Journal of Inorganic Biochemistry 136 (2014) 99–106

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Structural characterization of Cd2 + complexes in solution with DMSA and DMPS Elham Zeini Jahromi a, Jürgen Gailer a,⁎, Ingrid J. Pickering b, Graham N. George b,⁎⁎ a b

Department of Chemistry, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada Department of Geological Sciences, University of Saskatchewan, 114 Science Place, Saskatoon, Saskatchewan S7N 5E2, Canada

a r t i c l e

i n f o

Available online 4 December 2013 Keywords: Cadmium X-ray absorption spectroscopy Chelation therapy agents

a b s t r a c t Meso-2,3-dimercaptosuccinic acid (DMSA) and 2,3-dimercaptopropane-1-sulfonic acid (DMPS) are chelating agents which have been used clinically to treat patients suffering from Pb2+ or Hg2+ exposure. Cd2+ is a related environmental pollutant that is of increasing public health concern due to a demonstrated dose–response between urinary Cd level and an increased risk of diabetes. However, therapeutically effective chelating agents which enhance the excretion of Cd2+ from humans have yet to be identified. Here we present a structural characterization of complexes of DMSA and DMPS with Cd2+ at physiological pH using a combination of X-ray absorption spectroscopy, size exclusion chromatography and density functional theory. The results indicate a complex chemistry in which multi-metallic forms are important, but are consistent with both DMPS and DMSA acting as true chelators, using two thiolates for DMPS and one thiolate and one carboxylate for DMSA. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Cadmium is widely known as a toxic metal to which the potential health effects of exposure of human populations are of increasing concern [1]. The Centers for Disease Control and Prevention (CDC) of the United States indicated recently that around 12 million adults in the USA have urinary cadmium concentrations close to levels that might cause subtle kidney injury and decreased bone-mineral density [2]. Major sources of exposure can be linked to industrial uses of cadmium, and to the ingestion of traces of cadmium contained in tobacco through inhalation of cigarette smoke [3–7]. Occupational exposure arises predominantly from inhalation of cadmium-containing dusts or fumes, with the highest such exposures typically occurring in cadmium production and refining, nickel–cadmium battery manufacture, cadmium alloy production, mechanical plating, soldering or polyvinylchloride manufacture [6,8]. Intravenous cadmium selenide quantum dots are currently being investigated as alternatives to organic dyes for diagnostic purposes [9], and the deterioration of the CdSe lattice might yield free Cd2+[10]. Given the current level of human exposure, improved drug strategies to remove Cd2+ from humans are warranted. Chelators are entities that bind a metal ion with increased tenacity by dint of binding through more than one functional group. Chelation therapy is the drug use of these species to bind, mobilize and promote excretion of a heavy metal ion. To date, no chelating agent has been clinically approved for the treatment of Cd2 + intoxicated humans [11,12]. Given the known affinity of Cd2 + for chalconide donors [13] the vicinal dithiol chelator drugs meso-2,3-dimercaptosuccinic ⁎ Corresponding author. Tel.: +1 403 210 8899; fax: 4032899488. ⁎⁎ Corresponding author. Tel.: +1 306 966 5722; fax: 306 966 8593. E-mail addresses: [email protected] (J. Gailer), [email protected] (G.N. George). 0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jinorgbio.2013.10.025

acid (DMSA) and dimercaptopropane-1-sulfonic acid (DMPS) (Fig. 1) are potential candidates for treatment of cadmium intoxication [14,15]. DMSA and DMPS are commercially sold as Chemet® and Dimaval®, respectively. Several animal studies have shown that DMSA or DMPS can decrease toxicity, lower the cadmium body burden and increase urinary excretion of cadmium [16–20]. The non-chalconide chelator diethylenetriaminepentaacetic acid (DTPA) was effective in mobilizing Cd2+ to urine in rats only when intravenously administered immediately after Cd2+ [17]. Recent in vitro studies employing rabbit blood plasma have shown that pharmacologically relevant doses of DMSA and DMPS can mobilize Cd2+ from plasma proteins to a ~5 kDa Cd—species with an abstraction efficacy of 94% and 83%, respectively [21]. In previous work on complexes of DMSA and DMPS with Hg2+ we showed that in solution at physiological pH these vicinal dithiols are not in fact true chelators of Hg2+ [22], but instead act more like monofunctional thiolates. We report herein a structural study of the solution chemistry between Cd2+ and DMSA and DMPS using a combination of X-ray absorption spectroscopy (XAS), size exclusion chromatography in conjunction with inductively coupled plasma atomic emission spectroscopy (SEC-ICP-AES), and density functional theory (DFT).

