Cellular Fates of Manganese(II) Pentaazamacrocyclic Superoxide

Article pubs.acs.org/IC

Cellular Fates of Manganese(II) Pentaazamacrocyclic Superoxide Dismutase (SOD) Mimetics: Fluorescently Labeled MnSOD Mimetics, X‑ray Absorption Spectroscopy, and X‑ray Fluorescence Microscopy Studies Claire M. Weekley,†,‡ Isabell Kenkel,§ Rainer Lippert,§ Shengwei Wei,§ Dominik Lieb,§ Tiffanny Cranwell,† Jason L. Wedding,† Annika S. Zillmann,§ Robin Rohr,§ Milos R. Filipovic,§ Ivana Ivanović-Burmazović,*,§ and Hugh H. Harris*,† †

Department of Chemistry, The University of Adelaide, Adelaide, South Australia 5005, Australia Department of Chemistry and Pharmacy, University of Erlangen−Nuremberg, Egerlandstrasse 1, 91058 Erlangen, Germany

§

S Supporting Information *

ABSTRACT: Manganese(II) pentaazamacrocyclic complexes (MnPAMs) can act as small-molecule mimics of manganese superoxide dismutase (MnSOD) with potential therapeutic application in conditions linked to oxidative stress. Previously, the in vitro mechanism of action has been determined, their activity has been demonstrated in cells, and some representatives of this class of MnSOD mimetics have entered clinical trials. However, MnPAM uptake, distribution, and metabolism in cells are largely unknown. Therefore, we have used X-ray fluorescence microscopy (XFM) and X-ray absorption spectroscopy (XAS) to study the cellular fate of a number of MnPAMs. We have also synthesized and characterized fluorescently labeled (pyrene and rhodamine) manganese(II) pyane [manganese(II) trans-2,13-dimethyl-3,6,9,12,18-pentaazabicyclo[12.3.1]octadeca-1(18),14,16-triene] derivatives and investigated their utility for cellular imaging of MnPAMs. Their SOD activity was determined via a direct stopped-flow technique. XFM experiments show that treatment with amine-based manganese(II) pyane type pentaazamacrocycles leads to a 10−100-fold increase in the overall cellular manganese levels compared to the physiological levels of manganese in control cells. In treated cells in general, manganese was distributed throughout the cell body, with a couple of notable exceptions. The lipophilicity of the MnPAMs, examined by partitioning in octanol−buffer system, was a good predictor of the relative cellular manganese levels. Analysis of the XAS data of treated cells revealed that some fraction of amine-based MnPAMs taken up by the cells remained intact, with the rest transformed into SOD-active manganese(II) phosphate. Higher phosphate binding constants, determined from the effect of the phosphate concentration on in vitro SOD activity, were associated with more extensive metabolism of the amine-based MnPAMs to manganese(II) phosphate. In contrast, the imine-based manganese(II) pydiene complex that is prone to hydrolysis was entirely decomposed after uptake and free manganese(II) was oxidized to a manganese(III) oxide type species, in cytosolic compartments, possibly mitochondria. Complex stability constants (determined for some of the MnPAMs) are less indicative of the cellular fate of the complexes than the corresponding phosphate binding constants.

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INTRODUCTION

antioxidant activity. Although manganese can act as a neurotoxin,1 it is preferred in metal-based SOD mimetics over the more toxic iron and copper. There are three major classes of MnSOD mimetics: manganese(III) porphyrin compounds, manganese(III) salens, and manganese(II) pentaazamacrocycles (MnPAMs, also known as cyclic polyamines).2−5 MnPAMs had the highest SOD activity compared to representatives from other classes of MnSOD mimetics and were the first to enter clinical trials.4−7 In rat models of inflammation and ischemia−reperfusion injury, administration of M40403 (a MnPAM with cyclohexyl functional groups, also

Manganese plays a key role in the body as a cofactor of proteins involved in processes including metabolism and wound healing. It is essential for the function of the mitochondria in the form of the antioxidant enzyme manganese superoxide dismutase (MnSOD). Superoxide and other reactive species are byproducts of cellular respiration that can cause oxidative damage when they are not adequately controlled by antioxidant systems like MnSOD and its cytoplasmic counterpart CuZnSOD. The oxidative stress resulting from this redox imbalance is implicated in a number of diseases including cancer and inflammatory, cardiovascular, and neurodegenerative diseases. A wide array of small-molecule SOD mimetics have been designed with the aim of providing potentially therapeutic © 2017 American Chemical Society

Received: December 19, 2016 Published: May 11, 2017 6076

DOI: 10.1021/acs.inorgchem.6b03073 Inorg. Chem. 2017, 56, 6076−6093

Article

Inorganic Chemistry Scheme 1. MnPAMs That Have Entered Clinical Trials

Scheme 2. MnPAMs Used in This Study

known as GC4403; Scheme 1) prevented tissue damage7 and was shown to inhibit the inflammatory cascade.8 The two clinical trials involving M40403 have been suspended (NCT00033956) or terminated (NCT00101621), but the enantiomer of M40403, named GC4419 (Scheme 1), has entered phase 2 trials for protection against chemoradiationtherapy-induced oral mucositis (NCT02508389).9 Furthermore, studies on the safety, tolerability, and pharmacokinetics of the derivatives GC4702 and GC4711 (Scheme 1) are being performed (NCT03096756 and NCT03099824). MnPAMs are known as efficient catalysts for superoxide dismutation.4,10−13 Although they were considered selective toward superoxide, it is now known that manganese(II) pyane (Scheme 2) also disproportionates nitric oxide in vitro,14 in

cells,15 and in spermatozoa.16 This multifaceted antioxidant activity is a characteristic shared with MnSOD.17 The ability of MnPAMs to suppress superoxide, nitric oxide, and peroxynitrite generation15 and lipid peroxidation18 has been demonstrated in cells, but we lack an understanding of the intracellular fates of these complexes. Thus, we aim to determine (a) the uptake and distribution of manganese in cells treated with MnPAMs, (b) whether fluorescent tags can be used to track the intracellular distribution of MnPAMs, (c) whether the manganese ligand complexes remain intact in cells, and (d) how their cellular fate is related to their chemical form and physical properties. Synchrotron-based X-ray absorption spectroscopy (XAS) and X-ray fluorescence microscopy (XFM) techniques allow for 6077


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aluminum plates precoated with Merck 60 silica gel F254 and visualized by ultraviolet irradiation or by staining with aqueous acidic ammonium molybdate or aqueous acidic potassium permanganate solutions as appropriate. For further characterization, an ultrahighresolution (UHR) electrospray ionization (ESI) time-of-flight mass spectrometry (MS; Bruker Daltoniks, Bremen, Germany) maxis was used. Carlo Erba elemental analyzers 1106 and 1108 and a Bruker Avance DPX 300 NMR spectrometer were used for elemental and NMR analyses, respectively. UV/vis spectra were recorded on a HP 8452A diode-array spectrophotometer, and fluorescence measurements were performed with a FP-8200 spectrofluorometer (Jasco, Groß-Umstadt, Germany). Synthesis of Rhodamine B Succinimide (1).35,36

the determination of elemental speciation and distribution (for elements heavier than silicon) in intact cells that have been subjected to mild sample preparation that minimizes the risk of generating artifacts. These techniques, in combination, have proven useful in determining the fates of metal and semimetal complexes in biological samples.19−22 Thus far, XAS and XFM studies of manganese speciation and/or distribution in cells have been conducted mainly in the context of the neurotoxicity of MnCl2 and other manganese complexes known to be environmental contaminants.23−27 The intracellular distributions of porphyrin and acyclic MnSOD mimetics have previously been investigated using XFM, showing that manganese is distributed throughout the cell (similar to MnCl2) unless the ligands are designed to target subcellular compartments.28,29 The distributions of mitochondrially targeted zinc porphyrin photosensitizers were observed with fluorescence microscopy, and successful targeting of the inner mitochondrial membrane by one of the derivatives was implied by the effect of the drug on cytochrome c oxidase.30 Furthermore, liquid chromatography (LC)−mass spectrometer (MS)/MS methodology has been used extensively to determine the fates of manganese porphyrins in cells (mitochondria versus cytosol distribution)31 and in vivo both in organs and plasma32 and within the brain.33 The fates of six MnPAMs are investigated herein, including four SOD-active complexes and two SOD-inactive complexes (Scheme 2). Manganese(II) pyane has an in vitro catalytic SOD activity approaching that of M40403.6 Here we synthesized and characterized its fluorescently tagged derivatives, manganese(II) pyane−rhodamine B (rhodB) and manganese(II) pyane− pyrene, which provide an opportunity to correlate rhodamine and pyrene fluorescence with Mn Kα X-ray fluorescence. An additional derivative of manganese(II) pyane has also been studied: dimanganese(II) bipyane, a dinuclear analogue with a higher stability and approximately 2-fold greater in vitro SOD activity than the mononuclear manganese(II) pyane.34 The SOD-inactive compounds are manganese(II) pydiene, which contains two imine bonds in place of the amine bonds in manganese(II) pyane, and M40404, the SOD-inactive dimethylated derivative of M40403. Lipophilicity, thermodynamic stability, and phosphate-binding constants were determined for newly synthesized and related MnPAMs. The latter were obtained based on the effect of the phosphate concentration on the catalytic rate constant for superoxide dismutation and, consequently, were quantified only for SODactive complexes. To determine the impact of these modifications on cellular MnPAM uptake, distribution, and manganese speciation, we conducted XAS and XFM studies in SH-SY5Y human neuroblastoma and A549 human lung cancer cell lines, using untreated and MnCl2-treated cells as controls. The effects of thermodynamic stability versus phosphate binding on the cellular fates of these complexes are discussed.

