Structural Characterization of SmIII(EDTMP) - ACS Publications

Oct 5, 2015 - phonic acid) (153Sm-EDTMP, or samarium lexidronam), also known by its registered trademark name Quadramet, is an approved therapeutic ...
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Structural Characterization of SmIII(EDTMP) Y. Yang,†,‡ M. J. Pushie,‡,§ D. M. L. Cooper,§ and M. R. Doschak*,†,∥,⊥,# †

Pharmaceutical Orthopaedic Research Lab, 2-020J Katz Group Centre for Pharmacy & Health Research, Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E1 § Department of Anatomy & Cell Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5 ∥ Biomedical Engineering, ⊥Department of Dentistry, and #Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E1 Mol. Pharmaceutics 2015.12:4108-4114. Downloaded from pubs.acs.org by UNIV OF NEW ENGLAND on 10/24/18. For personal use only.

S Supporting Information *

ABSTRACT: Samarium-153 ethylenediamine-N,N,N′,N′-tetrakis(methylenephosphonic acid) (153Sm-EDTMP, or samarium lexidronam), also known by its registered trademark name Quadramet, is an approved therapeutic radiopharmaceutical used in the palliative treatment of painful bone metastases. Typically, patients with prostate, breast, or lung cancer are most likely to go on to require bone pain palliation treatment due to bone metastases. Sm(EDTMP) is a bone-seeking drug which accumulates on rapidly growing bone, thereby delivering a highly regionspecific dose of radiation, chiefly through β particle emission. Even with its widespread clinical use, the structure of Sm(EDTMP) has not yet been characterized at atomic resolution, despite attempts to crystallize the complex. Herein, we prepared a 1:1 complex of the cold (stable isotope) of Sm(EDTMP) under alkaline conditions and then isolated and characterized the complex using conventional spectroscopic techniques, as well as with extended X-ray absorption fine structure (EXAFS) spectroscopy and density functional structure calculations, using natural abundance Sm. We present the atomic resolution structure of [SmIII(EDTMP)−8H]5− for the first time, supported by the EXAFS data and complementary spectroscopic techniques, which demonstrate that the samarium coordination environment in solution is in agreement with the structure that has long been conjectured. KEYWORDS: Quadramet, lexidronam, DFT, EXAFS, molecular dynamics, solvation

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Sm(EDTMP), shown in Scheme 1, is a consensus,5 although to date, however, this complex has not been structurally

INTRODUCTION Samarium-153 ethylenediamine-N,N,N′,N′-tetrakis(methylenephosphonic acid) (153Sm-EDTMP, or samarium lexidronam), is also known by its registered trademark name Quadramet (Dupont Pharma),1,2 and is an approved therapeutic radiopharmaceutical for painful bone metastases that is employed when cancer has spread to the bone.3 Samarium-153 lexidronam is commonly indicated for end stage palliative treatment in diverse metastatic cancers such as lung cancer, prostate cancer, breast cancer, and osteosarcoma. Due to its inherent high bone affinity, the uptake of 153Sm-EDTMP is known to be superior to that of both 111In-EDTMP and 99mTcEDTMP4 and provides a means of delivering a highly sitespecific dose of radiation which kills adjacent cells and lessens pain following a short duration after infusion. Quadramet is commonly employed in the palliative care clinic, and clarification of its structure would significantly aid subsequent investigations of in vivo bioactivity, metabolism, and bioinorganic chemistry at the bone surface, as well as its potential interaction with proteins expressed by osteoblasts and osteoclasts. Such detailed characterization of this therapeutically important complex would help refine microdosimetry and improve current antitumor therapeutic use, while potentially reducing unwanted side effects. The complex depicted for © 2015 American Chemical Society

Scheme 1. Preparation of Na5[SmEDTMP]

characterized at atomic resolution. We therefore set out to structurally characterize the nature of the Sm3+ complex with EDTMP. The EDTMP ligand is also capable of coordinating a range of metals, including other lanthanides,6 and better understanding of those coordination preferences may guide Received: Revised: Accepted: Published: 4108

July 8, 2015 September 3, 2015 October 5, 2015 October 5, 2015 DOI: 10.1021/acs.molpharmaceut.5b00546 Mol. Pharmaceutics 2015, 12, 4108−4114

