Electron Paramagnetic Resonance Spectroscopy of Chromium Compounds

Chromium(V) concentration was determined quantitatively by comparison of IDH2 (where I = peak to trough signal intensity and DH = linewidth) with a K3CrO8 standard of known concentration. K3CrO8 was synthesized as described by Dalal et al. (54). A solution containing 200 mL nanopure H2O, 200 mL 25% KOH(aq), and 50 mL 50% CrO3 was cooled at -15 °C until a slurry was formed. To this solution 60 mL of 30% H2O2 was added dropwise while maintaining the temperature of the slurry at -15 °C. The reaction mixture was left in a cold room at 4 °C overnight, then filtered. The solid product was washed with ice-cold nanopure H2O, followed by 95% ethanol and air-dried, resulting in rose-colored crystals. Solid K3CrO8 is stable for several years when stored in a desiccator at -20 °C (55). Stock Cr(V) solutions made in 0.2 N KOH (pH > 13) containing ~0.3-0.5% H2O2 are stable for several weeks; solutions used in these studies were made fresh at least weekly and stored at 4 °C. Cr(V) concentration of stock solutions was in the 0.3-0.5 mM range. Working dilutions (0.3-300 µM range) of the stock solution were made fresh daily. Under the conditions employed in this study, 0.1 µM is about the lower limit of [chromium(V)]; lower concentrations (about 3-fold) are detectable, however the S/N ratio is too low for meaningful results. Standard K3CrO8 spectra have three features at 77 K: gz = 1.9974, DH = 4.10 G; gy = 1.9848, DH = 5.47G; gx = 1.9508, DH = 6.2 G. At 297 K, the spectrum of K3CrO8 in 0.2 N KOH displays a single chromium(V) signal at g = 1.971, with a 53Cr hyperfine value of a53Cr = 18.6 G.

For electron paramagnetic resonance (EPR) studies done in frozen solutions at 77 K, aliquots (350 µL) of the reactions were taken in quartz EPR tubes at various times and quenched by immersing the tubes in liquid nitrogen (77 K). EPR spectra of the aliquots were acquired at 77 K using a Bruker ESP-300 spectrometer with 100 kHz field modulation, 5.71 Gauss modulation amplitude, 1.0 x 105 receiver gain, 200-5000 Gauss sweep width, 5.12 ms time constant, 2 mW microwave power incident on cavity, 9.315 GHz microwave frequency, and a time of 37-41 minutes per spectrum (100 scans). A home-built frequency meter was used to measure microwave frequency, and chromium(V) concentration was determined by comparison with K3CrO8 standards of known concentrations.

For studies done at 297 K, aliquots (50 µL) of the solutions were taken in melting point capillaries which were in turn placed in quartz EPR tubes. EPR spectra were taken at 297 K using a Bruker ESP-300 spectrometer with 100 kHz field modulation, 1 Gauss modulation amplitude, 1.0 x 105 receiver gain, 100-200 Gauss sweep width, 5.12 ms time constant, 2 mW microwave power incident on cavity, 9.725 GHz microwave frequency, and a time of 3.5-8 minutes per spectrum (10-25 scans). A home-built frequency meter was used to measure microwave frequency, and DPPH (g = 2.0036 + 0.003) was used as a frequency reference standard. Chromium(V) concentration was determined by comparison with K3CrO8 standards of known concentrations (0.3-300µM; 5-fold lower dilutions result barely observable chromium(V) signals under the conditions employed in these studies).

All chromium(VI) and thiol solutions were freshly prepared in 0.050 M Tris-HCl buffer (treated with AG 50W-X8 ion exchange resin (4 g/L) for several hours and filtered before use), pH 7.0 at 37 °C. The 2 mL standard reaction mixture consisted of the following: 0.24 mM potassium dichromate (0.48 mM Cr(VI)), thiol, and calf thymus DNA where indicated. The thiols used in this study were b-mercaptoethanol, dithiothreitol, glutathione, or cysteine. The ratio of thiol to chromium was varied: 0,3,5,10 or 20 thiol per chromium (0-9.6 mM). The ratio of DNA-P to chromium (in those reactions involving DNA) was varied from 0 to 1 DNA-P per chromium (0-0.48 mM); for most reactions studied the ratio of Cr(VI):DNA:thiol was 1:0.1:20. All reactions involving DNA other than CT DNA, i.e., 5'-dGMP, 5'-dGTP, poly(dA), poly(dC), or poly(dG), were at a Cr(VI):DNA-P ratio of 1:0.1. All reactions were initiated by the addition of thiol. The pH of all stock solutions was 7.0 prior to start of reactions, and was not adjusted during the course of the reactions. Aliquots of reactions were taken at various times, and EPR spectra were obtained as described above.

