+ may have difficulty being completely engaged by the ligand, distorting the geometry of the complex with a perfect octahedral arrangement [22]. Identification of the complexes in aqueous solution by ESI-MS Electrospray mass spectrometry (ESI-MS) is a sensitive and versatile technique for the study of low-volatility ionic species in the gas phase [23], and is suitable for solutions containing pre-formed inorganic ions, including metal complexes and metalloids [24]. ESI-MS has been used to identify complexes formed from organic molecules, which naturally occur with toxic heavy metals, such as cadmium, mercury, lead and other less toxic metals, as well as those with some medicinal relevance, such as bismuth [25]. In the present study, conditions similar to those previously reported were adopted for sample preparation and analysis by mass spectrometry [26,27]. The authors describe that the increase in the metal:citrate molar ratio in aqueous solutions and acidic pH favors the formation of dinuclear and trinuclear oligomeric complexes, while low metal:citrate molar ratios and basic or physiological pH indicate the formation of mononuclear complexes. Therefore, the results obtained from the assays in the 2:1 metal:citrate ratio and pH 7 were chosen, since theoretical calculations were previously performed for mononuclear complexes and this pH range is related to the physiological conditions in which plants are inserted. ESI mass spectra for citrate:cadmium and citrate:lead solutions in the negative mode are respectively shown in Figs. 4 and 5. Citric acid (C6H8O7) is represented by H3Cit, while (Cit3) represents the completely ionized citrate with the molecular formula C6H5O3 7 . The spectra show peak aggregates assigned to anionic metal:citrate complexes, based on the m/z value and on the characteristic isotope distribution pattern for each species. As a result of the protonation that occurs during electrospray ionization, the species can be detected with different degrees of protonation [27]. The mass spectra showed intense signals at m/z 191, which are related to a mono-deprotonated molecule of citric acid (H2Cit) and, for both assays, there were predominantly mononuclear species. Mono-charged complexes were formed [Cd2+(Cit3)] (m/z 303) and [Cd2+(Cit3)(H2O)2] (m/z 339), the latter resulting from the formation with H2O adducts. It should be emphasized that each Cd2+ ion is distributed among its main isotopes (110Cd (12.49%); 111 Cd (12.80%); 112Cd (24.13%); 113Cd (12.22%); 114Cd (28.73%) and 116Cd (7.49%)), and that 114Cd represents the most intense peak. The formation of the coordinated complex to H2O molecules

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Table 2 Relevant bond lengths (Å) (Cd–O and Pb–O) between the oxygen atoms of the carboxylate groups and the metals. Structures optimized in the semi-empirical method (PM6). Bond lengths (Å) [Cd2+(Cit3)] Isomer 1

Isomer 2

Bond lengths (Å) [Cd2+(Cit3)2]4

Bond lengths (Å) [Cd2+(Cit3)3]7

Isomer 1

Isomer 1

Isomer 2

Isomer 2

Isomer 3

Isomer 4

Atoms

(Å)

Atoms

(Å)

Atoms

(Å)

Atoms

(Å)

Atoms

(Å)

Atoms

(Å)

Atoms

(Å)

Atoms

(Å)

Cd19AO1 Cd19AO2 Cd19AO18

2.11 2.17 2.22

Cd19AO1 Cd19AO2 Cd19AO18

2.20 2.15 2.19

Cd37AO1 Cd37AO2 Cd37AO4 Cd37AO6

2.37 2.35 2.34 2.37

Cd37AO1 Cd37AO3 Cd37AO5 Cd37AO6

2.37 2.32 2.30 2.40

Cd55AO18 Cd55AO35 Cd55AO54

2.37 2.35 2.32

Cd55AO7 Cd55AO19 Cd55AO38

2.36 2.32 2.34

Cd55AO34 Cd55AO35 Cd55AO38

2.43 2.35 2.32

Cd55AO17 Cd55AO20 Cd55AO22

2.31 2.31 2.31

[Pb2+(Cit3)]

[Pb2+(Cit3)2]4

Isomer 1

Isomer 2

Isomer 1

[Pb2+(Cit3)2]7 Isomer 2

Isomer 1

Isomer 2

Isomer 3

Isomer 4

Atoms

(Å)

Atoms

(Å)

Atoms

(Å)

Atoms

(Å)

Atoms

(Å)

Atoms

(Å)

Atoms

(Å)