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Molecular Pharmaceutics further chemical modification of the ligand to fine-tune its coordination chemistry. Herein, the cold (stable isotope) complex of Sm−EDTMP is prepared by directly chelating Sm3+ with EDTMP at a 1:1 ratio in aqueous solution at alkaline pH, and then characterized using mass spectrometry, NMR, fluorescence, and UV−vis spectroscopy. Extended X-ray absorption fine structure (EXAFS) spectroscopy is also employed to determine the coordination environment of the Sm3+ center in aqueous solution. Analysis and interpretation of spectroscopic data was complemented using computational chemistry methods, including density functional calculations and molecular dynamics simulations. Combining EXAFS spectroscopy with supporting structural characterization and computational methods have been successfully employed for alternate multidentate metal complexes previously,7−10 demonstrating the efficacy of this type of combined approach.

negative ion mode on an Agilent Technologies G1946A MSD instrument (Santa Clara, CA, USA) with the aqueous solution containing 50% methanol to aid ionization. No methanolcontaining adducts or complexes were identifiable in the results. X-ray Absorption Spectroscopy Data Collection. X-ray absorption spectroscopy (XAS) measurements were conducted at the Stanford Synchrotron Radiation Lightsource with the SPEAR storage ring containing 500 mA at 3.0 GeV, using the data acquisition program XAS Collect.13 Samarium LIII-edge data were collected on the structural molecular biology XAS beamline 7-3, employing a Si(220) double-crystal monochromator. Beamline 7-3 is equipped with a rhodium-coated vertically collimating mirror upstream of the monochromator with harmonic rejection accomplished by setting the mirror cutoff angle to 9 keV. Incident and transmitted X-ray intensities were monitored using nitrogen-filled ionization chambers with a sweeping voltage of 1.6 kV. X-ray absorption was measured as the Sm LIII fluorescence excitation spectrum using a germanium array detector (Canberra Ltd., Meriden, CT, USA).14 An aqueous sample of 2.5 mM SmIII(EDTMP)−8H at pH 8, containing 25% glycerol as a glassing agent to prevent ice diffraction, was loaded into a Br-free Delrin cell for Sm LIII EXAFS data collection. Removal of Br, which is present in many plastics (i.e., conventional sample cells) was necessary due to problematic Br Kα fluorescence, which otherwise contributed significantly to the background, arising from residual second harmonic photons from the monochromator. During data collection the sample was maintained at 10 K using an Oxford instruments liquid helium flow cryostat. A total of four spectra were accumulated with a k-range of 12.0 Å−1. Collecting higher k-range data was not possible due to Sm LII fluorescence, at 7312 eV. The energy of the incident beam was calibrated by reference to the absorption of a manganese metal foil measured before and after the series of Sm EXAFS scans, assuming a lowest energy inflection point of 6539.0 eV for the Mn K-edge. The energy threshold of the Sm EXAFS oscillations (k = 0 Å−1) was assumed to be 6735.0 eV. The presence of trace Fe in the Sm EXAFS spectrum, which would be apparent at 7112 eV, or ∼9.8 Å−1, was not evident. EXAFS Data Analysis. The EXAFS oscillations χ(k) were quantitatively analyzed by curve fitting using the EXAFSPAK suite of computer programs15 as previously described,16 using ab initio theoretical phase and amplitude functions calculated using the program FEFF version 8.25.17,18 No smoothing, filtering, or related operations were performed on the data. The EXAFS Fourier transform was phase-corrected using oxygen. Single scattering paths as well as a multiple scattering model used in the fitting procedure were obtained from a geometryoptimized DFT model of SmIII(EDTMP)−8H (see below). A scale factor (S02) of 1.0 was used for the Sm LIII EXAFS curve fitting procedure. Density Functional Calculations. Density functional theory (DFT) based calculations employed the Gaussian 09, revision D.01 suite of software.19 Spin-restricted geometry optimization calculations used an effective core potential for the Sm atom. Gaussian calculations were performed without symmetry constraints using the B3LYP hybrid functional method20−23 with a mixed basis set approach employed for geometry optimizations and subsequent harmonic frequency calculations, using the 6-311+G(d,p) basis set for C, O, N, P, and H atoms, with the MWB effective core potential (ECP) used for Sm. Bulk solvation of the gas-phase-optimized structure was modeled using the integral equation formalism