A bis-cysteinate chromium(III) complex was synthesized by the method of De Meester et al. (56), with minor modifications. Briefly, an aqueous solution (20.0 mL) of chromium(III) nitrate nonahydrate (0.166 M) and L-cysteine (0.5 M) was boiled for 5 minutes, and NaOH was added until the solution was blue (pH ~ 7). The hot solution was filtered and the filtrate was allowed to stand at room temperature for 1 day. As no crystals had formed, the solution was filtered again. The chromium concentration of the filtrate was determined by its absorbance at 372 nm (e = 4810 M-1 cm-1), after an aliquot was made alkaline with KOH and Cr(III) was oxidized to Cr(VI) with H2O2. The nature of the complex was confirmed by the electronic spectrum of the solution which had an absorbance band at 410 nm, e = 80.9 M-1 cm-1 that compared favorably to that reported for the bis-cysteinate chromium(III) (literature, e 410 = 81.7 M-1 cm-1 (57)).

All chromium(VI) and b-mercaptoethanol solutions were freshly prepared in 0.050 M Tris-HCl buffer (treated with AG 50W-X8 ion exchange resin (4 g/L) for several hours and filtered before use), pH 7.0 at 37 °C. The 2 mL standard reaction mixture consisted of the following: 0.24 mM potassium dichromate (0.48 mM Cr(VI)), b-mercaptoethanol (9.6 mM; 1:20 Cr:thiol ratio), calf thymus DNA (0.048 mM DNA-P; 10:1 Cr:DNA ratio), DMPO (100 mM), and either DES (0.48 mM), or DETAPAC (0.48 mM). All reactions were initiated by the addition of b-mercaptoethanol. The pH of all stock solutions was 7.0 prior to start of reactions, and was not adjusted during the course of the reactions. Aliquots of reactions were taken at various times, and EPR spectra were obtained as described above.

The DMPO used in these studies was purified by the method of Buettner et al. (58), with slight modifications. Briefly, 1 g DMPO in 10 mL of nanopure water was filtered though ca. 1 g of activated carbon, divided into 500 µL aliquots, and stored under argon at -20 oC in glass vials wrapped with foil. A solution of DMPO (100.0 mM, in 50.0 mM Tris-HCl, pH 7.0) alone gave no detectable EPR signal, indicating the absence of any radical species in stock DMPO solutions.

All chromium(VI) and thiol solutions were freshly prepared in 100.0 mM Tris-HCl buffer (treated with AG 50W-X8 ion exchange resin (4 g/L) for several hours and filtered before use), pH 7.0 at 25 °C. In the case of cysteine or cysteamine, the reactions were also performed at -15 °C by immersing reaction vessels in a CO2(s)/ethylene glycol bath. The 2 mL standard reaction mixture consisted of the following: potassium dichromate (1.8-100.0 mM Cr(VI)), DMPO (100.0 mM), and thiol or ascorbic acid (5.45-300.0 mM). The thiols used in this study were: cysteine, cysteamine, ethanethiol, propanethiol, dimercaptosuccinic acid (DMSA), dithiothreitol, and glutathione. The ratio of chromium(VI):thiol varied depending on the particular thiol used. All reactions were initiated by addition of thiol or ascorbate. Some reactions were carried out using cacodylic acid (100.0 mM) buffered solutions, or in unbuffered nanopure H2O. Aliquots of reactions were taken at various times, and EPR spectra were obtained as described above.

Sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V), sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (5.0-50.0 mM), potassium perchromate (5.0 mM), or sodium dichromate was reacted with b-mercaptoethanol, dithiothreitol, glutathione, oxidized glutathione, glycine, thioglycolic acid, oxalic acid, or ascorbic acid in various solvents (H2O, acetone, methanol, dimethylformamide, 50% acetic acid) at either 25 °C or -15 °C. All non-aqueous solvents were dried over 4 Å molecular sieves prior to use. The concentrations listed are for the stock reagent solutions; chromium and reducing agent solutions were mixed in a 1:1 ratio to initiate the reactions.

b-mercaptoethanol (100.0 mM) was added to sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (100.0 mM) in aqueous solution at 25 °C, with the initial pH = 5.9. b-mercaptoethanol (5.0-160.0 mM) was added to sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (5.0-10.0 mM; 1,2,20:1 thiol:Cr ratios) in 50% acetic acid solution at -15 °C, with the initial pH < 2.0. The reactions were also repeated with the ionic strength maintained at 1.5 via KCl. b-mercaptoethanol (20.0-1000.0 mM) was added to sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (10.0 mM; 2,20,100:1 thiol:Cr ratio) in dry acetone at 25 °C. Aliquots of the reactions were taken at various times, and EPR (297 K) spectra were obtained as described above.

Dithiothreitol (30.0 mM) was added to potassium perchromate, K3CrO8, (10.0 mM) in 0.2 N KOH(aq), at 25 °C, with an initial pH > 13.0. Dithiothreitol (20.0 or 200.0 mM) was added to sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (10.0 mM) in 50% acetic acid solution at 25 °C, with the initial pH < 2.0. Dithiothreitol (200.0 mM) was added to sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) (10.0 mM) in dry acetone at 25 °C. Dithiothreitol (15.0-200.0 mM) was added to sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (10.0 mM) in dry acetonitrile, methanol, or N,N-dimethylformamide at 25 °C. Dithiothreitol (50.0 mM) was added to sodium dichromate (10.0 mM) in dry methanol at 25 °C. Aliquots of the reactions were taken at various times, and EPR (297 K) spectra were obtained as described above.

Sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V) was synthesized following the method of Krumpolc and Rocek (46). Na2Cr2O7 (1.30 g, 5.0 mmol), made anhydrous by drying in vacuo at 100 °C until constant weight, was added to a solution consisting of 3.60 g (30.0 mmol) 2-hydroxy-2-methylbutyric acid in dry acetone (40.0 mL). The solution was allowed to stir at room temperature for 24 hours. Addition of 100 mL of hexane resulted in the formation of a dark brown oil. The oil was washed twice with 5 mL hexane, and dried in vacuo at room temperature, resulting in 1.931 g of dark red-brown crystals (60.0 % crude yield). The crude product was recrystallized by dissolving the product in acetone and precipitating a dark oil by addition of hexane. Upon standing, the oil formed dark red-brown crystals which were washed with hexane and dried in vacuo. The resulting red-brown crystals (1.528 g; 47.6% yield) were stored desiccated at -20 °C. The electronic spectrum (H2O) of the product compared favorably to the literature (46), l (e(experimental), e(46)): 350 nm (1200.0 M-1cm-1,1208.0), 491 nm (145.0 M-1cm-1, 164.0), 510 nm (143.0 M-1cm-1, 170.0), 646 nm (33.0 M-1cm-1, 34.3), 733 nm (38.0 M-1cm-1, 41.8), and 800 (29.5 M-1cm-1, 35.0).

Sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) was synthesized following the method of Krumpolc and Rocek (46). Na2Cr2O7 (0.65 g, 2.5 mmol), made anhydrous by drying in vacuo at 100 °C until constant weight, was added to a solution consisting of 1.98 g (15.0 mmol) 2-ethyl-2-hydroxybutyric acid in dry acetone (20.0 mL). The solution was allowed to stir at room temperature for 24 hours. Addition of 50 mL of hexane resulted in the formation of violet crystals. The crystals were washed twice with 5 mL hexane, dried in vacuo at room temperature, and recrystallized from acetone-hexane to yield 1.33 g of dark red-violet crystals (72.6% yield). The crystals were stored desiccated at -20 °C. The electronic spectrum (H2O) of the product compared favorably to the literature (46), l (e(experimental), e(46)): 485 nm (139.0 M-1cm-1, 160.0), 510 nm (151.0 M-1cm-1, 168.0), 633 nm (25.9 M-1cm-1, 28.6), 740 nm (37.5 M-1cm-1, 40.9), and 800 nm (34.5 M-1cm-1, 39.4).