2. Materials and methods 2.1. Chemicals Meso-2,3-dimercaptosuccinic acid (98%), sodium 2,3dimercaptopropanesulfonate monohydrate (95%), cadmium nitrate tetrahydrate (99.999%), tris(hydroxymethyl)-aminomethane (Tris, 99.9%), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, ≥99.5%) and phosphate-buffered saline (PBS) tablets were purchased

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injection of DMPS while monitoring sulfur as described below. The void and the inclusion volume define the chromatographic window. All chromatography experiments were carried out at room temperature using 0.1 M Tris-buffer pH 7.4 as the mobile phase with a flow rate of 1.0 mL min−1. Cd was monitored at the 226.502 nm emission line and S at the 180.731 nm emission line. The DMPS experiments were repeated with 0.15 M PBS buffer at pH 7.4 and were essentially identical to those obtained with Tris buffer. Fig. 1. Schematic structures of (A) meso-dimercaptosuccinic acid (DMSA) and (B) dimercaptopropanesulfonic acid (DMPS).

from Sigma-Aldrich. Samples for X-ray absorption spectroscopy were prepared at 5 mM Cd (final) in 100 mM HEPES buffer (pH 7.4) by mixing appropriate quantities of buffered solutions of DMSA/DMPS and Cd(NO3)2 to obtain DMSA/DMPS to Cd2+ molar ratios of 0.5, 1.0, 2.0, 4.0, and 8.0. Following incubation for 1 min at room temperature, the mixtures were loaded into acrylic 2 × 10 × 10 mm3 cuvettes and were frozen in liquid nitrogen. Samples for chromatography were prepared in an identical manner, but using PBS buffer (pH 7.4) unless otherwise stated. 2.2. X-ray absorption spectroscopy Measurements were carried out at Stanford Synchrotron Radiation Lightsource (SSRL) beamline 7-3, with the SPEAR storage ring containing 250 or 500 mA at 3.0 GeV, using a Si(220) double crystal monochromator with no specular optics in the beamline. The incident and transmitted X-ray intensities were monitored using nitrogen-filled gas ionization chambers (1.6 kV), while the Cd X-ray absorption was measured as the X-ray Kα fluorescence excitation spectrum using a germanium array detector with Soller slits and silver metal filters [23]. During data collection, the samples were maintained at a temperature of approximately 10 K using a liquid helium flow cryostat. For each sample, between two and six 35 min scans were accumulated, and the absorption of a standard Cd metal foil was measured simultaneously by transmittance. The energy was calibrated with reference to the lowest energy Kedge inflection of the metal foil, which was assumed to be 26,714.0 eV. The extended X-ray absorption fine structure (EXAFS) oscillations χ(k) were quantitatively analyzed by curve fitting using the EXAFSPAK suite of computer programs [24] as described by George et al. [25]. Fourier transforms were phase-corrected for Cd\S backscattering. The threshold energy (E0) was assumed to be 26,730.0 eV. Ab initio theoretical phase and amplitude functions were calculated using the program FEFF version 8.24 [26,27]. In EXAFS curve fitting analyses designed to determine effective coordination numbers N for Cd\S and Cd\O the Debye–Waller factors σ2 were held fixed in the refinements because of high mutual correlations between N and σ2. Thus, for these refinements, the offset to the energy threshold ΔE0 was held fixed at a value determined from fitting standard compounds of known structure at −11.17 eV, and the Cd\S Debye–Waller factor (σ2, 0.00320 Å2) were fixed at values determined from fitting the EXAFS of [Cd(SPh)4]2− [28,29], we note that this value agrees very well with the value calculated from vibrational spectra [28]. Similarly, the Cd\O Debye–Waller factor was fixed at 0.00466 Å2, determined for a 40 mM aqueous solution of Cd(NO3)2 measured in transmittance.

2.4. Molecular modeling Density functional theory molecular modeling used the program Dmol3 Materials Studio Version 6.1 [31,32]. The Becke exchange [33,34] and Perdew–Burke–Ernzerhof functional [35,36] were used to calculate both the potential during the self-consistent field procedure and the energy. Dmol3 uses numerically derived basis sets, and these included polarization functions for all atoms. Calculations were spin-unrestricted, and all-electron relativistic core potentials were used. No symmetry constraints were applied unless otherwise stated. Solvation effects were modeled using the Conductor-like Screening Model (COSMO) [37] with the dielectric constant of water (ε = 78.39). Convergence was said to be achieved when energies differed by less than 2 × 10 −5 Hartree, the maximum force was less than 0.004 Hartree Å−1 and the maximum displacement per iteration was less than 0.005 Å. A maximum step size of 0.3 Å was used. Non-bonded functional groups were given the protonation state according to the physiological pH of 7.4 used in this study. For DMSA the pKa values of the functional groups are 2.71 and 3.43 for two COOH groups and 9.65 and 12.05 for the SH groups [38]. For DMPS the values are b 1 for SO3H [39] and 8.53 and 11.62 for the two SH groups [40]. In cadmium chelate complexes the pKa values of groups not directly involved in binding the metal may be subtly perturbed, but at pH 7.4 the unbound carboxylate and sulfonate groups should be deprotonated and the thiol groups protonated. In density functional theory calculations comparing energies of mononuclear chelation conformers two free ligand molecules were added in order to ensure that equivalent numbers of atoms were present in each system, appropriately deprotonated to provide the same overall charge. 3. Results 3.1. X-ray absorption spectroscopy X-ray absorption spectroscopy can be used to give structural information on species in solution and was employed to gain structural insights into the interaction between Cd2+and DMSA or DMPS. Measurements were conducted in 100 mM HEPES buffer (pH 7.4), which was chosen both because it does not exhibit a strong tendency to form complexes with Cd2+ [41] and because, unlike phosphate, it does not change pH appreciably on freezing [42]. X-ray absorption spectra can be divided into two regions, the near-edge region which is sensitive to electronic structure of the element in question (e.g. oxidation states, covalency) and the extended X-ray absorption fine structure (EXAFS) region. The EXAFS part of the spectrum can be readily analyzed to provide accurate interatomic distances as well as more approximate coordination numbers and identities of the nearby atoms [43].