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MATERIAL AND METHODS Chemicals. Ethylenediamine tetra(methylenephosphonic acid) (H8EDTMP) was purchased from Santa Cruz Biotechnology, Santa Cruz, CA (95% purity), and SmCl3 (>99% purity) was purchased from Sigma-Aldrich, Carlsbad, CA. All chemicals were used without further purification. Preparation of Na5[Sm-EDTMP] was accomplished following previously published methods.11 Briefly, the pH of an aqueous solution containing 0.004 mol (1.74 g) of H8EDTMP was raised to pH 12 by the addition of aqueous NaOH (0.032 mol, 1.28 g) producing the EDTMP8− ligand, followed by the addition of 0.004 mol of SmCl3·6H2O (1.46 g); the light yellow reaction mixture was heated at 60 °C for 1 h (pH 7, using pH paper), as depicted in Scheme 1. The resulting white solid product was precipitated by dropwise addition of methanol, then filtered, and washed with additional methanol. ESI-MS+: m/z 717.8 {[152Sm-EDTMP−8H]·Na6}+ (see Figure S5). Quadramet is administered as-provided in solution and has a pH in the range of 7−8.5.12 Aqueous solutions prepared for spectroscopic characterization were at pH 8, achieved by addition of dilute aqueous NaOH. No buffers were used in any sample preparation. In instances where the sample preparation conditions were fundamentally incompatible with the experimental technique, any deviations or modifications from the above sample conditions are detailed in the relevant experimental sections below. Spectroscopic Characterization. Fluorescence spectra were collected on a Cary Eclipse fluorescence spectrophotometer (Varian, Inc., Palo Alto, CA). Aqueous samples were sonicated in order to remove air bubbles prior to characterization. All scans were performed at room temperature using a 245 nm excitation wavelength to initially determine emission wavelengths from 245 to 600 nm. The emission wavelengths were then fixed when verifying characteristic excitation wavelengths through a full range scan. Finally, emission wavelengths of analytes were defined under their corresponding excitation wavelengths, respectively. Fourier transform infrared (FTIR) spectroscopic characterization of the SmEDTMP product was performed using a Nicolet 8700 FTIR spectrometer (Thermo Fisher Scientific, US) in reflectance mode, with the complex prepared as a KBr pellet. 31P and 13C NMR spectra collected on a 600 MHz high resolution Bruker NMR spectrometer (Bruker, US) on the Sm(EDTMP) complex in aqueous solution. Electrospray ionization mass spectrometry (ESI-MS) data was collected in both positive and 4109

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Molecular Pharmaceutics variant of the polarizable continuum model, IEFPCM,24 with united atom radii defining the molecular cavity and a dielectric representing water (ε = 78.39), calculated at the same level of theory as above. The MWB ECP has been shown to be highly effective for modeling coordination chemistry involving lanthanides.25 Harmonic frequency calculations revealed no imaginary frequencies, confirming that the geometry-optimized structure was at a stationary point on the potential energy surface. Molecular Dynamics Simulations. In order to perform MD simulations containing the [SmIII(EDTMP−8H)]5− complex, the complex was parametrized for the OPLS-AA/L force field26,27 using bond lengths from EXAFS curve fitting for Sm− O (2.36 Å) as well as updated bond length and partial charge parameters for the phosphonate groups (at the B3LYP level of theory, as required for the OPLS-AA/L force field). The parametrization included supplementary structural parameters (bond, angle, and torsions) for the EDTMP ligand, while force constants for bonds (e.g., 106,000 kJ/mol/nm2 for Sm− Ophosphonate) and CHELPG charges were calculated at the B3LYP level using the mixed basis set approach detailed above. The parametrized model included bonding interactions only between the four phosphonate groups and the Sm3+ center and did not include any explicit bonds to solvent. The model did not include any explicit interactions for Sm−N bonding, due in part to the fact that the tertiary anime N atoms are sufficiently structurally constrained by defining the bonding interactions of the Sm−Ophosphonate, and are therefore unable to occupy any other position. Moreover, a well isolated ν(Sm−N) was not evident in the harmonic frequency output. Simulations (NPT) were performed using Gromacs, version 4.6,28,29 and the OPLSAA/L force field.18,19 The temperature was kept constant at 300 K (τT = 0.1 ps), by coupling the system to an external bath.30 Iterative updates of nearest-neighbor interactions used a grid-based group search algorithm and updated each step with a neighbor list of 2.5 nm. The simulation pressure was maintained at 1 bar with the Berendsen barostat (τP = 1.0 ps) with isotropic coupling and a compressibility of 4.5 × 10−5 bar−1. Long-range electrostatics were evaluated using the particle mesh Ewald (PME) method,31 with a cutoff of 1 nm. The linear constraint solver (LINCS) was used to constrain bond lengths within the Sm complex.32 A twin-range cutoff was used for van der Waals interactions, both set to 1 nm. A total of 7691 TIP4P solvent molecules33 were used and constrained using SETTLE.34 The complex and solvent were contained within a simulated dodecahedron with periodic boundary conditions and without additional counterions and simulated for 100 ns.