Atoms

(Å)

Pb19AO1 Pb19AO2 Pb19AO18

2.07 2.04 2.06

Pb19AO1 Pb19AO2 Pb19AO18

2.07 2.04 2.06

Pb37AO1 Pb37AO2 Pb37AO3 Pb37AO4 Pb37AO5 Pb37AO6

2.63 2.67 2.64 2.68 2.64 2.63

Pb37AO1 Pb37AO2 Pb37AO3 Pb37AO4 Pb37AO5 Pb37AO6

2.66 2.62 2.70 2.70 2.62 2.66

Pb55AO17 Pb55AO35 Pb55AO54

2.63 2.41 2.41

Pb55AO7 Pb55AO19 Pb55AO38

2.53 2.20 2.50

Pb55AO34 Pb55AO35 Pb55AO38

2.61 2.52 2.48

Pb55AO17 Pb55AO20 Pb55AO22

2.36 2.36 2.36

2:1 stoichiometry, as is the case of Cd2+, are formed by citrates with m/z 190 and 191, indicating that the metals may be coordinated to free carboxyl oxygen. The presence of citrate ions in aqueous solution relies on the ionic character of Pb2+ and leads to the formation of stable complexes. The tricarboxylate citrate ion is known to exist and react in three or four ionization states in biological media. As such, Pb2+ ions, which are interconnected through the terminal carboxylate group, can form oligomeric units [21].

(m/z 339) can be explained due to the large ionic radius of Cd (0.91 Å), since the coordination sphere of the metal favors coordination with solvent molecules [20]. Furthermore, in studies conducted with the complex [Cd2+(HCit2)(H2O)]n, concluded that, due to steric effects imposed by citrate in the formation of the crystal lattice of the complex, a coordination site is not occupied by carboxylates [5]. This allows a solvent molecule to enter the coordination sphere of Cd2+. The coordinated water molecule can participate in hydrogen interactions with citrate carboxylates, increasing the stability of the resulting structure. The other mononuclear species formed, [Cd2+(HCit2)(H2Cit)], is the adduct of two citrate molecules with different protonations, (HCit2) m/z 190 and (H2Cit) m/z 191, to an atom of Cd2+. For citrate:lead assays (Fig. 5), the relative intensities ranged considerably; however, peaks in stoichiometries 1:1 and 2:1 are also observed. The dominant species (100%) is related to the ion H2Cit m/z 191 and the peaks m/z 397 and 589 are assigned to the mono-charged complexes [Pb2+(Cit3)] and [Pb2+(HCit2) (H2Cit)], remembering that Pb2+ presents isotopic distribution (204Pb (1.4%); 206Pb (24.1%); 207Pb (22.1%) and 208Pb (52.4%)), and the most intense signal is related to 208Pb. Pb2+ complexes in a

Structural rearrangement and thermodynamic stability of the metal:citrate system by PM6 and B3LYP/LANL2DZ The combined use of spectrometric techniques and theoretical calculations is a powerful tool to investigate the chemical structure of a variety of compounds [4,22,28]. By ESI-MS analyses, it is possible to observe that, in part, the experimental results obtained differed from those obtained theoretically. However, molecular modeling studies were extremely important since, through such calculations, it was possible to look for the simulated complexes by ESI-MS assays.

[Cd2+(Cit3-)(H2O)2]-

[Cd2+(Cit3-)]-

(A) 2+

Relative Intensity (%)

75

H2Cit

191

(C)

(B) 3-

-

[Cd (Cit )]

100

[Cd2+(HCit2-)(H2Cit-)]-

303

2+

3-

-

[Cd (Cit )(H2O)2] 339

301

2+ 2- [Cd (HCit )(H2Cit )]

337 50

495

493

25 0 200

300

400

500

m/z Fig. 4. ESI mass spectrum of citrate:cadmium solutions in the negative mode, molar ratio 2:1 at pH = 7. (A), (B) and (C) isotopic distribution calculated for the respective complexes [Cd2+(Cit3)], [Cd2+(Cit3)(H2O)2] and [Cd2+(HCit2)(H2Cit)]. The spectrum is normalized to the most abundant peak (m/z = 303).