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Rhodamine B (3 g, 6.3 mmol) and N-hydroxysuccinimide (0.72 g, 6.3 mmol) were dissolved in dry acetonitrile (60 mL) and heated to 45 °C. N,N′-Dicyclohexylcarbodiimide (1.5 g, 7.5 mmol) in dry acetonitrile (40 mL) was added slowly. After additional stirring at 45 °C for 20 h, the solution was filtered over Celite and the solvent stripped off. The residue was dissolved in dichloromethane (DCM) and washed with a saturated NH4Cl solution. The organic phase was dried over Na2SO4 and the solvent removed under reduced pressure. Product 1 was obtained as a dark-green solid (1.6 g, 2.77 mmol, 44%). 1 H NMR (300 MHz, CDCl3, 25 °C): δ 1.32 (t, 12H, 3J = 7.1 Hz, H1), 2.77 (s, 4H, H12), 3.63 (q, 8H, 3J = 7.1 Hz, H2), 6.80 (d, 2H, 3J = 2.2 Hz, H7′ and H7″), 6.85 (d, 1H, 3J = 2.2 Hz, H4), 6.88 (d, 1H, 3J = 2.2 Hz, H5), 7.05 (s, 1H, H6), 7.09 (s, 1H, H3), 7.45 (d, 1H, 3J = 7.5 Hz, H8), 7.82 (t, 1H, 3J = 7.7 Hz, H9), 7.98 (t, 1H, 3J = 7.5 Hz, H10), 8.40 (d, 1H, 3J = 7.9 Hz, H11). 13C NMR (75 MHz, CDCl3, 25 °C): δ 12.5, 12.6, 26.8, 27.7, 29.3, 40.1, 44.5, 46.1, 51.5, 96.1, 113.6, 114.3, 128.6, 129.8, 130.3, 131.0, 131.9, 132.9, 132.8, 133.7, 158.3, 162.7, 172.9. Synthesis of Compound 2.37

N,N-Diisopropylethylamine (0.43 mL, 2.5 mmol) was added to a solution of sarcosine tert-butyl ester hydrochloride (0.45 g, 2.5 mmol) in dry acetonitrile (20 mL). After stirring for 10 min, a solution of 1 (0.72 g, 1.25 mmol) in dry acetonitrile (20 mL) was added slowly and the reaction mixture was heated to reflux for 4 h. After removal of the solvent, the residue was taken up in DCM and washed with brine. The organic layer was dried over Na2SO4 and the solvent stripped off. The crude compound was purified via column chromatography [SiO2, gradient elution with DCM/methanol (MeOH) for 1/0 → 15/1] to give compound 2 (0.38 g, 0.62 mmol, 50%). 1H NMR (300 MHz, CDCl3, 25 °C): δ 1.28 (m, 21H, H1/H14), 2.74 (s, 3H, H12), 3.62 (m, 8H, H2), 3.75 (s, 2H, H13), 6.55 (m, 2H, H4/H5), 6.72 (s, 1H, H6), 6.92 (d, 3J = 2.2 Hz, 2H, H7′ and H7″), 7.22 (s, 1H, H3), 7.37

EXPERIMENTAL PROCEDURES

Materials and Methods. All reactions were performed in flamedried glassware under a nitrogen atmosphere with dry solvents, unless otherwise noted. All reagents and starting materials were purchased from Sigma-Aldrich, Alfa Aesar, and Acros Organics, were of p.a. grade, and were used without further purification. All solvents used for syntheses were anhydrous, and deionized Millipore water was used for all measurements in aqueous media. Flash column chromatography was carried out using Merck 60 silica gel (230−400 mesh) under pressure, and all technical solvents were purified by rotary evaporation prior to use. Analytical thin-layer chromatography was performed on 6078


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Synthesis of N-(1-Pyrene)butyryloxysuccinimide (5).38

(m, 1H, H8), 7.67 (m, 2H, H9/H10), 8.16 (m, 1H, H11). 13C NMR (75 MHz, CDCl3, 25 °C): δ 12.6, 27.9, 28.0, 38.7, 39.4, 49.0, 49.1, 81.6, 96.0, 96.3, 106.6, 113.9, 114.0, 127.5, 129.6, 129.9, 130.0, 130.3, 132.2, 135.7, 155.2, 155.6, 158.0, 167.0, 168.9. Synthesis of Compound 3.37

1-Pyrenebutyric acid (0.20 g, 0.69 mmol) and N-hydroxysuccinimide (79 mg, 0.69 mmol) were dissolved in dry tetrahydrofuran (THF; 3 mL), and then a solution of N,N′-dicyclohexylcarbodiimide (0.17 g, 0.83 mmol) in THF (2 mL) was added slowly at 0 °C. The mixture was stirred overnight at room temperature. The white precipitate was removed from the reaction mixture via filtration. The residue was evaporated to dryness under vacuum and purified by column chromatography (SiO2 and DCM) to give 5 as a pale-yellow solid (0.21 g, 0.54 mmol, 78%). 1H NMR (300 MHz, CDCl3, 25 °C): δ 2.26 (m, 2H, H3), 2.75 (t, 2H, 3J = 7.2 Hz, H2), 2.83 (s, 4H, H5), 3.47 (t, 2H, 3J = 7.2 Hz, H4), 7.87 (m, 9H, H1). 13C NMR (75 MHz, CDCl3, 25 °C): δ 25.6, 26.4, 30.5, 32.2, 130.0, 134.8, 135.9, 168.5, 169.1. Synthesis of 2,6-Diacetylisonicotinonitrile (6).18

To a solution of compound 2 (0.32 g, 0.52 mmol) in anhydrous DCM (5 mL) was added trifluoroacetic acid (2.5 mL, 32.66 mmol). After stirring for 2 h at room temperature, the reaction mixture was evaporated to dryness in vacuo. The resulting red solid was washed with ether and neutralized with saturated NaHCO3. After extraction with chloroform, the organic layer was dried over MgSO4 and the solvent stripped off. The crude product was purified by column chromatography (SiO2, gradient elution with DCM/MeOH for 1/0 → 1/1) to give compound 3 (0.23 g, 0.42 mmol, 80%). 1H NMR (300 MHz, CD3OD, 25 °C): δ 1.33 (t, 12H, 3J = 7.1 Hz, H1), 2.86 (s, 3H, H12), 3.70 (m, 10H, H2/H13), 6.90 (s, 2H, H7′ and H7″), 7.10 (m, 2H, H4/H5), 7.24 (s, 1H, H6), 7.28 (s, 1H, H3), 7.47 (m, 1H, H8), 7.73 (m, 2H, H9/H10), 7.85 (m, 1H, H11). 13C NMR (75 MHz, CD3OD, 25 °C): δ 13.0, 34.7, 39.2, 57.0, 97.4, 97.5, 115.1, 115.2, 128.9, 130.8, 131.0, 131.2, 131.3, 131.6, 131.7, 132.3, 133.4, 133.5, 137.7, 157.4, 157.7, 159.6, 171.1, 175.1, 175.2. Synthesis of Compound 4.35,36

To a solution of isonicotinonitrile (3.6 g, 35 mmol) in 175 mL of H2SO4 (0.4 M) was added pyruvic acid (9.3 g, 105 mmol) and AgNO3 (0.48 g, 2.8 mmol) in 5 mL of water. The reaction mixture was cooled to approximately 5 °C, and ammonium persulfate (27 g, 120 mmol) was added slowly. The mixture was stirred at room temperature for 24 h. A sodium carbonate solution was added until the reaction mixture reached pH 10. After extraction with chloroform, the organic phase was dried over Na2SO4 and the solvent was removed under reduced pressure. Column chromatography (SiO2 and DCM) gave a mixture containing one byproduct. The crude product was recrystallized from ethyl acetate/n-hexane to give 6 as a colorless crystalline solid (1.5 g, 7.97 mmol, 23%). 1H NMR (300 MHz CDCl3, 25 °C): δ 2.74 (s, 6H, H1), 8.34 (s, 2H, H2). 13C NMR (75 MHz, CDCl3, 25 °C): δ 25.5, 115.4, 123.1, 126.2, 153.6, 197.3. Anal. Calcd for C10H8N2O2: C, 63.83; H, 4.29; N, 14.89. Found: C, 63.67; H, 4.31; N, 14.73. Synthesis of Manganese(II) Pyane−CH2NH2 (7).39,40

Compound 3 (0.25 g, 0.45 mmol) and N-hydroxysuccinimide (52 mg, 0.45 mmol) were dissolved in 4 mL of DCM with agitation. A solution of N,N′-dicyclohexylcarbodiimide (111 mg, 0.54 mmol) in 1 mL of dry DCM was added slowly to the reaction mixture. After continuous agitation at room temperature for 20 h, the white precipitate was removed as a byproduct from the reaction mixture via filtration. After removal of the solvent, the crude product was purified by column chromatography (SiO2, gradient elution with DCM/MeOH for 1/0 → 1/1) to give compound 4 (0.21 g, 0.32 mmol, 72%). 1H NMR (300 MHz, CD3OD, 25 °C): δ 1.34 (t, 12H, 3J = 7.1 Hz, H1), 2.93 (s, 3H, H12), 3.52 (s, 4H, H14), 3.71 (m, 8H, H2), 3.99 (s, 2H, H13), 6.72 (d, 2H, 3J = 2.0 Hz, H7′ and H7″, 6.88 (d, 1H, 3J = 2.2 Hz, H5), 6.91 (d, 1H, 3J = 2.1 Hz, H4), 7.17 (s, 1H, H6), 7.20 (s, 1H, H3), 7.30 (m, 1H, H8), 7.60 (m, 3H, H9/H10/H11). 13C NMR (75 MHz, CD3OD, 25 °C): δ 12.8, 39.0, 46.3, 48.8, 52.1, 96.4, 96.5, 144.1, 114.2, 114.4, 127.9, 130.2, 130.3, 130.4, 130.6, 132.1, 132.3, 135.6, 155.6, 155.8, 158.1, 168.7, 169.3.