compared to the metal-free ligand, reflecting the change in molecular structure, as well as the presence of fewer chemical forms (protonation states) following Sm coordination. The IR spectrum of Sm-bound EDTMP (Figure S1B) shows significant contribution from ν(O−H), compared to Figure S1A, which we attribute to water molecules strongly H-bonded to the highly charged [SmIIIEDTMP−8H]5− complex. Contributions from NH+ or OH(PO) were not detected in the Smbound EDTMP spectrum, whereas contributions from the methylene, C−N, and C−P moieties were observed for both. The δ(O−C) in the Sm complex is expected around 1050 cm−1;35 however, observation of this band could not be confirmed due to mutual overlap with the more strongly absorbing ν(PO) contribution. The peak at 1282 cm−1 with moderate intensity is attributed to a C−N bond stretch, which is influenced by association of the tertiary amine nitrogen with the Sm3+ center. 31 P and 13C NMR of EDTMP and [Sm(EDTMP)]. Further information regarding the structure of the Sm−EDTMP complexes was provided by NMR spectroscopy through detection of 31P (243 MHz) in D2O. The chemical shift (δ) of phosphorus in Na8EDTMP (Figure S2A) is at 9.844 ppm, indicating that all four atoms are in the same chemical environment, and the chemical shift is consistent with the expected range for phosphonates in aqueous solution. The triplet peaks in the spectrum (t, J = 10.449) arise from coupling with the adjacent methylene groups. After coordination of Sm3+, the 31P peak shifts downfield, to 28.118 ppm, with a 120 Hz width (Figure S2B). The large width of this new peak, compared to that of the free ligand (49 Hz), as well as the large downfield shift, is a result of phosphonate coordination to the Sm3+ center. The unique 31P chemical shift at 28.118 ppm of Sm−EDTMP indicates that each phosphonate is in a similar chemical environment. Further confirmation of complex formation was confirmed by 13C NMR, as illustrated in Figure S4. The free ligand demonstrated two chemical shifts at 51.01 and 53.45 ppm (the dd and d multiplet peaks in Figure S3A), which arise due to C− P and C−H couplings. These couplings disappear to become two singlets narrowly separated, at 48.81 and 48.84 ppm, following Sm complexation (Figure S3B). Fluorescence Spectroscopy of EDTMP and [Sm(EDTMP)]. The maximum emission wavelength of the EDTMP ligand in the Sm complex (shown in Figure S4B) was significantly different from that of the free ligand (Figure S4A). Excitation at 275 nm gives two broad maxima for the free chelator at 384 and 415 nm, while excitation at 375 nm of the Sm complex gives a sharp λmax at 420 nm (Figure S1B). The sharpness of the fluorescence emission peak for the Sm(EDTMP) complex, compared to the emission of the free ligand, is typical of such chelate complexes upon coordination of a rare earth metal.36 Furthermore, the optimal excitation wavelength of Sm-bound complex was 100 nm higher than that of the free ligand, which is also indicative of complex formation. ESI-MS Analysis of [Sm(EDTMP)]. The Sm-bound EDTMP complex gave rise to a large number of mass peaks in both positive and negative ion mode ESI-MS experiments; however, due to the anionic nature of the [SmIII(EDTMP−8H)]5− complex ion, yields in negative ion mode were significantly improved (see Figure S5A,C). The large number of peaks, in either ion mode, arise due to a combination of the seven stable isotopes of Sm present (excluding consideration of more minor elemental isotopes),