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[Pb2+(HCit2-)(H2Cit-)]-

[Pb2+(Cit3-)]-

(B)

Relative Intensity (%)

(A) 100 H2Cit

191 2+

3-

-

[Pb (Cit )]

75

397 2+ 2- [Pb (HCit )(H2Cit )]

50

589

25 0 200

300

400

500

600

m/z Fig. 5. ESI mass spectrum of citrate:lead solutions in the negative mode, molar ratio 2:1 at pH = 7. (A) and (B) isotopic distribution calculated for the respective complexes [Pb2+(Cit3)] and [Pb2+(HCit2)(H2Cit)]. The spectrum is normalized to the most abundant peak (m/z = 191).

Therefore, the complexes found by ESI-MS, [Cd2+(Cit3)] m/z = 303 and [Pb2+(Cit3)] m/z = 397, are related to the 1:1 stoichiometry and correspond to the structures proposed by theoretical calculations. The complexes of the 2:1 ratio were not formed through coordination with fully deprotonated citrates, according to the proposed theoretical structures. Some authors reported that the protonation that occurs during electrospray ionization may make the ligands be detected with different degrees of protonation [27]. The complexes found for this ratio, ([Cd2+(HCit2)(H2Cit)] and [Pb2+(HCit2)(H2Cit)]), had their structures readjusted through theoretical calculations and are shown in Fig. 6. Regarding the complexes simulated in the 3:1 condition, they were not detected in the ESI-MS conditions used in this study. This experimental result is in perfect agreement with the theoretical prediction since, according to the enthalpy results from the thermodynamic cycle, the preferred order for the formation of both metal complexes [Cd2+ and Pb2+] is 1:1 > 2:1 > 3:1. Thus, the complex [Cd2+(Cit3)] is 30.81 kcal mol1 more stable than [Cd2+(Cit3)2]4 which, in turn, is about 500.00 kcal mol1 more stable than [Cd2+(Cit3)3]7. In relation to Pb2+ complexes, the energy difference between the species [Pb2+(Cit3)] and [Pb2+(Cit3)2]4 is 71.85 kcal mol1, while [Pb2+(Cit3)2]4 is more stable than [Pb2+(Cit3)3]7. Some dinuclear and trinuclear species with a higher number of ligands were also observed, but were not addressed in this study. From the experimentally obtained results, new theoretical calculations were performed. The energy minimums of the complexes [Cd2+(HCit2)(H2Cit)] and [Pb2+(HCit2)(H2Cit)]) at the 2:1 ratio and [Cd2+(Cit3)] and [Pb2+(Cit3)] at the 1:1 ratio were calculated by the PM6 semi-empirical method and DFT, using the B3LYP functional and the LANL2DZ basis. In Fig. 6, the most stable Cd2+ and Pb2+ complexes are presented for the 2:1 ratio. The energy results obtained by the B3LYP/LANL2DZ and PM6 methods for the complexes in 1:1 and 2:1 conditions are given in Table 3. Energies calculated for the formation reactions of the complexes were given in DH0. These values can be approximated to DG0, since DS0 is considered negligible in conformational changes [14].

Geometry optimization and the obtention of energy minimums is one of the main bottlenecks in the application of quantum chemistry to complex molecules. For these molecules, it is even more important to search for more economical alternatives to obtain energy minimums, especially when it is necessary to perform a conformational search over several degrees of freedom. The PM6 semi-empirical method, recently submitted by Stewart’s research group, carries several improvements over the older members of the semiempirical family (AM1, PM3, etc) and is a good option to perform the calculations of molecules of interest [29]. On the other hand, DFT methods are commonly used to model systems with these compounds, and due to the fact that they present more accurate energy values [22,30]. To compare the results of the complexes after ESI-MS assays, energy minimums were obtained using the PM6 and the B3LYP/LANL2DZ methods. The energy values (DH0(aq.)) of the complexes obtained from the B3LYP functional together with the LANL2DZ basis were more exoenergetic (or less endoenergetic) than those found by PM6. However, for both methods, the stability of the complexes follows a trend, that is, the lowest-energy isomers in PM6 are also the most stable in B3LYP/LANL2DZ. Organomercury compounds reported showed insignificant differences between the geometric parameters obtained by the B3LYP/LANL2DZ method, as well as by the PM3 semi-empirical method [31]. In another study conducted with organotin, the PM6 semi-empirical method reproduced DfH0 more accurately than DFT methods [30]. According to the author, this performance is expected from PM6, since this method is parameterized with experimental values. The most stable structures obtained theoretically for the complexes [Cd2+(Cit3)] (energy variation between the most stable isomer and the least stable: PM6 DDH0(aq.) = 13.47 and B3LYP DDH0(aq.) = 7.92 kcal mol1) and [Pb2+(Cit3)] (energy variation between the most stable isomer and the least stable: PM6 DDH0(aq.) = 0.07 and B3LYP DDH0(aq.) = 7.63 kcal mol1) are in accordance with the experimental results reported by various authors [1,5,7,21] who, for the same species at physiological pH, found