Compound 6 (0.32 g, 1.7 mmol) and MnCl2·2H2O (0.33 g, 2 mmol) were dissolved in 50 mL of MeOH under a nitrogen atmosphere and heated to 50 °C. Triethylenetetraamine (0.29 g, 2 mmol) dissolved in MeOH (10 mL) was added dopwise. The reaction mixture was refluxed over a period of 2 h. The solvent was removed under reduced pressure to obtain the imine intermediate as a deep-red solid (0.72 g, 99%), which was used without any further purification. The residue was dissolved in dry ethanol (50 mL) and cooled to 0 °C. Sodium borohydride (1.1 g, 28 mmol) was added slowly. The reaction mixture 6079


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and water. The aqueous layer was extracted with chloroform (3 × 3 mL), and the combined organic phases were washed with brine, dried over MgSO4, and concentrated in vacuum to afford a deep-red solid. The crude product was purified by column chromatography (neutral Al2O3, gradient elution with DCM/MeOH for 1/0 → 15/1) to give 9 as a deep-red solid (0.12 g, 0.12 mmol, 65%). Anal. Calcd for 9·4H2O (C47H72Cl3MnN9O7): C, 60.76; H, 6.63; N, 10.63. Found: C, 60.08; H, 6.76; N, 10.77. UHR-ESI-MS (positive-ion mode): m/z 285.8176 ([M − 3Cl]3+). Calcd: m/z 285.8165. Determination of the SOD Activity and Phosphate Binding Constant via a Stopped-Flow Technique. The SOD activity was tested by a direct method using a stopped-flow technique as described elsewhere.6 Experiments were carried out on a Biologic SFM-400 instrument, using syringes 1−3, combined with an Energetiq LDLS ENQ EQ-99-FC laser-driven light source and a J&M TIDAS diodearray detector (integration time = 0.5 ms; λ = 180−724 nm). The source of superoxide was commercially available KO2 dissolved in dry dimethyl sulfoxide (DMSO; [O2•−] ≈ 1−2 mM). Complexes were used in at least four different concentrations [between 0.45 and 4.5 μM in a 60 mM N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid (HEPES) buffer, pH 7.4 and 8.1, and a 50 mM sodium phosphate buffer, pH 7.4]. For phosphate dependence, buffers (60 mM total concentration) were prepared as a mixture of HEPES and phosphate with an increasing amount of phosphate and a decreasing amount of HEPES (for details, see the Supporting Information and Table S2). The ionic strength of the buffers was kept constant by the addition of sodium chloride (Table S2), and the pH was adjusted to 7.4. An aqueous complex solution was mixed in a 9/1 ratio with the superoxide solution in DMSO by using a high-density mixer. The superoxide concentration always exceeded the complex concentration in at least a 10-fold excess to ensure catalytic conditions. Millipore water was used for the preparation of buffer solutions, and the buffer was treated for at least 12 h with a Chelex 100 sodium exchange resin before use. Data analysis was performed using BioKine V4.66 software. Each kobs value is a mean value of at least 10 values. kcat values were determined from the slope of the kobs versus [SODm] plot. For the binding constant of phosphate, the obtained catalytic rate constants (kcat) were plotted versus the respective hydrogen phosphate (HPO42−) concentration and fitted using eq 1 (see the Supporting Information).

was refluxed for 4 h, and the solvent was removed under reduced pressure. A solution of LiCl (2.5 g, 59 mmol) in MeOH (55 mL) was added, and the mixture was thoroughly stirred until no more gas evolved. After removal of the solvent, the residue was dissolved in water (25 mL) and extracted with chloroform (2 × 50 mL). The organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure. Product 7 was obtained as a pale-yellow solid (0.62 g, 1.43 mmol, 85%). Anal. Calcd for 7·2MeOH (C18H38Cl2MnN6O2): C, 43.55; H, 7.72; N, 16.93. Found: C, 43.46; H, 7.67; N, 16.97. UHR-ESI-MS (positive-ion mode): m/z 396.1591 ([M − Cl]+). Calcd: m/z 396.1595. Synthesis of Manganese(II) Pyane−Pyrene (8).

A solution of 5 (0.11 g, 0.27 mmol) in dry DCM (2 mL) was added dropwise to a solution of compound 7 (0.12 g, 0.27 mmol) in DCM (2 mL). The resulting mixture was stirred overnight at room temperature under an argon atmosphere; a precipitate was formed during the reaction. The solvent of the suspension was removed under reduced pressure to give the crude product as a deep-yellow solid, which was washed with DCM (2 × 5 mL). The residue was purified by column chromatography (SiO2, gradient elution with DCM/MeOH for 1/0 → 15/1) to give 8 as a pale-yellow solid (0.12 g, 0.17 mmol, 63%). Anal. Calcd for 8·EtOAc (C40H52Cl2MnN6O3): C, 60.76; H, 6.63; N, 10.63. Found: C, 60.08; H, 6.76; N, 10.77. UHR-ESI-MS (positive-ion mode): m/z 666.2650 ([M − Cl]+). Calcd: m/z 666.2640. Synthesis of Manganese(II) Pyane−Rhodamine B (9).

kcat = k 0 + (k∞ − k 0)

K[HPO4 2 −] 1 + K[HPO4 2 −]

(1)

The binding constants for dimanganese(II) bipyane were obtained by fitting the data using eq 2 (see the Supporting Information). kcat = (k 0 + k1K1[HPO4 2 −]) +

k 2K1K 2[HPO4 2 −]2 1 + K1[HPO4 2 −]

(2)

Rhodamine B and pyrene itself were tested for SOD activity as a control in a 60 mM HEPES buffer (pH 7.4). Potentiometric Titration. pMn values and stability constants for M40403 and M40404 were determined by potentiometric titrations at 25 °C, as previously published.34 Partition Coefficient Determinations. Phosphate-buffered saline (PBS; 10 mM, pH 7.4)-saturated 1-octanol and 1-octanol-saturated PBS were obtained by extensive mixing of equal amounts of both solutions for 5 min and then separating, centrifuging each phase for 10 min at 1000g, and discarding any residual minor phase. For every MnPAM, the extinction coefficient (ε) at their local maxima was determined in 1-octanol-saturated PBS by recording the absorbance of standard solutions and calculation of a calibration curve (see Table S1). The respective MnPAMs were dissolved in a defined concentration [MnPAM]0 in 1-octanol-saturated PBS (5 mL) and mixed with equal volumes of PBS-saturated 1-octanol. The two-phase mixture was shaken for 60 min at room temperature (300 rpm) on a plate shaker, the phases were separated via centrifugation (10 min, 25 °C, 1000g), and a UV/vis spectrum of the aqueous phase was recorded. Under

Compound 4 (0.12 g, 0.19 mmol) was dissolved in a mixture of dry DCM (2 mL) and dry DMF (1 mL). To this solution was added dropwise compound 7 (82 mg, 0.19 mmol) dissolved in DCM (2 mL) and N,N-diisopropylethylamine (0.07 mL, 0.38 mmol). The resulting mixture was stirred overnight at room temperature under an argon atmosphere. The reaction mixture was poured on a mixture of ether 6080


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Inorganic Chemistry Scheme 3. Reaction Schemes Describing the Activation of Rhodamine B (Top) and Pyrene (Bottom)

washed with PBS (3 times) and a cold 100 mM (isotonic) ammonium acetate solution and, finally, dipped very briefly in Milli-Q water.41,42 Samples were allowed to air dry, covered, before fluorescence imaging and were stored and desiccated until XFM. XFM mapping of the individual cells was performed at the Advanced Photon Source beamline 2-ID-D (Chicago, IL). Maps were collected at 0.5 μm resolution with a 10.1 keV beam focused through a zone plate and an order sorting aperture. The fluorescence signal was collected for 0.5 s/point using a single-element silicon drift energy dispersive detector (Vortex EX, SII Nano-technology, Northridge, CA), at 90° to the incident beam. The integrated fluorescence spectra were extracted from the images and fit with modified Gaussians to determine the average elemental content (μg/cm2), and quantification was performed by comparison to the corresponding measurements on the thin-film standards NBS-1832 and NBS-1833 (National Bureau of Standards, Gaithersburg, MD). Analysis was performed using MAPS software.43 Further imaging of 8-, 9-, and manganese(II) pydiene-treated cells was conducted at the Australian Synchrotron, Clayton, Victoria, Australia. The beam was tuned to an incident energy of 12.7 keV using a Si(111) monochromator and focused to ∼2 μm using KirkpatrickBaez mirrors. A 384-element silicon array detector (Maia 384; Brookhaven National Laboratory, Upton, NY, and CSIRO, Clayton, Victoria, Australia)44 in 180° backscatter geometry was used to collect the fluorescence signal from the samples mapped at 0.8 μm resolution with a dwell time of 4 or 16 ms/point. The fit to a representative fluorescence spectrum was used to generate a dynamic analysis matrix that was used to project the images.

consideration of the previously determined extinction coefficients, the concentration of MnPAM in each phase was recalculated. The partition coefficient (log P) was calculated using eq 3. Data are mean ± standard deviation of three determinations.

log P = log

[MnPAM]octanol [MnPAM]water

(3)

Cell Culture. SH-SY5Y human neuroblastoma cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 nutrient solution (F12) supplemented with fetal bovine serum (5% v/v), penicillin (100 mg/mL), streptomycin (100 units/mL), L-glutamine (2 mM), and nonessential amino acids (100 units/mL) at 310 K in a 5% CO2-humidified incubator. A549 cells were cultured as above in DMEM media. Cells were subcultured every 3−5 days. Manganese(II) pentaazamacrocycle stock solutions were prepared and serially diluted in Milli-Q water before the addition to the cells. XFM. SH-SY5Y human neuroblastoma cells were seeded at approximately 200000 cells/well in a 6-well plate for 24 h at 310 K in a 5% CO2-humidified incubator. Cells were grown in complete DMEM/F12 media on silicon nitride membranes, 2 mm × 2 mm area and 500 nm thickness, manufactured by the Melbourne Centre for Nanofabrication (Clayton, Victoria, Australia). In one instance, the manganese(II) pydiene treatment medium was replaced after 23 h with a 100 nM fluorescent mitochondrial tracker (Mitotracker Deep Red, Invitrogen in serum-free DMEM) for 45 min. At the end of the treatment time, the cells were washed with PBS and then fixed with a 3.7% paraformaldehyde solution (PBS solution, pH 7.2) for 15 min at 37 °C in a 5% CO2-humidified incubator. The samples were then 6081