2.3. Size exclusion chromatography Size exclusion chromatography in conjunction with inductively coupled plasma atomic emission spectroscopy (SEC-ICP-AES) was carried out as previously reported [22,30]. A 1.0 cm × 30 cm Superdex Peptide column with a fractionation range of 7.0–0.1 kDa served as the stationary phase and was equipped with a Rheodyne injection valve (500 μL loop). The void volume, equivalent to 430 s, was determined by injection of Blue Dextran and by following the C emission line at 193.091 nm. The inclusion volume, equivalent to 1176 s, was determined by the

3.1.1. Near-edge spectra Fig. 2 shows the Cd K near-edge spectra that were obtained during the titration of Cd(NO3)2 with increasing DMSA (panel A) or DMPS (panel B). Both show a variation in near-edge structure as a function of increasing ratio of chelating agent to Cd2+, with the prominent peak on top of the edge for the Cd(NO3)2 decreasing in intensity. The spectra change noticeably from 0 to 0.5 for both DMSA and DMPS, with the two molar ratio 0.5 spectra with the different agents being quite similar. At higher molarities the effect of the two chelating agents differs. The DMPS

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Fig. 2. X-ray absorption spectroscopy titration of Cd2+ with DMSA and with DMPS. (A, B) The Cd K near-edge spectra; (C, D) the EXAFS Fourier transforms (phase-corrected for Cd\S backscattering). The ratios of DMSA:Cd2+ and DMPS:Cd2+ are indicated on the figure. Insets to panels A and B show the results of linear combination fits of each near-edge spectra using DMPS molar ratios 0.0 (▲), 1.0 (●) and 8.0 (■) as standards.

spectral series shows systematic changes through 2.0 and thereafter only subtle differences through 8.0. The highest molar ratio spectra show a much decreased intensity at the top of the edge and resemble spectra previously reported for [Cd(SR)4]2− species [28]. In contrast, for DMSA the spectra change slightly between 0.5 and 1 and then only subtly up to the highest molar ratio of 8.0. The spectra for the highest molar ratios of DMSA show the most similarity with the spectrum of DMPS: Cd2+ molar ratio of 1.0. Principal component analysis (PCA) can estimate the number of discrete components required to fit a set of data [44]. PCA of the near-edge spectral series (Fig. 3), together with examination of Malinowski's indicator function IND [44] indicated that at least three components were needed to fit each series. The indicator function IND is expected to give a minimum value for the required number of components [44], and for both DMPS and DMSA IND shows a minimum on inclusion of three components, although this is better defined for DMPS than for DMSA. Linear combination fits of the individual spectra used three components, chosen to be the spectrum of Cd(NO3)2 in the absence of chelating agents (molar ratio 0.0), and the DMPS:Cd2+ molar ratios 1.0 and 8.0. DMPS:Cd2+ 8.0 was chosen to be the most extreme end member species observed in either series. DMPS:Cd2+ 1.0 was chosen as the third component since this ratio showed a single species in the size exclusion chromatography measurements (see below) and also showed the worst residual when the near-edge spectrum was fit with the two end-member species alone. The results of the near-edge linear combination fits are shown as insets to Fig. 2 and in Table 1. For both chelating