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Fig. 6. Most stable complexation forms in the gas phase, obtained for the ratio 2:1 (A) [Cd2+(HCit2)(H2Cit)] and (B) [Pb2+(HCit2)(H2Cit)].

Table 3 Enthalpy values (kcal mol1) for the formation reactions of the complexes in solution (DDH0(aqueous)) among citrate (Cit3), (HCit2), (H2Cit) and the metals Cd2+ and Pb2+. Structures optimized in the semi-empirical (PM6) and DFT (B3LYP/LANL2DZ) methods. Models

PM6 DDH0(aq.)

B3LYP/LANL2DZ DDH0(aq.)

[Cd2+(Cit3)] Isomer 1 Isomer 2

0.00 13.47

0.00 7.92

[Cd2+(HCit2)(H2Cit)] Isomer 1 Isomer 2 Isomer 3 Isomer 4

0.00 30.11 9.03 29.96

0.00 44.24 2.51 57.84

similarities such as 1:1 citrate:metal stoichiometry, same 1 charge and triple deprotonation of citrate bonded to the metals Cd2+ e Pb2+. For the species with 2:1 stoichiometry, the complex [Cd2+(HCit2)(H2Cit)] (energy variation between the most stable isomer and the least stable: PM6 DDH0(aq.) = 30.11 and B3LYP DDH0(aq.) = 57.84 kcal mol1), as well as [Cd2+(Cit3)2]4, found in first section of theoretical calculations of the study, presented tetrahedral geometry, although the d10 electron configuration of Cd2+ favors the stability of the complex in the octahedral coordination [32]. However, when ligands are less bulky, such as some organic acids, Cd2+ complexes can adopt a tetrahedral coordination environment [19,20]. Studies in 2:1 and 4:1 ligand:Cd2+ stoichiometric conditions, showed that the complexes formed had a tetrahedral geometry [33]. The most stable structure found for the complex [Pb2+(HCit2) (H2Cit)] (energy variation between the most stable isomer and the least stable: PM6 DDH0(aq.) = 6.37 and B3LYP DDH0(aq.) = 43.47 kcal mol1) showed an irregular arrangement, similar to trigonal bipyramidal, which has the coordination number 5. As stated earlier, due to its large atomic radius, Pb2+ may have difficulty being completely engaged by the ligand, distorting the geometry of the complex with a perfect octahedral arrangement [22]. Furthermore, Pb2+ compounds can have a variety of coordination numbers (between 2 and 12), depending on the number of nearby ligands [34]. The wide variety of configurations for Pb2+ reflects the stereochemical activity of the unpaired electron pair, which can be described by interactions of the 6s antibonding orbital of Pb2+, resulting in structural distortions to minimize unfavorable interactions [35]. Vibrational assignments FTIR assays for the metal complexes, as well as analyzes by ESIMS, were performed at pH 7. The experimental spectra (FTIR) of free citrate at pH 7 and those related to its speciation as a function of pH variation are shown in Fig. 7. Experimental and calculated

Models

PM6 DDH0(aq.)

B3LYP/LANL2DZ DDH0(aq.)