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Inorganic Chemistry Scheme 4. Reaction Scheme Describing the Coupling Reaction of the Activated Fluorescence Markers 4 and 5 and 7

Quantification was performed by corresponding measurements and analysis on a manganese foil standard. Analysis was performed using GeoPIXE software. Fluorescence Microscopy. Fluorescent images of cells prepared for XFM analysis were collected using an Olympus BX51 microscope. Pyrene was excited at wavelengths of 330−385 nm, and emission was collected at a wavelength of 420 nm. Rhodamine was excited at wavelengths of 530−550 nm, and emission was collected at a wavelength of 575 nm. Mitotracker Deep Red was excited at wavelengths of 600−650 nm, and fluorescent emission was collected at wavelengths of 665−740 nm. Confocal Fluorescence Microscopy. SH-SY5Y cells were plated at 10000 cells/well in 8-well glass slide chambers and allowed to adhere for 24 h. Cells were treated with 8 (20 μM) or 9 (200 μM) for 24 h. Immediately prior to imaging, conditioned media were removed,

and the cells were washed twice in PBS and then covered in phenol red-free DMEM. Confocal fluorescence images were collected using a Leica SP5 confocal microscope. Pyrene was excited with an argon laser at 405 nm, and emission was collected from 413 to 550 nm. Rhodamine B was excited at 561 nm, and emission was collected from 569 to 700 nm. Control cells were excited at the same wavelengths, but no fluorescent emission was observed. XAS. Bulk cell pellets (∼106 cells) from treated cultures were prepared for XAS. Cells were grown to ∼85% confluency in 75 cm2 flasks in complete media and were treated with manganese(II) pentaazamacrocycles or MnCl2 (300 μL addition at 100× final concentration) in fresh complete media for 4 or 24 h. Cells were collected by gentle scraping and centrifugation. The supernatant was removed, and the cells were rinsed by resuspension in PBS (3 × 5 mL) and centrifugation before the pellet was collected, frozen in dry ice, 6082


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Table 1. Catalytic SOD Rate Constants (kcat) and Binding Constants for HPO42− [K(HPO42−)] of Fluorescently Labeled MnPAMs and Related MnSOD Mimetics kcat × 107 (M−1 s−1)

a

complex

HEPES at pH 7.4

HEPES at pH 8.1

phosphate at pH 7.4

K(HPO42−)a (M−1)

manganese(II) pyane M40403 dimanganese(II) bipyane manganese(II) pyane−rhodB manganese(II) pyane−pyrene manganese(II) pydiene manganese(II) pyane−CH2NH2 M40404

2.73 ± 0.04 4.91 ± 0.066 4.70 ± 0.0229 1.20 ± 0.10 0.95 ± 0.05 inactive 1.71 ± 0.30 inactive



90 ± 10 1200 ± 200 K1 = 13 300 ± 7300, K2 = 440 ± 120 540 ± 140 1 400 ± 200 n/a 880 ± 200 n/a

6

6

6

Binding constants were determined in buffer mixtures of HEPES and sodium phosphate; for details, see the Supporting Information and Table S2

and freeze-dried overnight (−83 °C, 1.2 mbar). The cell culture media from the 4 h treatments were retained and also freeze-dried. XAS spectra were collected at the XAS beamline (Australian Synchrotron, Clayton, Victoria, Australia). The X-ray beam was monochromated by diffraction through a pair of Si(111) crystals. Harmonic rejection was achieved by setting the cutoff of a rhodium-coated mirror to 15 keV. Spectra were collected in fluorescence mode at ∼5 K where possible, or at room temperature for lower-concentration samples, using a 100-element solid-state germanium detector (Canberra-Eurisys) at 90° to the incident beam. X-ray absorption near-edge spectra (XANES) were collected at the following energy ranges: 6339−6519 eV (3 eV steps) in the preedge region; 6519−6589 eV (0.25 eV steps) in the XANES region; 6589−7091 eV (0.01 Å−1 steps in k space to 7 Å) in the postedge region. Extended Xray absorption spectra (EXAFS) were collected as described for XANES spectra, except the preedge region was collected in 6 eV steps and the postedge EXAFS region was extended to 12 Å−1 (with 0.035 Å−1 steps in k space). An elemental manganese foil standard was used to calibrate the energy scale to the first peak of the first derivative of the manganese edge (6539 eV). Multiple spectra were collected from each sample in order to increase the signal-to-noise ratio by averaging. There were no signs of photodamage. All analysis of XAS spectra including calibration, averaging, and background subtraction was performed using the EXAFSPAK software package (G. N. George, SSRL, http://www-ssrl.slac.stanford.edu/ exafspak.html). Principal-component analysis was used to identify the total number of components that make up the XANES spectra collected from treated cells. Linear combination fits of these experimental spectra with model compound spectra (selected to represent the range of species likely to be present in cells) were performed over the region 6520−6650 eV, also using EXAFSPAK. Components smaller than 1% were rejected, as were those with a calculated estimated standard deviation larger than the size of the component. Most manganese model compound spectra were collected during these experiments, with the remainder were provided by Enzo Lombi (University of South Australia). The EXAFS oscillations were analyzed by curve fitting using EXAFSPAK software based on the FEFF7 code.45

Determination of the SOD Activity, Phosphate Binding, Stability Constants, and Lipophilicity. The SOD activity of the newly synthesized fluorescently labeled MnPAMs was determined using a well-established stopped-flow technique.6 The obtained catalytic rate constants, kcat, are summarized in Table 1 together with those for other studied or related complexes (for additional data, see Table S3). The introduction of a rhodamine B or a pyrene moiety to the pyane system results in a relatively small decrease in the activity of the complex toward the catalytic removal of superoxide. A methylamine-functionalized manganese(II) pyane (7), being the parent compound of the two fluorescently labeled MnPAMs, was tested as well because it may be generated in vivo by hydrolysis of the corresponding amide linkers in the fluorescently labeled MnPAMs. All tested complexes show good SOD activity in a HEPES buffer; however, the application of a phosphate buffer induces a very strong decrease of the catalytic activity (up to 1 order of magnitude, e.g., see M40403; Table 1). In our previous work,6 the formation of adducts between MnPAMs [M40403 and manganese(II) pyane] and HPO42− is demonstrated. By the application of high-resolution ESI-MS, different phosphate adducts could be identified. The effect of the association of MnPAMs with HPO42− on kcat was used to determine the corresponding binding constant K(HPO42−). Therefore, we performed the measurements of the SOD activity as a function of the phosphate concentration (see the Supporting Information). A HEPES buffer was mixed with an increasing amount of sodium phosphate, whereas the ionic strength and pH value was kept constant. For each condition (1−50 mM total phosphate concentration), the catalytic rate constant for superoxide dismutation was determined (Tables S4−S9). The obtained kcat values were plotted versus the HPO42− concentration and fitted using eq 1 or eq 2 described in Experimental Procedures and the Supporting Information (demonstrated in Figure 1 in the case of 9; for other complexes, see Figures S2−S6). The binding constants obtained are summarized in Table 1. The lipophilicity of the studied MnPAMs was examined by partitioning in an octanol−buffer system, and the data are summarized in Table 2. The highest hydrophilicity was observed for manganese(II) pydiene (the π character of the imine bonds makes them more hydrophilic than the amine bonds), 7 (the amine group is probably protonated, which increases the charge and hydrophilicity of the complex), and dimanganese(II) bipyane (its high overall charge, 4+, contributes to its hydrophilicity). The reference manganese(II) pyane complex exhibits a slightly lower hydrophilicity (because of the factors mentioned above). Although the rhodamine

■

RESULTS Synthesis of Fluorescently Labeled MnPAMs. For the purpose of this study, two derivatives of manganese(II) pyane, a well-characterized MnSOD mimetic that belongs to the MnPAM family and that also dismutates NO,6,15,39 were synthesized with rhodamine B and pyrene functionalities, respectively (Scheme 2). Their syntheses are summarized in Schemes 3 and 4. The general methodology for the complex characterization is given in Experimental Procedures. For the UV/vis characteristics and fluorescence spectra, see Table S1 and Figure S1, respectively. 6083


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Table 3. pMn Values and Stability Constants for the Studied and Related MnPAMs Determined by Potentiometric Titration at 25 °C in Water pMna

complex manganese(II) pydiene manganese(II) pyane M40403 M40404 dimanganese(II) bipyane

3.5 4.9 5.9 5.9 6.1

± ± ± ± ±

log KMLb 29

0.1 0.229 0.2d 0.2d 0.229

10.5 11.0 10.9 11.0

± ± ± ±

log KM2Lc

29

0.2 0.229 0.2d 0.2d 8.8 ± 0.329

pMn = −log [Mn ]free (values calculated for ccomplex = 1 mM and pH 7.4). bKML = [ML]/[M][L]. cKM2L = [M2L]/[M]2[L]. dThis work. a

stability, as indicated by the smaller concentration of free MnII ions compared to manganese(II) pydiene and manganese(II) pyane. Importantly, as will be demonstrated and discussed below, the amounts of free Mn(II) present in the solutions of these complexes, i.e., their stability constants, are not directly correlated with their speciation in cells. It seems that phosphate binding, which is related to a partial transformation of the complexes within cells into manganese(II) phosphate (see below), is more critical for the cellular fate of complexes. Manganese Uptake and Distribution. The uptake and distribution of manganese in SH-SY5Y human neuroblastoma cells treated with MnPAMs was determined by XFM. Previous work has shown that the proliferation of A549 human lung cancer cells is unaffected by MnCl2 concentrations of up to 2 mM for 24 h,47 and the MnPAMs were well-tolerated by A549 cells treated with up to 1 mM manganese(II) pyane. Although SH-SY5Y cells were more sensitive, 500 μM concentrations were generally well-tolerated, with the exception of the more toxic compounds, 8 and M40404. Treatment concentrations that resulted in strong uptake without cell death were selected and are reported in Table 4.