agents, the molar ratio 0.5 showed very similar composition, with the DMSA and DMPS spectra respectively showing (54 ± 1) and (49 ± 2)% contribution from the DMPS:Cd2+ 1.0 spectrum, with the balance coming from the 0.0 spectrum. For DMPS:Cd2+ molar ratios greater than 1.0, the 0.0 spectrum did not contribute and DMPS:Cd2+ 8.0 was the major component; molar ratio 2.0 showed some DMPS:Cd2+ 1.0 while higher molar ratios showed more than 99% of the end member DMPS:Cd2+ 8.0 spectrum. In the DMSA series, similarly fit with the DMPS:Cd2+ 1.0 and 8.0 spectra, molar ratio 1.0 showed an approximate 80/20 split between the 0.0 and 1.0 spectra, while ratios 2.0 and above fitted with 3 components, showing an average of about 10, 70 and 20 for the 0.0, 1.0 and 8.0 molar ratio spectra, respectively. 3.1.2. Extended X-ray absorption fine structure (EXAFS) Panels C and D of Fig. 2 show the EXAFS Fourier transforms, phasecorrected for Cd\S backscattering, from the same samples shown in Fig. 2A and B. All of the Fourier transforms show a single prominent peak, corresponding to the first shell coordination of Cd2+, the apparent interatomic distance of which increases between molar ratios 0.0, 0.5 and 1.0 for both DMSA and DMPS. For molar ratios 1.0 to 8.0, the peaks appear relatively invariant, although those of DMSA are somewhat broader and less intense than those of DMPS. All data were analyzed by quantitative curve fitting, the numerical results of which are presented in Table 2. In order to minimize correlations between variables, the EXAFS data sets for all the DMSA:Cd2+ and DMPS:Cd2 + samples were fit using the same constraints. It was

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E. Zeini Jahromi et al. / Journal of Inorganic Biochemistry 136 (2014) 99–106 Table 2 EXAFS curve-fitting results for DMSA:Cd2+ and DMPS:Cd2+ complexes.a R (Cd\S)

N (Cd\O)b

R (Cd\O)

Fc

NNE (Cd\S)d

DMSA:Cd 0.5 1.0 (3) 1.0 1.8 (3) 2.0 2.0 (2) 4.0 2.3 (4) 8.0 2.2 (2)

2.581 (15) 2.553 (7) 2.548 (4) 2.551 (8) 2.540 (3)

3.0 (3) 2.2 (3) 2.0 (2) 1.7 (4) 1.8 (2)

2.340 (15) 2.339 (17) 2.334 (13) 2.34 (3) 2.335 (13)

0.6605 0.4968 0.3499 0.5812 0.2970

1.0 1.7 2.2 2.0 2.4

DMPS:Cd 0.5 0.9 (3) 1.0 1.8 (2) 2.0 3.1 (2) 4.0 3.5 (3) 8.0 3.8 (2)

2.556 (15) 2.547 (5) 2.529 (3) 2.523 (5) 2.521 (3)

3.1 (3) 2.2 (2) 0.9 (2) 0.5 (3) 0.2 (2)

2.334 (13) 2.347 (15) 2.33 (3) 2.31 (8) 2.3 (2)

0.6073 0.4031 0.3083 0.3982 0.2642

1.1 2.0 3.4 4.0 4.0

N (Cd\S)

a Coordination numbers (N), interatomic distances (R, Å) and goodness of fit (F). Three times the estimated standard deviation in the last digit(s), derived from the diagonal elements of the covariance matrix, is shown in parentheses for all refined parameters. See Materials and methods for fixed values of Debye–Waller factors. b The sum of coordination numbers for Cd\S and Cd\O were constrained to be 4.0 in each case. rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi  2 6 6 2 c F ¼ ∑k χ ðkÞcalc −χ ðkÞexpt =∑k χ ðkÞexpt : d Coordination numbers for Cd\S (NNE) were predicted from the near-edge fit results assuming molar ratios 0.0, 1.0 and 8.0 have 0, 2 and 4 sulfur ligands, respectively.

Fig. 3. The eigenvalue-weighted eigenvectors (components) from principal component analysis of the near-edge spectra of the X-ray absorption spectroscopy titration of Cd2+ with DMPS and with DMSA. We note that these are not physical components, but merely mathematical constructs which can completely represent the data sets. The insets show Malinowski's indicator function (IND) plotted against factor number showing that the data can be adequately represented by 3 components for DMPS, and by 2–3 components for DMSA.

assumed that both Cd\S and Cd\O ligands are present in each mixture, the proportions of which could vary depending on the nature of the complexes present and their proportions in each sample. The coordination numbers for both Cd\S and Cd\O were refined, assuming that Cd\S and Cd\O coordination numbers always summed to an ideal value of 4.0, indicated from detailed fits of the key data sets in the titration (see below). Both Cd\S and Cd\O interatomic distances were allowed to vary independently in each fit.