[Pb2+(Cit3)] Isomer 1 Isomer 2

0.07 0.00

7.63 0.00

[Pb2+(HCit2)(H2Cit)] Isomer 1 Isomer 2 Isomer 3 Isomer 4

0.00 6.37 0.92 6.26

0.00 42.01 7.57 43.47

vibrational spectra for the most stable complexes, [Cd2+(Cit3)], [Cd2+(HCit2)(H2Cit)], [Pb2+(Cit3)] and [Pb2+(HCit2)(H2Cit)], are shown in Figs. 8 and 9, respectively. Vibration frequencies and intensities were calculated using the PM6 semi-empirical method, as well as DFT B3LP/LANL2DZ. In general, calculated wave numbers become higher than experimental wave numbers, due to factors such as neglect of anharmonicity, electron correlation and deficiencies in the basis set [36]. Comparing the experimental values with the calculated vibrational modes (Table 4), it is observed that the DFT B3LYP/LANL2DZ method provides a better quantitative performance in the prediction of vibrational frequencies than the PM6 semi-empirical method [37]. Furthermore, for B3LYP/ LANL2DZ, there is a good correlation between experimental and calculated vibrational frequencies: R2 = 0.9989–0.9994 for Cd2+ complexes and R2 = 0.9949–0.9988 for Pb2+ complexes, while for PM6 the values are R2 = 0.9383–0.9019 for Cd2+ complexes and R2 = 0.9113–0.9377 for Pb2+ complexes. The correlation graphs for the complexes are presented in the Supplementary Material. The experimental spectra for the free citrate at pH 7 and at different pH values are shown in Fig. 7. For the complexes [Cd2+(Cit3)] and [Cd2+(HCit2)(H2Cit)], and [Pb2+(Cit3)] and [Pb2+(HCit2)(H2Cit)], the experimental and calculated spectra by the PM6 and B3LYP/LANL2DZ methods are shown in Figs. 8 and 9, respectively, for the lowest-energy stereoisomers. A detailed description of the main experimental and calculated vibrational assignments for the stereoisomers of the lowest-energy complexes by the B3LYP/LANL2DZ and PM6 methods is shown in Table 4. CAH vibrational modes The characteristic bands of chemical groups which are useful for the identification of the molecular structure often involve coupled vibrations. According to the experimental vibrational assignments presented in Table 4, bands in the region from 1027.05 to 1280.54 cm1 may be attributed to the angular deformation of CH [38] in the twisting (dtwist(HCH)), rocking (drocking(HCH)),

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IR intensity

IR intensity

EXPERIMENTAL

B3LYP/LANL2DZ

PM6

(A)

(A) 4000 3500 3000 2500 2000 1500 1000

500

4000

3500

3000

2500

2000

1500

1000

500

Wavenumber (cm-1)

Wavenumber (cm-1)

IR intensity

5,0 4,0 3,0 2,0 1,0

IR intensity

EXPERIMENTAL

pH 9,0 8,0 7,0 6,0

B3LYP/LANL2DZ

PM6

(B) (B) 1800

1600

1400

1200

4000

1000

Wavenumber (cm-1) Fig. 7. (A) IR citrate pH 7 and (B) IR citrate at different pH values. In figure (B), the scale is shown from 1000 to 1800 cm1, in order to facilitate the visualization of the main bands.

IR intensity

EXPERIMENTAL

B3LYP/LANL2DZ

PM6

(A) 4000

3500

3000

2500

2000

1500

1000

500

Wavenumber (cm-1)

IR intensity

EXPERIMENTAL

B3LYP/LANL2DZ

PM6

(B) 4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm ) Fig. 8. (A) Experimental IR spectra, B3LYP/LANL2DZ and PM6 for the complex [Cd2+(Cit3)]. (B) Experimental IR spectra, B3LYP/LANL2DZ and PM6 for the complex [Cd2+(HCit2)(H2Cit)].

wagging (dwagg(HCH)) and scissors (dsciss(HCH)) modes. All these vibrations are in accordance with the frequencies calculated by

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm ) Fig. 9. (A) Experimental IR spectra, B3LYP/LANL2DZ and PM6 for the complex [Pb2+(Cit3)]. (B) Experimental IR spectra, B3LYP/LANL2DZ and PM6 for the complex [Pb2+(HCit2)(H2Cit)].