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RESULTS Fourier Transform Infrared Spectroscopy of EDTMP and [Sm(EDTMP)]. The characteristic IR bands of EDTMP and Sm-bound EDTMP complexes are shown in Table S1 and Figure S1. The observed absorption bands for EDTMP (Figure S1A) are assigned to the protonated quaternary N atom, OH, the PO of the free phosphonate moieties, and the methylene groups, respectively, as well as contributions from water. The bands in the range of 950−1300 and 400−700 cm−1 are assigned to P−O arising from the phosphonate moieties. In the case of the investigated Sm−EDTMP complex, an OH contribution at roughly 1700 cm−1 is still observed, likely representing residual water. By comparison, the Sm−EDTMP complex gave rise to fewer apparent absorption bands 4110

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corrected EXAFS Fourier transform (Figure 2) is at 2.35 Å (R + Δ), corresponding to 4 Sm−O scattering interactions as well

ranging from 3.07% to almost 26.75% natural abundance, the large number of possible salt adducts and possible protonation states that may form, and the presence of single and multiply charged species. Although negative ion mode gave the best total ion yields overall, the data in positive ion mode provided the simplest combination of peaks to interpret. Figure S5B shows the dominant Sm(EDTMP) complex in positive ion mode, which is confirmed to be {[152Sm(EDTMP)−8H]·Na6}+, based on its mass and isotope ratio (inset plot in Figure S5B). Related [152Sm(EDTMP)−8H] ions with varying charge, salt, or protonation state could be found in both positive and negative ion mode, and this parent ion provided the rationale for construction of a computation structural model. Density Functional Structure Calculations for [SmIII(EDTMP)−8H]5−. A structural model of the Sm3+ complex with EDTMP was constructed in the fully deprotonated form, based on data from ESI-MS and NMR experiments (see Supporting Information). The geometry-optimized structure (Figure 1) resulted in a D2d-distorted coordination complex,

Figure 2. EXAFS spectrum and EXAFS Fourier transform of SmIII(EDTMP)−8H at pH 8 (solid line), fitted using scattering paths (dotted line) based on the DFT structure with additional singlescattering paths to solvent. The Fourier transform is phase-corrected using oxygen.

Figure 1. Geometry optimized DFT structure of [SmIII(EDTMP)−8H]5−, used to fit the EXAFS data. Bonds to the remote N atoms, beyond 2.7 Å, are not explicitly drawn. Additional coordination from solvent is not explicitly modeled. Relevant Sm···X atom separations are shown with the distance closest to the atom of interest (Sm−O bond lengths are shown in bold).

as contributions from an additional backscattering atom at 2.51 Å, which may be due to a bound water O atom, as well as scattering to the two EDTMP N atoms at a distance of 2.68 Å. The Sm−O and Sm···N scattering interactions, at 2.35 and 2.68 Å, respectively, are in good agreement with the distances obtained from the DFT calculations (∼2.36 and 2.70 Å, respectively). The longer-range Sm···N interactions partially cancel the backscattering contribution from some of the phosphonate O atoms, decreasing their apparent contribution in the EXAFS spectrum, as well as the magnitude of their contribution in the EXAFS Fourier transform. The EXAFS Fourier transform also consists of a significant contribution due to multiple scattering arising predominantly from the more distant atoms of the phosphonate moieties, as well as minor contributions arising from Sm···C scattering to each of the methylene moieties within the EDTMP ligand. The EXAFS curve fitting results indicate that the structure obtained for [SmIII(EDTMP−8H)]5− using the DFT-calculated structure (Figure 1) provides the best fit when supplemented with an additional light-atom component (modeled as an oxygen) in the primary coordination sphere, which we attribute to one or more highly ordered waters of solvation. A summary of trial EXAFS curve fitting models, as well as the best fit to the experimental data, is summarized in Table 1. Previous EXAFS characterizations of Sm3+ complexes have reported similar Sm−O backscattering distances at ∼2.32 Å,37

formed by the Sm center and a single oxygen atom from each of the phosphonic acid donors, with trans O−Sm−O angles of 137° and 147°. The average Sm−O bond length was found to be 2.36 Å, while the amine groups were at significantly longer distances (2.70 Å) from the Sm center. DFT calculations involving small numbers of explicit water molecules reveal that these waters do not remain coordinated to the Sm3+ center and instead form more stabilizing Hbonding interactions with the anionic phosphonate groups, even in the presence of a reaction field used to mimic the longrange stabilization due to solvent (structures with explicit waters not shown). It is likely that a large number of explicit solvent molecules would be required to solvate the phosphonate groups first, before coordination at the Sm3+ would be thermodynamically favorable and Sm3+ solvation could be evaluated at this level of theory. The coordinates from the [SmIII(EDTMP)−8H]5− complex were subsequently used to calculate theoretical Sm EXAFS scattering interactions for curve fitting to the experimentally obtained complex in frozen aqueous solution. EXAFS Curve Fitting for [Sm(EDTMP)] in Aqueous Solution. The primary backscattering peak in the phase4111