Table 1 Cd K near-edge spectra least squares fitting results.a Chelator:

DMPS

DMSA

Component: Molar ratio a

b

c

a

b

c

0 0.5 1.0 2.0 4.0 8.0

0.0 0.54 (1) 1.0 0.33 (3) 0.0 0.0

0.0 0.0 0.0 0.67 (3) 0.99 (1) 1.0

1.0 0.50 (2) 0.18 (1) 0.13 (2) 0.12 (2) 0.04 (1)

0.0 0.50 (2) 0.82 (2) 0.64 (8) 0.77 (8) 0.71 (5)

0.0 0.0 0.0 0.23 (7) 0.11 (7) 0.25 (5)

1.0 0.46 (1) 0.0 0.0 0.01 (1) 0.0

a The components used for the fit are those representing the observed spectroscopic extremes, as discussed in the text. Component a is Cd(NO3)2 in the absence of chelating agents (molar ratio 0.0), b is the DMPS:Cd2+ molar ratios 1.0 as SEC indicated essentially a single component in this case, and c for molar ratio 8.0 DMPS:Cd2+. As discussed in the text, c was chosen to be the most extreme end member species observed in either series. Fits of these components therefore are not valid analyses and gave the expected single species with vanishingly small errors. The table gives fractions with the values in parentheses being the 99% confidence limits in the last digit (3× the estimated standard deviation, obtained from the diagonal elements of the covariance matrix).

Table 2 shows that for DMSA, within the accuracy of the data, the mean number of Cd\S interactions in the first coordination shell increases from 1 to 2 between molar ratios 0.5 and 1.0. For the remaining DMSA molar ratios of 1.0 through 8.0 the mean number of Cd\S contacts is constant at 2, which corresponds to each Cd2+ coordinated by two sulfurs and two oxygens. In contrast the DMPS series shows an increase in Cd\S coordination number to approach a final value of 4.0, corresponding to Cd2 + 4-coordinate by sulfur with negligible contribution from Cd\O. Table 2 also shows the Cd\S coordination numbers (NNE) estimated from the near-edge fits, showing excellent agreement with those derived from the EXAFS fits. The determined Cd\O distances are the same within the accuracy of the data at 2.34 Å for all molar ratios and both chelating agents. The Cd\S distances, however, do show a significant variation as a function of molarity for DMPS, with those for the highest molar ratios being 2.52 Å, slightly but significantly shorter than those for lower ratios of 2.55 Å. The DMSA series exhibits the longer value of 2.55 Å for all molar ratios between 1.0 and 8.0. A significantly longer value of 2.58 Å observed for the Cd\S distance for molar ratio 0.5 has greater uncertainty as the contribution of the Cd\S shell is much smaller for this composition. There are a large number of crystallographically characterized cadmium compounds reported in the literature. Examination of the Cambridge Structural Database [45] indicates coordination numbers between 2 and 8. Two coordinate cadmium complexes are restricted to oranometallic species, such as dimethylcadmium (CH3\Cd\CH3) which show linear coordination geometries. Setting these aside, the lowest coordination number relevant to our work is 3. These species typically have trigonal planar type coordination geometry. Much more common is the fourcoordinate pseudo-tetrahedral coordination geometry. Less common again are five coordinate species, which tend to have square-based pyramidal type geometries and six, seven and eight coordinate species can be considered rare. For cadmium sulfur complexes the Cd\S bond-length changes systematically from 2.48 Å for 3-coordinate species, 2.54 Å for 4-coordinate pseudo-tetrahedral species, 2.63 Å for 5-coordinate, 2.71 Å for 6-coordinate, 2.76 Å for 7-coordinate and 2.84 Å for 8-coordinate. 3.2. Size exclusion chromatography 3.2.1. DMPS Size exclusion chromatography (SEC) of the solution with a DMPS: Cd2+ molar ratio of 1.0 (Fig. 4A) revealed that Cd and S co-eluted with

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3.2.2. DMSA The SEC results obtained for DMSA:Cd2+ are shown in Fig. 4B. The SEC of DMSA:Cd2+ molar ratio of 1.0 shows two peaks containing both Cd and S at retention times of 742 and 831 s and no free DMSA. The shorter retention time peak is probably from a complex related to that observed with a DMPS molar ratio of 1.0, containing a tri-metallic core with bridging DMSA. The longer retention time peak corresponds to a smaller species, possibly containing a di-metallic core, consistent with the shorter retention time relative to the peak attributed to a mono-metallic complex with DMPS (Fig. 4A). Similarly to DMPS, the SEC results of higher molar ratios show a multiple peak containing both Cd and S that appears similar for molar ratios of 2, 4 and 8. In addition the higher molar ratios show increasing amounts of a peak containing S but not Cd, attributable to free DMSA at a retention time of 952 s. The multiple peak contains at least three features with retention times close to 706, 790 and 840 s. As with DMPS this clearly indicates a complex mixture of species which changes only slightly in composition between molar ratios of 2.0 and above. 3.3. Molecular modeling

Fig. 4. A: Size exclusion/ICP-AES chromatography of different DMPS:Cd2+ ratios, as indicated on the figure. B: Size exclusion/ICP-AES chromatography of different DMSA:Cd2+ ratios, as indicated on the figure.