the DFT and semi-empirical methods. The B3LYP calculations predicted CAH deformations between 1030.00 and 1288.69 cm1, while values in the range from 1014.65 to 1297.42 cm1 were found in PM6. CAO stretching Carboxylic acids are characterized by a CAO stretching, present in the region of 1240 cm1 [38]. The experimental frequencies for this group are concentrated in the region from 1027.05 to 1248.26 cm1 and correspond to the ones theoretically obtained, which are between 1030.00 and 1244.02 cm1 for B3LYP/LANL2DZ, and between 1014.65 and 1242.62 cm1 for PM6. The bands relative to this stretching may be overlapping, largely in the range of 1150–1300 cm1, and this vibration was confirmed at 1278 cm1 using the DFT method [39]. CAC stretching For complexes formed around the central metal cation, finding vibrational couplings that describe single bonds such as CAC and CAO makes physical sense [40]. CAC stretching modes have been assigned in the region from 1027.05 to 1259.46 cm1 for the experimentally obtained species, while the calculated frequencies ranged, respectively, between 1030.00 and 1254.07 cm1 using B3LYP/LANL2DZ, and between 1019.96 and 1256.11 cm1 according to PM6 calculations. The experimental assignment is correlated with those obtained theoretically and is consistent with the results, in which CAC stretching modes were present at 1026–1031 cm1 for the metal complexes of Co2+, Cu2+ e Zn2+, obtained by experimental and theoretical methods with B3LYP/LANL2DZ [41]. COO stretching The carboxylate ion gives rise to two bands, one of which, between 1650 and 1550 cm1, is intense and related to asymmetric stretching, and the other, weaker, around 1400 cm1, is from symmetric stretching. The C@O stretching region is very sensitive

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A.C. Bertoli et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 271–280 Table 4 Main experimental and calculated frequencies (cm1), and vibrational assignments of the complexes by the semi-empirical (PM6) and DFT (B3LYP/LANL2DZ) methods. Approximate assignments

dtwist(HCH) + m(CO) drocking(HCH) + m(CC) dtwist(HCH) + m(CC) dsciss(HCH) drocking(HCH) + m(CC) + m(CO) dwagging(HCH) + d(OH) msym(COO) + dsciss(COH) msym(COO) masym(COO) masym(COO) m(OH)

[Cd2+(Cit3)]

[Cd2+(HCit2)(H2Cit)] Approximate assignments

[Pb2+(Cit3)] Exp.

PM6

B3LYP

PM6

B3LYP

1027.29 1092.94 1186.07 1259.46 1280.54 1395.17 1415.96 1457.76 1560.51 1638.66 3350.68

1021.00 1090.58 1163.57 1256.11 1274.96 1354.99 1382.89 1438.39 1735.91 1749.52 2552.51

1062.44 1146.72 1196.89 1254.07 1288.69 1322.99 1401.56 1477.89 1617.72 1633.89 3651.27

1019.96 1090.47 1181.05 1252.52 1269.14 1400.28 1411.28 1441.49 1650.27 1713.46 2544.20

1030.00 1099.26 1159.53 1240.60 1277.60 1388.99 1407.65 1462.42 1572.95 1608.19 3519.65

Exp.

PM6

B3LYP

PM6

B3LYP

1027.05 1078.98 1095.98 1207.78 1248.26 1280.53 1391.44 1445.87 1573.87 1638.70 3350.46

1030.21 1085.60 1139.52 1222.88 1242.62 1297.42 1383.29 1396.31 1751.26 1793.32 2722.45

1033.16 1055.50 1103.63 1175.20 1244.02 1273.35 1353.17 1420.90 1616.53 1646.53 3464.68

1014.65 1082.52 1100.33 1207.46 1241.98 1280.36 1406.46 1413.43 1771.17 1776.49 2558.56

1034.65 1073.11 1110.25 1194.72 1236.03 1280.26 1388.43 1438.63 1619.02 1638.31 3442.75

drocking(HCH) + m(CC) + m(CO) drocking(HCH) + m(CC) dtwist(HCH) + d(OH) drocking(HCH) + m(CC) + d(OH) dwagging(HCH) msym(COO) + dsciss(COH) msym(COO) msym(COO) masym(COO) masym(COO) m(OH)

[Pb2+(HCit2)(H2Cit)]

dtwist: twisting deformation; drocking: rocking deformation; dwagging: wagging deformation; dsciss: scissors deformation; msym: symmetric stretching; masym: asymmetric stretching.