DOI: 10.1021/acs.molpharmaceut.5b00546 Mol. Pharmaceutics 2015, 12, 4108−4114

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Molecular Pharmaceutics Table 1. Selected EXAFS Curve Fitting Resultsa ΔE0

F

−8.8(9) −8.9(8) −8.9(8)

0.5824 0.6247 0.7260

−5.3(7)

0.5443

2.367(3) 0.0020 −6.7(5) 2.640(7) 0.0022 3.544(4) 0.0025 Full Multiple Scattering Model Model 4 (Based on DFT Model) Sm−O 4 2.362(4) 0.0017(2) +2.0(4) Sm···O 1 2.52(2) 0.0017 Sm···N 2 2.68(1) 0.0020 Sm···C 2 2.82(2) 0.0025 Sm···C 2 3.07(1) 0.0022 Sm···C 2 3.34(2) 0.0023 Sm···P 2 3.556(8) 0.0021 Sm···P 2 3.85(1) 0.0042 Sm···P···O 4 3.52(5) 0.0043

0.4504

path

N

Model 1 (Oxygens Sm−O 4 Sm−O 5 Sm−O 6 Model 2 Sm−O 4 Sm···N 2 Model 3 Sm−O 4 Sm···N 2 Sm···P 4

R

σ2

Single Scattering Models Only) 2.351(5) 0.0021 2.351(4) 0.0021 2.352(4) 0.0021 2.370(4) 2.629(9)

0.0020 0.0021

Figure 3. Radial distribution function for Sm3+ and solvent O atoms from MD simulations of [SmIII(EDTMP−8H)]5−. Solvent is strongly retained in the primary coordination sphere (Sm−O ∼ 2.3 Å), with additional shells of solvent ordered out to ∼8 Å by the highly charged complex.

0.3061

conducive to supporting additional water molecules in an orientation that would allow further coordination directly to the Sm3+ center. There is also no evidence of any such transient coordination of waters to the metal center during the simulation. The highly anionic complex strongly orders multiple concentric shells of solvent, out to 8−10 Å from the Sm3+ center, as evidenced by the radial distribution function (RDF) shown in Figure 3.

a

Coordination numbers N, internuclear separations R (Å), Debye− Waller factors σ2 (Å2), and threshold energy shift ΔE0 (eV), derived from EXAFS curve fitting. The fit error parameter F is defined as F = {∑k6[χ(k)calc − χ(k)expt]2/∑k6χ(k)expt2}1/2, with the summation being over data points included in the fit. Values shown in bold represent the best fit obtained. Values in parentheses are the estimated standard deviations obtained from the diagonal elements of the covariance matrix. The k-range of the data fitted was from 1.0 to 12.0 Å−1.

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DISCUSSION Spectroscopic characterization of the SmEDTMP complex reveals that the structure of the 1:1 complex agrees with the consensus structure that has long been proposed in the literature, and also clarifies the role of the coordinating atoms (primarily via phosphonate O-donors, with additional longrange interactions with the EDTMP tertiary amines). The EXAFS data also required inclusion of additional backscattering atoms, which could be justified based on highly ordered solvent molecules observed in MD simulations, and which appear to lack any well-defined solvent coordination directly to the metal center despite its relatively low number of coordinated atoms. A general survey of the CSD reveals that aqueous Sm3+ complexes are typically 8-coordinate, but there are examples of 7- and 6coordinate complexes, although these are most typical when the metal center is bound by bulky ligand substituents which hinder further coordination. Quadramet is supplied in solution with a stated pH range of 7−8.5.12 In all experiments herein pH 8 was maintained, without addition of buffering agents. Sawada et al. have investigated the formation constants for various lanthanoid− EDTMP complexes (including Sm3+) and found the first two protonation constants are close to pH 7.42 It is anticipated that the initial protonation sites are most likely at the tertiary amines; however, due to the multidentate nature of the Sm− EDTMP complex, protonation is unlikely to result in loss of the metal except at low pH. Previously characterized metal-bound EDTMP complexes from the CSD reveal examples of additional ligands coordinated to the metal center beyond the EDTMP donor ligands. For example, Nd and Eu EDTMP complexes have been