a retention time of 676 s. The fact that no substantial additional Cd or S peaks were detected can be rationalized in terms of a virtually quantitative formation of a 1:1 Cd2+–DMPS complex. Size estimation for this solution species indicates an equivalent molecular weight of about 1 kDa. The EXAFS results for this molar ratio suggest a four-coordinate Cd2+– DMPS complex with two sulfur ligands from DMPS and two oxygen ligands, probably from water (Fig. 5A, B). Hence the size of this solution species likely corresponds to a cyclic arrangement of CdnDMPSn where each Cd is coordinated to thiols from different DMPS molecules and two water molecules, possibly a tri-metallic species with n = 3 (Fig. 5b). The SEC analysis of solutions with higher molar ratios shows increasing free DMPS with increasing molar ratio as a sulfur peak at long retention times (1076 s) plus a more complicated multiple peak containing both Cd and S between 715 and 834 s which is remarkably similar at molar ratios 2, 4 and 8. This Cd\S set of peaks contains at least three features, indicating that a mixture of species is present. The species with the longest retention time of 834 s contributing to this multiple peak corresponds to a comparatively small complex, possibly to a mono-metallic species in which the Cd atom is coordinated to four S atoms from two DMPS molecules in a true bis-chelate complex (Fig. 5c). The species with the shortest retention time of 715 s is likely related to the species observed for a molar ratio 1.0 with a retention time of 676 s, but with the higher sulfur coordination observed by EXAFS, possibly a tri-metallic species with external DMPS chelation of Cd (Fig. 5e). It seems plausible that intermediate poly-metallic forms may give rise to the additional complexity of the Cd\S multiple peak.

Since DMSA and DMPS each have two thiol groups, their mode of chelation is often assumed to be through both sulfurs. However, both molecules have additional functional groups – two carboxylate groups for DMSA, and a sulfonate group for DMPS – which could provide alternative chelation conformations. Both DMSA and DMPS can therefore potentially bind Cd2 + in a number of different ways. When the multimetallic forms are considered the number of permutations becomes very large. Therefore the mono-metallic complexes were compared as a computationally tractable alternative. Density functional theory energy minimized geometry optimization calculations were used to compare the difference in energies of chelating through the alternative functional groups. The schematic structures and their relative energies are shown in Fig. 6. 3.3.1. DMPS In the case of DMPS, the two thiolate groups and the additional sulfonate group give three conformations for DMPS to function as a bidentate chelator to mono-metallic complexes. Binding with the sulfonate group in addition to one thiolate gives two possible Cd(O)2(S)2 coordinations, forming 6- or 7-membered rings, depending on which thiolate is binding. However, the conventional bis-thiolate chelation forming Cd(S)4 coordination with a 5-membered ring is energetically favored by more than 150 kJ mol−1 (Fig. 6). This is consistent with the observation of 4 Cd\S interactions in the EXAFS of higher molar ratios of DMPS, although poly-metallic complexes probably also contribute. 3.3.2. DMSA The two thiolate and two carboxyl groups of DMSA give four conformations in which DMSA could function as a bidentate chelator in mono-metallic complexes (Fig. 6). The conventional bis-thiolate chelation with two molecules yields Cd(S)4 coordination with 5-membered rings. Binding with one thiolate and one carboxylate from each molecule gives two additional possibilities, both with Cd(O)2(S)2, giving 5- or 6-membered rings depending on whether the carboxylate is proximal or vicinal to the thiolate. Finally, binding with both carboxylates gives a 7-membered ring chelate with Cd(O)4 coordination. Density functional theory calculations show that this latter structure is strongly disfavored with respect to the other three (Fig. 6). The remaining three structures are relatively close in energy to each other, suggesting that any one or more could be favored depending on the detailed conditions within the solution. This suggests that more permutations of oxygen and sulfur coordination are likely in the case of DMSA relative to DMPS, which is in broad agreement with the EXAFS results, which show a mixed coordination O, S species predominating at molar ratios of 2.0 and above.

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Fig. 5. Schematic structures of mono and poly-metallic clusters involving Cd2+ and DMPS. A shows the mono-metallic species representing the smallest 1:1 complex, B shows a tri-metallic species with three bridging DMPS ligands. The complex a represents a true chelate complex whereas b does not. The complexes C c–E are the series [Cdn(DMPS)2n]4n− for n = 1 to 3, respectively.

3.3.3. Multi-metallic complexes Geometry optimization of the larger multi-metallic forms (e.g. Fig. 5) indicated that these structures are chemically feasible. Generalizing both DMSA and DMPS as the prototypical vicinal dithiol chelator ethane dithiol (EDT) (HS\CH2CH2\SH), we find a number of stable forms of different metal ion content. For example, bi-metallic structures with two μ2 bridging thiolates from one EDT and two additional EDT each

chelating the two Cd2 + are predicted to have a Cd⋯Cd separation of 3.4 Å. The Cd\S bond-lengths to μ2 bridging thiolates are predicted to be about 0.12 Å longer than terminal non-bridging chelator thiols. This is in excellent agreement with available crystallographic information [46,47]. Comparing geometry optimized energies for the complexes [Cd(EDT)2]2−, [Cd2(EDT)4]4− and [Cd3(EDT)6]6−, the most stable is the mono-metallic complex, but only by 23 kJ/mol per Cd, with higher

Fig. 6. Density functional theory calculated differences in energies of the various chelation conformers for monometallic complexes (A) Cd(DMPS)2and (B) Cd(DMSA)2. The variation in charges of the different DMSA species was corrected for by computing the difference in energy between DMPS and DMSA with protonated and de-protonated thiols.