Figure 1. SOD catalytic rate constants kcat as a function of the HPO42− concentration of 9, fitted in eq 1 (pH 7.4; 60 mM total HEPES/ phosphate buffer concentration).

Table 2. log P Values Calculated for All MnPAMs for Partitioning between n-Octanol and PBS Buffer at pH 7.4 complex manganese(II) pydiene manganese(II) pyane−CH2NH2 dimanganese(II) bipyane manganese(II) pyane manganese(II) pyane−rhodB M40403 M40404 manganese(II) pyane−pyrene

log P −2.28 −2.06 −2.01 −1.61 −1.17 −0.38 0.36 1.50

± ± ± ± ± ± ± ±

II

0.23 0.08 0.20 0.13 0.28 0.04 0.07 0.01

moiety carriers a positive charge, 9 is less hydrophilic that manganese(II) pyane because of the presence of an extended organic ligand. The introduction of the cyclohexyl moieties in M40403 and M40404 contributes to an increased lipophilicity, whereas the lower lipophilicity of M40403 compared to M40404 is related to the absence of methyl groups in the former. As expected, the highest lipophilicity is observed for 8 because of the presence of a very hydrophobic extended organic substituent without charge. The two complexes with the highest lipophilicity, M40404 and 8, exhibit the greatest cellular uptake (see below). Interestingly, the highly hydrophilic manganese(II) pydiene also shows a great accumulation of manganese in the cells (see below), but this can likely be attributed to its lack of steric bulk. In addition to phosphate binding, we determined stability constants for M40404 and, for comparison, M40403 as well. These values, together with the literature values for other studied complexes, are summarized in Table 3. A direct comparison of the stability constants for the dinuclear and mononuclear complexes is not straightforward.46 Therefore, we also calculated pMn (−log [Mn]free) values at pH 7.4 and [complex] = 1 mM (Table 3). On the basis of the pMn values, the imine complex manganese(II) pydiene shows the lowest stability in solution, as expected, whereas its amine analogue manganese(II) pyane exhibits more than 1 order of magnitude smaller concentration of free Mn II ions in solution, demonstrating its higher stability. The dinuclear analogue dimanganese(II) bipyane, as well as complexes M40404 and M40403 with cyclohexyl groups, shows the same higher

Table 4. Total Manganese Areal Density (μg/cm2) of SHSY5Y Cells Treated with MnSOD Mimetics, As Determined by XFMa compound control (water) MnCl2 manganese(II) pyane manganese(II) pyanec dimanganese(II) bipyane manganese(II) pyane− rhodBc manganese(II) pyane− pyrene M40404c manganese(II) pydiene

treatment concn (μM)

n

total manganese (×10−3 μg/cm2)

200 200 200 200

5 6 5 9 5

0.5 (0.2) 7 (3)b 5 (2)b 1.7 (0.5)b 11 (2)b

200

10

9 (2)b

20

6

18 (8)b

50 200

7 5

50 (20)b 62 (6)b

a

Data are presented as the mean (standard deviation) of n cells. Significantly different from the control at the P < 0.05 significance level (two-tailed t test assuming unequal variance). cSamples were prepared and imaged on a second occasion. b

The intracellular manganese content, a proxy for cellular uptake of the MnPAMs, was measured by XFM and is reported as the elemental content over the cell area (Table 4). After 24 h, the greatest accumulation of manganese (∼100-fold over the control cells) was observed in cells treated with the SODinactive M40404 (50 μM) and manganese(II) pydiene (200 6084


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Figure 2. Optical micrographs (top left) and XFM elemental distribution maps of cells treated with (a) vehicle (Milli-Q), (b) MnCl2, (c) M40404, (d) manganese(II) pyane, (e) manganese(II) pydiene, (f) dimanganese(II) bipyane, (g) 8, and (h) 9. The maximum intensity of each elemental map was scaled, where necessary, for clarity. Cells presented here are representative of the entire treatment group, with the number of cells imaged (0.5 μm step size; 0.5 s dwell time) per treatment indicated in Table 3.

μM). On the other hand, cells treated with the SOD-active pyane-based pentaazamacrocycles experienced a 10−20-fold increase in the manganese content compared to controls, as did cells treated with MnCl2. Notably, manganese levels in 8treated cells were on the same order as those of the rest of the pyane-based pentaazamacrocycles despite a 10-fold lower treatment concentration. The high toxicity of this complex is explained by its high lipophilicity, resulting in efficient uptake. Elemental maps of individual MnPAM-treated cells, showing elemental distributions typical of the entire sample set, are shown in Figure 2. In vehicle control (Figure 2a) and MnCl2treated cells (Figure 2b), manganese is found throughout the cell body, with higher intensities associated with the thicker nuclear region (defined by the high abundance of phosphorus and, usually, zinc). This manganese distribution is also observed

in cells treated with manganese(II) pyane (Figure 2d), dimanganese(II) bipyane (Figure 2f), and 9 (Figure 2h). For comparison, manganese has been reported to be concentrated in the cytoplasm and Golgi apparatus of MnCl2-treated primary midbrain dopaminergic neuron cells and dopaminergic PC12 cells, respectively.26 Whether manganese was found in the nucleus or the cytoplasm of cells treated with porphyrin-based MnSOD mimetics was found to depend on the porphyrin-ring substituents.28 In manganese(II) pydiene-treated (Figure 2e) and 8-treated (Figure 2g) cells, manganese accumulated in the cytoplasm. In the former, the manganese was highly compartmentalized and colocalized with iron and zinc and, to a lesser extent, copper, which suggests a significant impact of manganese(II) pydiene on the homeostasis of these transition metals. Similarly, 6085


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Inorganic Chemistry dimanganese(II) bipyane treatment causes the redistribution and colocalization of iron and zinc. Finally, manganese in M40404-treated cells is mostly found in the nucleus, with some accumulation in the perinuclear region (white arrow, Figure 2c). The distribution of manganese in manganese(II) pydienetreated cells underwent further investigation by the preparation of samples labeled with a fluorescent mitochondrial tracker. A comparison of the fluorescence from this probe with the Mn Kα fluorescence revealed a similar distribution, with the highest levels of manganese and Mitotracker fluorescence found in a ring around the nucleus (Figure 3). This suggests a mitochondrial localization of manganese from manganese(II) pydiene treatment, although it does not rule out localization in other perinuclear organelles.

Figure 4. Distribution of the rhodamine tag, which does not correspond with the distribution of manganese in SH-SY5Y cells treated with 200 μM 9 for 24 h. (a) Fixed cells imaged by both fluorescence microscopy (rhodamine tag, left) and low-resolution XFM (manganese, right). (b) Confocal fluorescence microscopy image of the rhodamine tag (red) in living cells (bright field) treated with 9. (c) High-resolution elemental maps of manganese in fixed cells; see also Figure 2h.

accumulation of both pyrene and manganese provides evidence that complex 8 remains largely intact. This cytosolic accumulation of manganese also indicates that the addition of the pyrene tag alters the cellular distribution of the complex, which is now excluded from the nucleus. Speciation of Manganese in MnPAM-Treated Cells. Here, we describe how XAS can provide information about the manganese oxidation state and the atoms to which it is bound by a comparison to model compound spectra (XANES) and by a priori determination of the distances between the central manganese and backscattering atoms in its first and second coordination shells (EXAFS).48 In combination, XANES and EXAFS reveal the speciation of manganese in cells treated with MnPAMs and can help to answer the question of whether the MnPAM complexes remain intact in cells or, alternatively, manganese is released from the PAM for coordination by endogenous ligands. To this end, a model compound library of manganese XANES spectra was built containing a variety of Mn−N/O species in the MnII, MnIII, and MnIV states likely to be found in cells (Figure 6). The Mn K-edge XANES spectra of the MnPAMs bear a strong resemblance to each other and to other models of MnII bound to organic ligands via O and/or N atoms (e.g., MnEDTA and MnEGTA). The choice of pure water or PBS as the solvent had some impact on the

Figure 3. Comparison of manganese and zinc distributions with the distribution of Mitotracker in the same cells treated with manganese(II) pydiene. The manganese distribution is shown with an intensity scale (top) and overlaid on the zinc map (center) for comparison with the fluorescence microscopy image of the Mitotracker Deep Red tag (bottom).