and the number of bound atoms for Sm3+, like most lanthanides, tends toward coordination numbers greater than 6. A search of the Cambridge Structural Database38,39 found the structure JEVVOV,40 which is a phosphonate-bound Sm3+ complex, where the Sm center is bound by 6 phosphonate O atoms, at 2.31 Å ± 0.02 Å. In an analogous complex, Campello et al.41 have reported a small molecule crystal structure (BABGUI) where the Sm3+ center is bound by four phosphonate O atoms at 2.35 Å ± 0.06 Å, with four additional longer range N atoms belonging to the chelator backbone at 2.631 Å ± 0.001 Å, similar to the distances reported herein. Overall, the EXAFS spectrum is composed of scattering interactions involving the EDTMP ligand, as well as apparent association with solvent. Campello et al. have reported structures for related phosphonic acid bound Sm3+ species which demonstrate Sm-coordinated waters in the solid state,33 with water molecules oriented in a coordinating fashion toward the Sm center at ∼2.5 to 2.6 Å, as well as a similarly long Sm··· N separation imposed by the structure of the chelate complex. Solvation Environment of [SmIII(EDTMP−8H)]5− Revealed by Molecular Dynamics. Results from a 100 ns MD simulation of [SmIII(EDTMP−8H)]5− in explicit aqueous solvent reveals that multiple water molecules are retained close to the metal center. An average of three to four water molecules are bound in a bridging manner between the coordinating phosphonates, at a Sm···Owater distance of 2.3−2.6 Å (see representative structure, inset in Figure 3). The H-bonding environment is composed of the phosphonic acid moieties themselves, and these bridging waters do not appear to be 4112

DOI: 10.1021/acs.molpharmaceut.5b00546 Mol. Pharmaceutics 2015, 12, 4108−4114

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Innovation, the Natural Sciences and Engineering Research Council of Canada, the University of Saskatchewan, the Government of Saskatchewan, Western Economic Diversification Canada, the National Research Council Canada, the Canadian Institutes of Health Research, and the Saskatoon Health Region.

characterized with carbonate bound to the metal centers (YIGVAN and SERQOW, respectively). Additionally, an analogous Eu complex (HENLAO) has been shown with a water and an OH bound at ∼2.4 Å; however, these are at 52° from one another and there is additional density between the two ligands, suggesting that the site may be singly occupied, either with fluxional occupancy, or with variability in the position of the bound ligand due to local H-bonding interactions with the coordinating phosphonates.43 In an alternate Tb complex (HENLES), the metal center binds a hydroxy anion at 2.35 Å and a water molecule at 2.47 Å, although, like the Eu complex above, there is additional electron density between the two atom positions.34 Alternatively, these complexes may contain fractional occupancy of an alternate species, such as a coordinating carboxylate, which would partially fit the observed electron density. Through a combination of FTIR spectroscopy as well as EXAFS spectroscopy and modeling in MD simulations, the Sm−EDTMP complex demonstrates the propensity to strongly retain solvent, which we have identified is likely bound between the coordinating phosphonate groups. These solvent molecules are oriented in a manner such that they are unable to coordinate to the metal center, but where the O atoms lie at distance from the Sm3+ center consistent with the primary coordination sphere bond lengths (i.e., close to the Sm···N distance in the formed complex).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.5b00546. IR data; FTIR, 31P- and 13C NMR, and fluorescence spectra; ESI-MS (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ‡

Y.Y. and M.J.P. contributed equally to the work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was undertaken, in part, thanks to funding from the Canada Research Chairs program (D.M.L.C.) and further supported by a grant from the Sylvia Fedoruk Canadian Centre for Nuclear Innovation and Innovation Saskatchewan (awarded to D.M.L.C. and M.R.D.). Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, 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 Center for Research Resources, Biomedical Technology Program (P41RR001209). Computational aspects of this research were enabled by support provided by WestGrid (www.westgrid.ca) and Compute Canada Calcul Canada (www.computecanada. ca), as well as the BioXAS compute cluster at the Canadian Light Source, which is supported by the Canada Foundation for 4113

DOI: 10.1021/acs.molpharmaceut.5b00546 Mol. Pharmaceutics 2015, 12, 4108−4114

Article

Molecular Pharmaceutics

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