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metal contents corresponding to progressively higher energies, each separated by ~20 kJ/mol per Cd. We note that these calculations only include enthalpies, and that with energy separations as close as these a small entropic component could easily alter which complex is most stable. The conformational flexibility of [Cdn(EDT)n]n− complexes is also evident from our calculations, with Cd⋯Cd separations computed to range between 5 to 7 Å, so that these would be very difficult to observe using EXAFS, which requires approximately rigid interatomic distances for best observation [43]. 3.4. Combined density functional theory and EXAFS structures We selected two DMPS data sets for detailed EXAFS analysis. Fig. 7 shows EXAFS curve-fitting results for the DMPS:Cd2 + molar ratio of 1:1 and 8:1, together with the EXAFS Fourier transforms. The EXAFS of the 8:1 molar ratio sample clearly shows four sulfur donors, plus more subtle features attributable to the outer shell carbon atoms of the DMPS ligand. There is no indication of long-range Cd\Cd interactions expected for polymeric forms, although this could be explained by the presence of a number of different conformations as discussed in Section 3.3.3. The EXAFS derived bond-lengths show excellent correspondence with those from the density functional theory calculations. The EXAFS of the 1:1 sample shows clear evidence of mixed coordination by two Cd\O and two Cd\S, with the former presumably arising from water donors. Again, the EXAFS derived bond-lengths show match well with those from the density functional theory calculations. The structures of three representative complexes are shown in Fig. 8. 4. Discussion

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with two quite different retention times elute for each of the higher molar ratios supports the presence of more than one form in these solutions. The EXAFS excludes species with pairs of Cd bound through μ2 bridging thiolates as these would give short Cd⋯Cd distances easily observable by EXAFS. PCA of the DMPS series near-edge data indicates that three components are required to fit the spectra. Our observations are consistent with the formation of a different species for molar ratio 1.0. The SEC result for this ratio shows a single species of high relative molecular mass for which all of the Cd and S co-elute, while EXAFS indicates two sulfur and two oxygen ligands. This is consistent with the formation of a Cdn(DMPS)n(H2O)2n complex, probably with n = 3, in which the DMPS has bis-thiolate coordination. 4.2. DMSA:Cd2+complexes In the case of the DMSA:Cd2+ series, our observations support the formation of bidentate chelate complexes, in the form of multi-metallic complexes, and using more than one functional group. Comparison of mono-metallic species using DFT calculations suggests that for DMSA, chelation through two thiolates, or through one thiolate and either the proximal or vicinal carboxylate, all have fairly similar energies. Our EXAFS observations suggest that the mixed thiolate-carboxylate chelation predominates at all compositions above 1.0, although the minority presence of a bis-thiolate chelation is also supported by the near-edge data. Similar to DMPS, the SEC indicates the presence of complexes containing more than one Cd2 + ion and multiple chelator molecules. A species corresponding to Cd4(DMSA)4 previously has been suggested to be the most stable polymetallic form [38].

4.1. DMPS:Cd2+ complexes

4.3. Implications for in vivo DMSA and DMPS chelation

The DMPS:Cd2+ series provides strong evidence that DMPS can form a bidentate chelator, with bis-thiolate coordination, for higher molar ratios. For these samples the EXAFS shows four Cd\S interactions, which are consistent with Cd2+ binding either two bidentate DMPS or multi-metallic ring-shaped forms where DMPS bridges between Cd atoms as indicated by the SEC results. The observation from the nearedge that some 67% of the 8.0 molarity end member Cd coordination is present already in the DMPS:Cd2+ sample with molar ratio 2.0 argues that chelate complexes with four sulfur donors do indeed form. DFT calculations also strongly support the formation of chelate complexes with binding through the two thiolate groups, and the possibility of multi-metallic forms. Conversely, the SEC observation that Cd/S species

In view of the fact that in biological tissues, the molar ratio of chelator to metal is relatively high, [Cd(DMSA)2]4− and [Cd(DMPS)2]4− are likely the complexes that are formed in vivo. In order for these species to be excreted from the kidneys, they need to be recognized by molecular pumps to be ultimately excreted in the urine. The renal anion transporters which are thought to enhance the excretion of complexes, such as those formed between Hg2+ and DMPS [1] likewise may be involved in the excretion of negatively charged chelate Cd2+ complexes to urine [48,49]. Since Cd2+ in the blood circulation may be potentially translocated to organs, it is important to devise strategies to transform plasma protein-bound Cd2 + into a complex that can be readily excreted via