To address the question of how well the rhodamine B and pyrene fluorescent tags track the intracellular distributions of the MnPAMs, the cellular distributions of manganese and fluorescent tags were compared for 9 and 8. Rhodamine B and pyrene fluorescences in both fixed and live cells were clearly confined to the cytosol (Figures 4 and 5, respectively). In 9treated cells, the cytosolic concentration of rhodamine fluorescence is at odds with the distribution of manganese throughout the cells, which indicates that either the rhodamine tag is cleaved from the pentaazamacrocycle or the MnPAM complex is no longer intact in the cell. By contrast, the cytosolic 6086


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Figure 5. Distributions of the pyrene tag and manganese, which are similar in SH-SY5Y cells treated with 200 μM 9 for 24 h. (a) Fixed cells imaged by both fluorescence microscopy (pyrene tag, left) and low-resolution XFM (manganese, right). (b) Confocal fluorescence microscopy image of the pyrene tag (blue) in living cells (bright field) treated with 9. (c) High-resolution elemental maps of manganese in fixed cells; see also Figure 2g.

manganese(II) pyane spectrum, as expected from its ability to bind phosphate. The absorption edge in the spectrum of MnCl2 in water [MnII(H2O)62+] was observed at higher energy than those of the MnPAMs. The spectrum of manganese in an amorphous phosphate complex (MnPi) had a reduced peak intensity relative to that of other MnIIO species, a lower energy rising-edge shoulder, and an absorption edge at higher energy compared to those of the MnPAMs. MnIIIO species are readily distinguished from MnIIO species by their higher edge energy and broader, less intense peaks. Low-intensity preedge peaks were observed in the spectra of all model compounds. The speciation of manganese in treated cells was first determined from XANES collected from SH-SY5Y cells treated with 200 μM manganese complexes for either 4 or 24 h (Figure 7). The XANES spectra were fit, by a linear combination, with selected model compound spectra to determine the composition of the manganese species in each sample. Principalcomponent analysis indicated that three models would be sufficient to describe the collected samples. Manganese(II) pyane (mixed MnIIN/MnIIO) or MnCl2 [in solution existing as manganese(II) aqua species, i.e., MnIIO], MnPi (MnIIOP), and MnIII2O3 (MnIIIO) were selected to represent the range of manganese environments likely to be found in the cells, based on information from target transformation. Good fits were obtained for most spectra (Table 5), including that from manganese(II) pydiene-treated cells for which MnIII2O3 was an adequate MnIIIO model (Figure S7). Some of the higher residuals are accounted for by noise in the sample spectra. The manganese in MnPAM-treated cells retains the spectral MnII−N/O character of the MnPAMs to varying degrees, with partial transformation into species whose XANES spectra are well-represented by the spectrum of manganese(II) phosphate (MnPi). The formation of MnPi occurs in the cell culture media (∼0.8 mM phosphate) with 30−40% of manganese present as MnPi in the media containing manganese(II) pyane and dimanganese(II) bipyane that was collected from cells

Figure 6. Library of manganese model compound spectra. Spectra were collected from 3−5 mM frozen solutions in water or PBS (M40404) with 30% glycerol, except for MnPi and manganese(III) acetate, which were collected from solids diluted in cellulose. The spectra of MnIIO, MnIII2O3, and MnIVO2 were collected from solids diluted in boron nitride.

treated for 4 h. However, MnPi formation is even more extensive in the cells, perhaps because of higher phosphate concentrations throughout the cells or in certain subcellular compartments. Cells treated with manganese(II) pydiene, where MnIII species were dominant, were the exception. This oxidation must occur inside cells because manganese in media harvested from treated cells contains only MnII species. The dinuclear dimanganese(II) bipyane was more extensively metabolized compared to other compounds (despite the fact that its pure solution exhibits the highest stability against the release of free MnII; Table 3), with a majority of the manganese having amorphous phosphate character after both 4 and 24 h of incubation with cells. This can be explained by the fact that complex stability constants and pMn values only reflect the thermodynamics in pure solutions, and if other species are present, the extent and nature of interactions with them will predominantly affect manganese speciation in solution. This includes interactions with endogenous phosphates for which dimanganese(II) bipyane has the highest binding constant (Table 1) compared to that of other studied complexes, meaning that phosphate binding facilitates transformation of the complex into manganese(II) phosphate. It should be 6087


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character, with the majority of the manganese modeled as manganese(II) pyane. By way of comparison, M40404 has the same thermodynamic stability as dimanganese(II) bipyane but is much more resistant to the formation of manganese(II) phosphate within cells. Although the phosphate binding constant could not be determined for M40404, its low values may be expected as a result of steric hindrance (the presence of both methyl and cyclohexyl substituents) and its lower conformational flexibility. EXAFS spectra were obtained from MnPi, MnCl 2 , manganese(II) pyane, dimanganese(II) bipyane, M40404, and manganese(II) pydiene (Figure 8). The amplitude of the EXAFS from the amorphous MnPi was significantly lower than that of the more rigid MnPAMs. Two prominent peaks were observed in the Fourier transforms of each of the MnPAM spectra at around 2.25 and 3.1 Å. These peaks broadened and their intensities diminished in a phosphate buffer, indicating a loss of purity associated with the formation of manganese(II) phosphate. Fitting of the EXAFS spectra (Table 6) showed that the two peaks in the Fourier transforms of the MnPAMs were the result of Mn−N and Mn−O bonds (EXAFS analysis is unable to distinguish backscatters of similar atomic number such as N and O; as such, our fits to MnPAMs are reported as Mn−N/O) with average lengths between 2.26 and 2.28 Å and interactions between the central Mn atom and C atoms in the pentaazamacrocyclic rings, with average distances between 3.09 and 3.19 Å. The addition of these second-shell Mn−C bonds (that have previously been observed in the Mn K-edge EXAFS spectra of organic manganese compounds49) substantially improved the fits over those featuring Mn−N/O alone. These fits are in agreement with the crystal structures of manganese(II) pyane and manganese(II) pydiene, which show a seven-coordinate manganese bound to five N and two O atoms (from coordinated water molecules) with average bond lengths of 2.30 Å50 and 2.28 Å,51 respectively. Coordination numbers for Mn−N/O bonds fitted to the MnPAMs and other manganese compounds vary between 7 and 9 (fixed at whole or

Figure 7. Mn K-edge X-ray absorption spectra of MnPAMs in solution and of SH-SY5Y cells treated with the following compounds: (a) MnCl2, (b) manganese(II) pyane, (c) dimanganese(II) bipyane, (d) M40404, and (e) manganese(II) pydiene. The spectra shown are MnPAM (or MnCl2) in water (black solid line) and in media retrieved from treated cells after 4 h (black dotted line) and cells treated with MnPAMs for 4 h (blue solid line) and 24 h (red solid line). The red dotted line in part b is the spectrum collected from cells treated with 9 for 24 h.

pointed out that manganese(II) phosphate is less soluble than MnPAMs, which shifts the equilibrium upon phosphate binding toward its formation. At the other extreme, the manganese in M40404-treated cells largely retained its pentaazamacrocyclic

Table 5. Results of a Linear Combination Fitting of Mn K-Edge Spectra of SH-SY5Y Cells Treated with MnCl2 or MnPAMs and of Separated and Freeze-Dried Cell Culture Media (∼0.8 mM Phosphate) Post-treatment percentage of components fitted treatment

treatment concn (μM)

treatment time (h)

MnCl2 (media, m) MnCl2 (cells, c) MnCl2 (c) manganese(II) pyane (m) manganese(II) pyaned (c) manganese(II) pyane (c) dimanganese(II) bipyane (m) dimanganese(II) bipyane (c) dimanganese(II) bipyane (c) manganese(II) pyane−rhodB (c) M40404 (c) M40404 (c) manganese(II) pydiene (m) manganese(II) pydiene (c) manganese(II) pydiene (c)

200 200 200 100 100 200 200 200 200 200 200 100 200 200 200

4 4 24 4 4 24 4 4 24 24 4 24 4 4 24

manganese(II) pyanea 43(2) 16(1) 31(1) 71(1) 41(2) 62(2) 59(1) 12(1) 27(1) 51(1) 58.1(1) 87.8(8) 64(1)

MnPib 57(2) 85(1) 69(1) 30(1) 61(2) 42(2) 42(1) 84(3) 74(1) 48(1) 42.0(1) 12.7(8) 36(1) 14(2) 13(1)

MnIII2O3c

total fraction

residual (×10−3)

5.7(1)

1.00 1.01 1.00 1.01 1.02 1.04 1.01 1.01 1.00 0.98 1.00 1.01 1.00 1.06 1.03

1.27 0.81 0.97 0.56 1.50 1.75 0.50 0.25 1.1 1.09 0.51 0.35 0.60 1.73 1.50

92(2) 91(1)

Manganese(II) pyane in Milli-Q water or MnCl2 in Milli-Q water for MnCl2-treated cells (MnII−O/N). bMnPi = amorphous manganese(II) phosphate (MnIIOP). cProvided by Enzo Lombi. dSpectrum collected from A549 cells.

a

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Analysis of the EXAFS spectra in addition to the XANES spectra provides further insight into and confirmation of manganese speciation in cells. EXAFS spectra were readily collected from all but the manganese(II) pyane-treated SHSY5Y cells (Figure 9). The low uptake of manganese(II) pyane

Figure 8. EXAFS (left) and Fourier transforms (right) of manganese compounds: (a) MnPi, (b) MnCl2 (water), (c) manganese(II) pyane (water), (d) manganese(II) pyane (PBS), (e) dimanganese(II) bipyane (water), (f) M40404 (PBS), and (g) manganese(II) pydiene (water).

half-integer values), but given the estimated 20% uncertainty48 in coordination numbers determined by EXAFS fitting and somewhat higher than the usual Debye−Waller factors, these values are in agreement with the crystal structures. Second-shell Mn−C distances in the crystal structures range between 3.08 and 3.23 Å, in agreement with the distances fit to the EXAFS spectra, reported below. The Mn−O distances fitted to the spectrum of MnCl2, at 2.155 Å, are shorter than those in the MnPAMs, as are the first-shell Mn−O bonds in MnPi (2.19 Å). Second-shell Mn−O bonds (3.13 Å) were also fit to the MnPi spectrum.

Figure 9. EXAFS (left) and Fourier transforms (right) of SH-SY5Y or A549 cells treated with MnCl2 or MnPAMs for 24 h: (a) MnCl2, (b) manganese(II) pyane (A549 cells, black) and manganese(II) pyane (SH-SY5Y cells, gray), (c) dimanganese(II) bipyane, (d) M40404, and (e) manganese(II) pydiene.

by the cells resulted in lower-quality EXAFS [better-quality EXAFS was collected from manganese(II) pyane-treated A549 cells, suggesting better uptake of manganese(II) pyane by this cell line], with 9-treated cells generating a similar, albeit slightly less, noisy spectrum.