to several factors, such as physical state, hydrogen bonding, electronic substituent effect, ring tension, etc. [42]. For the complexes featured, bands of lower intensity between 1391.44 and 1457.76 cm1, experimentally found, were assigned to symmetric stretching (maymCOO ). The most intense C@O bands, related to asymmetric stretching (maymCOO ), were present in the region from 1560.51 to 1638.70 cm1. The experimental assignments for both stretching modes resembled the theoretical frequencies found by the DFT method (1353.17–1646.53 cm1). However, for the semi-empirical method, the values were overestimated, especially for asymmetric stretching (1354.99– 1793.32 cm1). Studies with organotins using the methods B3LYP, PM6 and B3PW91, have predicted that, in general, theoretical approaches tend to overestimate vibration frequencies [30]. OAH stretching The hydroxyl function is probably one of the most dominant characteristics among infrared frequencies. In most chemical environments, the hydroxyl group does not exist in isolation, and vibration can be generally coupled, resulting in extensive hydrogen bonding. These groups may be linked within the same molecule (intramolecular hydrogen bond) or between neighboring molecules (intermolecular hydrogen bond). The impact of the hydrogen bond is the production of a significant broadening in the absorption band. In compounds such as carboxylic acids, which exhibit extremely strong hydrogen bonds, one characteristic is the presence of this bond at lower frequencies [38,39]. The mOH stretches present in the complexes obtained experimentally showed very intense and broad bands, characteristic of OAH groups in the range of 3350 cm1. Like other assignments, frequencies calculated by DFT for this group were very close to the experimental frequencies. OAH stretches for B3LYP/LANL2DZ were present between 3442.75 and 3519.65 cm1, and in the region from 2544.20 to 2722.45 cm1 for PM6, which does not correspond to experimental OAH stretches. Low-intensity vibrational modes from CAOAH bonds were observed in the same symmetric stretching region msym(COO), between 1322.99 and 1406.46 cm1. A characteristic band of the spectrum, which involves the formation of complexes from carboxylic acids, is derived from the angular deflection of the OAH group (dOH) in hydrogen bonding. The low-intensity band appeared between 1159.53 and 1297.42 cm1 [40]. Hydrogen bonding In general, a hydrogen bond is formed when the hydrogen atom from a AAH covalent bond of a proton donor molecule interacts with a pair of electrons from an atom X, which is a proton receptor. In carboxylic acids, the strength of hydrogen bonds is discussed especially for OAH  O interactions. These interactions are shown

to be of great importance in systems, in order to elucidate structure-property relationships [43]. Through theoretical calculations performed in this study for both methods, semi-empirical and DFT, it was possible to observe vibrations related to hydrogen bonds for the species [Cd2+(HCit2) (H2Cit)] and [Pb2+(HCit2)(H2Cit)]. Intense bands for the Cd2+ complex were found at 2184.37 cm1 using PM6, and 2880.19 cm1 according to B3LYP/LANL2DZ. Regarding the Pb2+ complex, they were in the region of 2102.64 cm1 for PM6 and very intense, at 3003.54 cm1, for B3LYP/LANL2DZ. These vibrations are OAH  O interactions between atoms of carboxylic groups, which may or may not be coordinated to the metal and are related to intramolecular hydrogen bonds for the species [Cd2+(HCit2)(H2Cit)] and intermolecular for [Pb2+(HCit2)(H2Cit)]. These interactions are shown in Fig. 6 between the atoms O(1)AH(40)  O(25) and O(10)AH(37)  O(26) for the complexes of Cd2+ and Pb2+, respectively. The intramolecular bond H(40)  O(25) is distant 1.66 Å in PM6, while the DFT calculation predicts the reduction of the same bond length to 1.57 Å. The intermolecular distance of the bond H(37)  O(26) is 1.63 Å for PM6 and, in DFT, the bond was elongated to 1.66 Å. Conclusions The combined use of ESI-MS, FTIR-ATR and computational techniques proved to be a powerful tool for the structure elucidation of the complexes Cd- and Pb-Citrate. Among the proposed ratios, the stoichiometries 1:1 and 2:1 were found for metal:citrate. The results of ESI-MS and DH0(aq.) suggest that the complexes [Cd2+(Cit3)] and [Pb2+(Cit3)] can be formed, preferably to [Cd2+(HCit2)(H2Cit)] and [Pb2+(HCit2) (H2Cit)]. The formation of mononuclear species in the negative mode was favored in the ratio 2:1 metal:citrate and pH 7. The calculations performed for geometry and thermodynamic stability of the complexes show that the results obtained by the DFT method are not better than the data from the semi-empirical method. However, the vibrational frequencies in B3LYP/LANL2DZ best describe the experimental results. Understanding the toxicity of Cd2+ and Pb2+ in biological systems implies a thorough knowledge of their chemical behavior in aqueous solution and the species formed. The metal complexes formed with a physiologically relevant ligand, such as citrate, showed a structural diversity in the species investigated. Acknowledgments The authors would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the Grant provided; Laboratório de Modelagem Molecular of Universidade Federal de

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