Fig. 7. Detailed EXAFS analysis for samples with DMPS:Cd2+stoichometries of 8:1 (A) and 1:1 (B), showing EXAFS oscillations (left), and Cd\S phase-corrected EXAFS Fourier transforms (right). Data and best fits are shown as solid and dashed lines, respectively. Best fit EXAFS parameters were (a) 4 Cd\S at 2.518 (4) Å, σ2 = 0.0033 (2) Å2, ΔE0 = −11.9 (1.2) eV, F = 0.25117, (b) 2 Cd\S at 2.547 (8) Å, σ2 = 0.0038 (6) Å2, 2 Cd\O at 2.352 (15), σ2 = 0.0041 (14) Å2, ΔE0 = −11.3 (1.5) eV, F = 0.37166 (see text and Table 2 for definitions).

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Fig. 8. Representative density functional theory energy minimized geometry optimized structures for (A) [Cd(DMPS)2]4−, (B) [Cd(DMPS)(OH2)2]− and (C) [Cd3(EDT)6]6− (EDT is 1,2ethanedithiol) containing the representative core structure [Cd3(EDT)3]0. The structures A and B are shown on the same relative scale, and C at a slightly smaller scale for convenience of display. In A the average Cd\S bond-length is 2.57 Å and in B the average Cd\S is 2.48 Å and Cd\O 2.39 Å. In the case of C the structure was constrained to have D3 point group symmetry and has a Cd⋯Cd separation of 6.98 Å (Cd\S of 2.58 Å). When symmetry constraints are lifted from C a large number of possible conformers can result with Cd⋯Cd separations ranging from 7.1 to 5.2 Å, which would explain why no long range Cd⋯Cd is observed by EXAFS.

the kidneys. Considering that Cd represents a pollutant of growing concern, the studies presented herein represent an important first step in the context of developing more effective chelating agents for this toxic heavy metal. Acknowledgments E.Z.J. is a fellow of the Canadian Institutes of Health Research— Training grant in Health Research Using Synchrotron Techniques (CIHRTHRUST). G.N.G. and I.J.P. are Canada Research Chairs. Research at the University of Calgary is funded by NSERC (J.G.). Research at the University of Saskatchewan is funded by grants from NSERC (G.N.G., I.J.P.), and from CIHR (G.N.G., I.J.P.) and SHRF (G.N.G., I.J.P.). Portions of this research were carried out at the Stanford Synchrotron Radiation Light source, a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the U.S. Department of Energy Office of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS, NCRR or NIH. References [1] J. Godt, F. Scheidig, C. Grosse-Siestrup, V. Esche, P. Brandenburg, A. Reich, D.A. Groneberg, J. Occup. Med. Toxicol. 1 (2006) 22–27. [2] Fourth National Report on Human Exposure to Environmental Chemicals, Centers of Disease Control and Prevention (CDC), Atlanta, GA, USA, 2009. [3] J.R. Edwards, W.C. Prozialeck, Toxicol. Appl. Pharmacol. 238 (2009) 289–293. [4] S. Satarug, S.H. Garrett, M.A. Sens, D.A. Sens, Environ. Health Perspect. 118 (2010) 182–190. [5] J.P. Buchet, J.P. Buchet, R. Lauwerys, H. Roels, A. Bernard, P. Bruaux, F. Claeys, G. Ducoffre, P. de Plaen, J. Staessen, A. Amery, P. Lijnen, L. Thijs, D. Rondia, F. Sartor, A. Saint Remy, L. Nick, Lancet 336 (1990) 699–702. [6] J. Huff, R.M. Lunn, M.P. Waalkes, L. Tomatis, P.F. Infante, Int. J. Occup. Environ. Health 13 (2007) 202–212. [7] H.I. Afridi, T.G. Kazi, A.G. Kazi, F. Shah, S.K. Wadhwa, N.F. Kolachi, A.Q. Shah, J.A. Baig, N. Kazi, Biol. Trace Elem. Res. 144 (2011) 164–182. [8] W.J. Crinnion, J.Q. Tran, Altern. Med. Rev. 15 (2010) 303–310. [9] A.R. Edmund, S. Kambalapally, T.A. Wilson, R.J. Nicolosi, Toxicol. in Vitro 25 (2011) 185–190. [10] A.M. Derfus, W.C.W. Chan, S.N. Bhatia, Nano Lett. 4 (2004) 11–18. [11] M. Blanusa, V.M. Varnai, M. Piasek, K. Kostial, Curr. Med. Chem. 12 (2005) 2771–2794. [12] O. Andersen, Mini-Rev. Med. Chem. 4 (2004) 11–21. [13] K. Yamasaki, K. Suzuki, Proc. Int. Conf. Coord. Chem. 8th (1964) 357–360.

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