Table 6. Structural Parameters from Best Fits to EXAFS Spectra of Model Manganese Complexes in Water, unless Otherwise Indicated compound MnPi (solid)

b

fit no.

bond

coordination number (N)a

distance (R)

Debye−Waller factor (σ2)

−ΔE (eV)

F factor (%)

1

Mn−O Mn−O Mn−O Mn−N/O Mn−N/O Mn−C Mn−N/O Mn−C Mn−N/O Mn−C Mn−N/O Mn−N/O Mn−C

2 2 9 7 7 7 7 7 8 8 9 8 8

2.191(6) 3.13(1) 2.155(7) 2.259(5) 2.257(3) 3.175(5) 2.257(5) 3.15(2) 2.280(3) 3.191(4) 2.276(9) 2.258(6) 3.085(7)

0.002(5) 0.002(1) 0.0089(5) 0.0093(4) 0.0093(3) 0.0077(5) 0.0040(4) 0.010(2) 0.0087(3) 0.0066(5) 0.0089(7) 0.0072(5) 0.0029(6)

7.5(0.9)

68

14(1) 12.2(6) 12.6(3)

59 51 33

14.0(7)

50

12.3(3)

34

12(1) 14.5(6)

70 58

MnCl2c manganese(II) pyaned

1 1 2

dimanganese(II) bipyane

1

M40404 (PBS)d

2

manganese(II) pydieneb

1 2

The coordination number was held constant after floating to determine the optimum value to the nearest integer. bFit to 10.5 Å−1 in k space. cFit to 9.8 Å−1 in k space. dIn water. Fit to 12 Å−1 in k space. a

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Inorganic Chemistry Table 7. Parameter Fits to the EXAFS Spectra of SH-SY5Y Cells Treated with Manganese Complexes fit no.

bond

coordination number (N)a

distance (R)

Debye−Waller factor (σ2)

MnCl2b

1

manganese(II) pyaneb,c manganese(II) pyane−rhodBd

1 1 2

dimanganese(II) bipyaneb

1

M40404d

1 2

manganese(II) pydiened

1

Mn−O Mn−O Mn−N/O Mn−N/O Mn−N/O Mn−C Mn−O/N Mn−O/N Mn−N/O Mn−N/O Mn−C Mn−O

4 3 4.5 4 4 4 3.5 4 9 8 8 3.5

2.131(4) 3.117(9) 2.24(1) 2.260(7) 2.260(6) 3.19(2) 2.165(4) 3.160(9) 2.267(5) 2.280(3) 3.191(4) 1.893(8)

0.0074(3) 0.008(1) 0.0024(1) 0.0016(5) 0.0016(5) 0.002(1) 0.0025(4) 0.0035(9) 0.0097(4) 0.0087(3) 0.0066(5) 0.0090(6)

treatment compound

−ΔE (eV) 12.5(5) 8(2) 10(1) 11(1)

F factor (%) 40 89 81 78

10.5(7)

61

12.8(6) 12.3(3)

53 34

11(1)

67

The coordination number was held constant after floating to determine the optimum value to the nearest 0.5. bFit to 9.8 Å−1 in k space. cA549 cells. d Fit to 12 Å−1 in k space. a

XANES spectra for this sample. Finally, the fit to the EXAFS of manganese(II) pydiene-treated cells confirms the oxidation of MnII to MnIII after cellular uptake with the dramatically shortened Mn−N/O bond length (1.89 vs 2.26 Å), loss of the second-shell Mn−C interaction, and reduced coordination number compared to the solution structure of manganese(II) pydiene.

The N/O coordination number of manganese in MnCl2treated cells is half that of MnCl2 in solution, but the bond length remains at ∼2.15 Å (Table 7). Combined with the substantial MnPi component fit to the XANES spectra, this indicates that changes to the local coordination environment of MnII involve the partial replacement of water ligands with phosphate. The high noise levels in EXAFS spectra collected from manganese(II) pyane-treated cells made analysis challenging. The Mn−N/O bond length of 2.24 Å is consistent with the aqueous manganese(II) pyane structure, but attempts to fit a Mn−C component were unsuccessful. A slight improvement in the fit was obtained when fitting both the Mn−N/O and Mn− C components to the spectrum of 9-treated cells, giving bond lengths consistent with those of the aqueous manganese(II) pyane spectrum. However, the EXAFS spectra collected from manganese(II) pyane- and 9-treated cells have EXAFS amplitudes that increase beyond 8 Å−1 in k space, which is opposite to what is observed in the MnPAM standard spectrum but is consistent with the MnPi standard spectrum. This subtle difference alludes to the mixed manganese(II) pyane/MnPi character indicated by fits to the XANES spectra. XANES and EXAFS results, in combination with our knowledge of the chemical properties of MnPAM complexes, lead us to conclude that this mixed manganese(II) pyane/MnPi character is due to a combination of intact MnPAM, the formation of MnPAM− phosphate adducts, and finally the release of manganese from the pentaazamacrocycles to form manganese(II) phosphate. It should be noted that while we believe that the MnPAM− phosphate adducts are important intermediates between the MnPAM and the formation of manganese(II) phosphate, it is difficult to directly identify and distinguish them from the parent complexes using XAS techniques. The extent of conversion to MnPi in dimanganese(II) bipyane-treated cells is greater than that observed when cells are treated with either manganese(II) pyane or 9. This is reflected in the EXAFS spectrum (Figure 9c) that is fit with a first-shell Mn−N/O bond length of 2.17 Å, which is ∼0.1 Å less than that found in the parent MnPAM standards and similar to that found in the MnPi standard. The reduced total amplitude of the EXAFS spectrum observed in the dimanganese(II) bipyane-treated cells is also in keeping with the formation of MnPi. By contrast, in cells exposed to M40404 treatment, the EXAFS spectrum is almost identical with the M40404 model spectrum, which is supported by similar results from the

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DISCUSSION

We have characterized the speciation and distribution of manganese in cells treated with a number of different MnPAMs based on manganese(II) pyane, including newly synthesized fluorescently labeled derivatives. The chemical form and physical properties of the MnPAMs, especially their phosphate-binding constant and lipophilicity, influence the cellular fate of these complexes. Treatment with the manganese(II) pyane-derived pentaazamacrocycles resulted in a 10−20-fold increase in the cellular manganese levels compared to physiologically present manganese in control cells. Manganese uptake was correlated with the lipophilicity of the MnPAMs, except for the hydrophilic, but less sterically bulky, manganese(II) pydiene. Of the two different tags added to manganese(II) pyane, pyrene was observed to alter the distribution of manganese from that of manganese(II) pyane alone, leading to exclusion of the complex from the nucleus and indicating that the pyrene tag is not a useful marker for the parent pyane complex. Conversely, treatment with 9 resulted in the same manganese distribution and speciation as the manganese(II) pyane treatment. However, the cytosolic localization of the rhodamine tag suggests that while the manganese(II) pyane core persists largely intact, the rhodamine tag may be cleaved because of hydrolysis of the amide linker and does not enter the nucleus with the rest of the complex. It seems that the positive charge on the rhodamine B tag makes the amide bond cleavage more favorable in comparison to that in 8. Importantly, the hydrolysis product 7 has SOD activity similar to that of manganese(II) pyane. The SOD-inactive M40404 and manganese(II) pydiene are both more readily taken up by cells than the SOD-active manganese(II) pyane-based pentaazamacrocycles. M40404 is hardly metabolized in cells and exhibited some accumulation in a perinuclear region. Manganese(II) pydiene is readily oxidized to a MnIIIO species. In addition to their inherent lack of SOD activity, these complexes were expected to have a low 6090


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Inorganic Chemistry interaction with phosphate, which was borne out in the analysis of XANES and EXAFS spectra and may be related to their unique manganese distributions compared to the MnPAMs that interact with phosphates. For manganese(II) pydiene, hydrolysis of imine bonds leads to decomposition of the macrocycle and release of MnII. We have previously observed that manganese(II) pydiene decomposes upon reaction with NO.10 The addition of peroxynitrite and superoxide to manganese(II) pydiene in vitro leads to decomposition of the ligand, the release of MnII and its oxidation by an excess of oxidizing agents. This can be visualized by decay of the UV/vis absorption bands characteristic for manganese(II) pydiene (Figure S10). A comparison with the fate of manganese in cells that are treated with aqueous MnII indicates that manganese(II) pydiene circumvents “normal” manganese processing in the cells and suggests that some physicochemical property of manganese(II) pydiene is recognized, leading to compartmentalization, followed by ligand decomposition and the release of MnII into an oxidizing environment (such as that known to exist in the mitochondria) where MnIII is formed; outcompeting the formation of manganese(II) phosphate. The pro-oxidative environment in mitochondria speaks in favor of the localization of MnIII species in this organelle. The metabolism of manganese(II) pydiene to a stable MnIII complex in cells is surprising. Gunter et al. conducted XAS studies of a number of cell lines treated with MnCl2 and found that MnIII must be at or below the experimental detection limit (about 2.5% of total manganese), concluding that any formation of MnIII is rapidly followed by the reduction to MnII.23−25 They later showed that MnIII could be transported into cells via the transferrin mechanism.52 To our knowledge, the formation of MnIII species in cells exposed to manganese(II) pydiene is the first example of the generation of stable MnIII species inside cells from the oxidation of an exogenously administrated MnII compound. Knowledge that stable MnIII species can form in cells treated with MnII may be useful in elucidating the relative contributions of MnII and MnIII species to neurotoxicity, which remain uncertain.53−55 Evidence from XANES and EXAFS suggests that 50−85% of manganese(II) pyane, 9, and M40404 are found in cells in the intact form, i.e., as MnII bound to the macrocyclic ligand. The remainder is metabolized over time into manganese(II) phosphate, modeled here by a solid amorphous manganese(II) phosphate (MnPi). The greatest MnPi component, 75−85%, was found in cells treated with dimanganese(II) bipyane even though this complex possesses high thermodynamic stability. M40404, however, with a stability similar to that of dimanganese(II) bipyane, remains largely intact in cells. This illustrates the fact that stability constants per se do not reflect the cellular fate of the complexes and that the thermodynamics and kinetics of their interactions with other species present in the environment are critical. Binding of phosphate to MnPAMs weakens the interaction between MnII and the macrocyclic ligand, which triggers ligand dissociation and the formation of manganese(II) phosphate. This also explains why the formation of manganese(II) phosphate does not correlate with the complex stability constant but rather with the phosphate binding constant. On the basis of the determined K(HPO42−) binding constants (Table 1), strong phosphate binding is expected for dimanganese(II) bipyane, which facilitates its transformation to less soluble MnPi. Importantly, manganese(II) phosphate is SOD-active as well, with the catalytic rate constant of ca. 5 × 106 M−1 s−1 at pH 7.4.6,56 Interestingly, kcat

for dimanganese(II) bipyane in the presence of phosphate, per single manganese center, is exactly the same as that for manganese(II) phosphate (Table 1). By way of comparison, manganese(II) pyane that is only ca. 50% transformed into MnPi in cells exhibits somewhat higher SOD activity than dimanganese(II) bipyane in the presence of phosphate. M40403 also has a prominent phosphate binding constant, and, consequently, in the presence of phosphate, its kcat significantly decreases. Thus, one may predict that within cells it will be partially converted into MnPi. The same fate is expected for GC4419 because, as the enantiomer of M40403, it should have the same stability constant and reactivity features toward nonchiral species. Consequently, in terms of the SOD activity or reactivity with other reactive oxygen and nitrogen species, M40403 and GC4419 should be identical. Potentially different uptake and biological effects may come from their different interactions with chiral molecules, e.g., proteins and/ or sugars. We have previously reported that phosphate anions play an important role in the activity of manganese SOD mimetics in general, and of MnPAMs in particular, which is crucial for understanding and comparing their effects under physiological conditions.6 Here we have demonstrated that phosphate binding is related to the transformation of MnPAMs into MnPi in cells, which, depending on the extent of the complex− phosphate interactions, can level their SOD activity in cellular environments to that of MnPi. Thus, the role of the ligand is in tuning manganese uptake in cells and controlling its cellular distribution.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b03073. UV/vis and fluorescence characteristics of new complexes, SOD activity experimental details and results, details of the HPO42− binding constant equations and fits, plots of fitted XANES and EXAFS spectra, elemental area densities of cells as determined by XFM, and decomposition of manganese(II) pydiene by the reaction with peroxynitrite (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (I.I.-B.). Phone: +49 9131 85-25428. Fax: +49 9131 85-27345. *E-mail: [email protected] (H.H.H.). Phone: 61-0883135060. Fax: 61-8-83134358. ORCID

Claire M. Weekley: 0000-0002-7616-2706 Ivana Ivanović-Burmazović: 0000-0002-1651-3359 Hugh H. Harris: 0000-0002-3472-8628 Present Address ‡

C.M.W.: Department of Chemistry, The University of Chicago, 929 East 57th Street, Chicago IL 60637. Notes

The authors declare no competing financial interest. 6091


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(11) Riley, D. P.; Schall, O. F. Structure−Activity Studies and the Design of Synthetic Superoxide Dismutase (SOD) Mimetics as Therapeutics. Adv. Inorg. Chem. 2006, 59, 233−263. (12) Ivanović-Burmazović, I.; van Eldik, R. Metal complex-assisted activation of small molecules. From NO to superoxide and peroxides. Dalton Trans. 2008, 49 (39), 5259−18. (13) Ivanović-Burmazović, I.; Filipović, M. R. Reactivity of manganese superoxide dismutase mimics toward superoxide and nitric oxide-Chapter 3: Selectivity versus cross-reactivity. Adv. Inorg. Chem. 2012, 64, 53−95. (14) Filipović, M. R.; Duerr, K.; Mojović, M.; Simeunović, V.; Zimmermann, R.; Niketić, V.; Ivanović-Burmazović, I. NO Dismutase Activity of Seven-Coordinate Manganese(II) Pentaazamacrocyclic Complexes. Angew. Chem., Int. Ed. 2008, 47 (45), 8735−8739. (15) Filipović, M. R.; Koh, A. C. W.; Arbault, S.; Niketić, V.; Debus, A.; Schleicher, U.; Bogdan, C.; Guille, M.; Lemaître, F.; Amatore, C.; Ivanović-Burmazović, I. Striking Inflammation from Both Sides: Manganese(II) Pentaazamacrocyclic SOD Mimics Act Also as Nitric Oxide Dismutases: A Single-Cell Study. Angew. Chem., Int. Ed. 2010, 49 (25), 4228−4232. (16) Otasevic, V.; Korac, A.; Vucetic, M.; Macanovic, B.; Garalejic, E.; Ivanović-Burmazović, I.; Filipović, M. R.; Buzadzic, B.; Stancic, A.; Jankovic, A.; Velickovic, K.; Golic, I.; Markelic, M.; Korac, B. Is Manganese (II) Pentaazamacrocyclic Superoxide Dismutase Mimic Beneficial for Human Sperm Mitochondria Function and Motility? Antioxid. Redox Signaling 2013, 18 (2), 170−178. (17) Niketíc, V.; Stojanović, S.; Nikolić, A.; Spasić, M.; Michelson, A. M. Exposure of Mn and FeSODs, but not Cu/ZnSOD, to NO leads to nitrosonium and nitroxyl ions generation which cause enzyme modification and inactivation: an in vitro study. Free Radical Biol. Med. 1999, 27 (9−10), 992−996. (18) Lieb, D.; Kenkell, I.; Miljković, J. L.; Moldenhauer, D.; Weber, N.; Filipović, M. R.; Gröhn, F.; Ivanović-Burmazović, I. Amphiphilic Pentaazamacrocyclic Manganese Superoxide Dismutase Mimetics. Inorg. Chem. 2014, 53 (2), 1009−1020. (19) Weekley, C. M.; Aitken, J. B.; Vogt, S.; Finney, L. A.; Paterson, D. J.; de Jonge, M. D.; Howard, D. L.; Witting, P. K.; Musgrave, I. F.; Harris, H. H. Metabolism of selenite in human lung cancer cells: x-ray absorption and fluorescence studies. J. Am. Chem. Soc. 2011, 133 (45), 18272−18279. (20) Weekley, C. M.; Shanu, A.; Aitken, J. B.; Vogt, S.; Witting, P. K.; Harris, H. H. XAS and XFM Studies of Selenium and Copper Speciation and Distribution in the Kidneys of Selenite-Supplemented Rats. Metallomics 2014, 6, 1602−1615. (21) Wu, L. E.; Levina, A.; Harris, H. H.; Cai, Z.; Lai, B.; Vogt, S.; James, D. E.; Lay, P. A. Carcinogenic Chromium(VI) Compounds Formed by Intracellular Oxidation of Chromium(III) Dietary Supplements by Adipocytes. Angew. Chem. 2016, 128 (5), 1774−1777. (22) MacDonald, T. C.; Korbas, M.; James, A. K.; Sylvain, N. J.; Hackett, M. J.; Nehzati, S.; Krone, P. H.; George, G. N.; Pickering, I. J. Interaction of mercury and selenium in the larval stage zebrafish vertebrate model. Metallomics 2015, 7, 1247−1255. (23) Gunter, T. E.; Miller, L. M.; Gavin, C. E.; Eliseev, R.; Salter, J.; Buntinas, L.; Alexandrov, A.; Hammond, S.; Gunter, K. K. Determination of the oxidation states of manganese in brain, liver, and heart mitochondria. J. Neurochem. 2004, 88 (2), 266−280. (24) Gunter, K. K.; Aschner, M.; Miller, L. M.; Eliseev, R.; Salter, J.; Anderson, K.; Hammond, S.; Gunter, T. E. Determining the oxidation states of manganese in PC12 and nerve growth factor-induced PC12 cells. Free Radical Biol. Med. 2005, 39 (2), 164−181. (25) Gunter, K. K.; Aschner, M.; Miller, L. M.; Eliseev, R.; Salter, J.; Anderson, K.; Gunter, T. E. Determining the oxidation states of manganese in NT2 cells and cultured astrocytes. Neurobiol. Aging 2006, 27 (12), 1816−1826. (26) Dučić, T.; Barski, E.; Salome, M.; Koch, J. C.; Bähr, M.; Lingor, P. X-ray fluorescence analysis of iron and manganese distribution in primary dopaminergic neurons. J. Neurochem. 2013, 124 (2), 250−261. (27) Carmona, A.; Roudeau, S.; Perrin, L.; Veronesi, G.; Ortega, R. Environmental manganese compounds accumulate as Mn(ii) within

ACKNOWLEDGMENTS We thank Dr. Ian Musgrave (The University of Adelaide) for sharing his cell culture facilities. Use of the Advanced Photon Source at Argonne National Laboratory was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract DE-AC02-06CH11357. We thank Dr. Barry Lai for facilitating the beamtime. Parts of this research were undertaken at the X-ray Fluorescence Microprobe and XAS beamlines at the Australian Synchrotron, Clayton, Victoria, Australia. We thank Drs. Daryl Howard and Simon James (XFM) and Drs. Peter Kappen, Chris Glover, and Bernt Johannessen (XAS) for their assistance. Fluorescence and confocal fluorescence microscopy was conducted with the assistance of Dr. Agatha Labrinidis at Adelaide Microscopy at The University of Adelaide, an AMMRF facility. We acknowledge travel funding provided by the International Synchrotron Access Program, managed by the Australian Synchrotron and funded by the Australian Government, research funding from the Australian Research Council (Grant DP140100176 to H.H.H.) and from the NHMRC (CJ Martin Overseas Fellowship to C.M.W.). We also acknowledge funding support by a “Medicinal Redox Inorganic Chemistry” Emerging Field intramural grant from the FAU Erlangen−Nuremberg.

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Inorganic Chemistry

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Cellular Fates of Manganese(II) Pentaazamacrocyclic Superoxide

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