Ratiometric quantitation of redox status with a molecular Fe2 magnetic resonance probe

We demonstrate the ability of a molecular Fe2 complex to enable magnetic resonance-based ratiometric quantitation of redox status.


Table of Contents
Experimental Section S3 Table S1 | Crystallographic data for LFe2(etidronate)·7H2O S8 Figure S1 | CV of 1 in pH 7.4 buffer solution S9 Figure S2 | Variable temperature Mössbauer spectra of 2 S10 Figure S3 | Mössbauer spectrum of 1 at 80 K S11 Figure S4 | UV-vis-NIR spectra of 1 and 2 in neutral D2O S12 Figure S5 | Diffuse reflectance spectrum of 2 S13 Figure S6 | Variable temperature dc susceptibility data for 1 and 2 S14 Figure S7 | NMR spectra of 1 in neutral H2O and D2O S15 Figure S8 | NMR spectra of 2 in neutral H2O and D2O S16 Figure S9 | CEST spectra for 1 and 2 in pH 7.4 buffer S17 Figure S10 | Omega plot of 1 in pH 7.4 buffer S18 Figure S11 | Omega plot of 2 in pH 7.4 buffer S19 Figure  S3 Experimental Section General considerations. Unless otherwise specified, chemicals and solvents were purchased from commercial vendors and used without further purification. Deuterated solvents were purchased from Cambridge Isotope Laboratories. Bovine blood plasma was obtained from commercial sources (Sigma Aldrich lot P4639). When necessary for moisture sensitive experiments, glassware was flame dried or stored in an oven at 150 °C for at least 4 hours, followed by cooling in a desiccator. Air-and water-free manipulations were carried out using standard Schlenk techniques. Acetonitrile was dried using a commercial solvent purification system from Pure Process Technology and stored over 4 Å molecular sieves prior to use. Water was obtained from a purification system from EMD Millipore.  Figure S43). After stirring for 12 h, the ensuing red solution was dried under reduced pressure to give a red solid. The solid was stirred in DMF (10 mL) for 30 min, and was then collected by filtration and washed with THF (10 mL) and Et2O (10 mL). The residue was dried under reduced pressure for  Figure S43).

X-ray structure determination.
A single crystal of LFe2(etidronate)·7H2O was directly coated with Paratone-N oil and mounted on a MicroMounts TM rod. The crystallographic data were collected at 100 K on a Bruker APEX II diffractometer equipped with MoKα sealed tube source. Raw data were integrated and corrected for Lorentz and polarization effects using Bruker APEX2 v. 2009.1. 2 The program SADABS was used to apply absorption correction. 3 Space group assignments were determined by examining systematic absences, E-statistics and successive refinement of the structure. Structures were solved by SHELXT 4 using direct methods and refined by SHELXL within the OLEX interface. (3) Partially occupied solvent H2O molecules that were potentially hydrogen bonded were modeled isotropically. Thermal parameters for all other non-hydrogen were refined anisotropically. Crystallographic data and the details of data collection are listed in Table S1.

H NMR experiments.
Variable temperature 1 H NMR spectra were collected on an Agilent DD MR-400 system (9.4 T) system. The T1 of H2O was obtained by fitting H2O intensities from experiments with an array of relaxation times implemented in the program vnmr. Linewidth analyses were obtained in the program MNOVA.
CEST experiments. Variable temperature CEST experiments were performed on an Agilent DD MR-400 system (9.4 T) system. In a typical experiment, samples containing 100% 1, 100% 2 or mixture of the two at a desired ratio in buffer containing 100 mM NaCl and 100 mM of HEPES at pH desired were used for CEST experiments. Z-spectra (CEST spectra) were obtained according to the following protocol. NMR spectra were acquired using the presaturation pulse applied for 7 s at a power level of 24 μT. The saturation frequency offsets were screened with a step increase of 1 ppm. The obtained NMR spectra were plotted as normalized water intensity S5 against frequency offset to produce a Z-spectrum. Direct saturation of the water signal was set to 0 ppm. D2O was placed in an inner capillary to lock the sample. Exchange rate constants were calculated based off a reported method. 5 The B1 values are calculated based on the calibrated 90degree pulse on a linear amplifier. The NMR spectra were acquired at various presaturation powers ranging from 14 to 24 μT applied for 7 s. To correct for baseline, reported values of %CEST are the difference in percent H2O signal reduction between applied on-resonance and off-resonance pre-saturations.
Solid state magnetic measurements. Magnetic measurements were carried out using a Quantum Design MPMS-XL SQUID magnetometer. Powder samples were sealed in 2 mL polyethylene bags. Dc susceptibility data were collected from 1.8 to 300 K at applied dc fields of 1, 1.5 and 2 T. Dc susceptibility data were corrected for diamagnetic contribution from the sample holders and from the sample (estimated using Pascal's constants 6 ). The temperature dependent magnetic susceptibility data for 1 (10-300 K) and 2 (1.8-300 K) and were model using spin Hamiltonian Ĥ = −2J(ŜFe1·ŜFe2), 7 where J is the magnetic superexchange coupling constant; and ŜFe1 and ŜFe2 are the spin operators for the Fe ions. The best fits of the data give g = 2.20(3) and 2.00(4) for 1 and 2, respectively.

Solution magnetic measurements.
Magnetic moments of metal complexes were carried out using Evan's method 8 at 310 K. In a typical experiment, compounds (about 4 mM) were dissolved in a mixture of 0.5 w/w % of DMSO in D2O. A capillary containing same solvent mixture (without the to-be-characterized compound) was inserted into each NMR sample as reference. Diamagnetic correction was carried out based on the empirical formula of each compound (as determined by elemental analysis) using Pascal's constants. 6 Electrochemical measurements. Cyclic voltammetry was carried out in a standard onecompartment cell inside a nitrogen glove box at room temperature, equipped with a platinum working electrode, a platinum wire as counter electrode and a SCE reference electrode using a CHI 760c potentiostat. The analyte solution was with 100 mM NaCl and 100 mM HEPES buffered at pH 7.4. The voltammogram was converted and shown as values referred to the normal hydrogen electrode (NHE), using a literature conversion factor. 9 Open circuit potentials were measured by the built-in technique "open circuit potential -time" within the CHI660E electrochemical workstation software. The open circuit potential readings were recorded 10 minutes after the experiment started, at which time the reading was stabilized.
Mössbauer spectroscopy. Zero-field 57 Fe Mössbauer spectra were obtained at various temperatures with a constant acceleration spectrometer and a 57 Co/rhodium source. Prior to measurements, the spectrometer was calibrated at 295 K with α-iron foil. Samples were prepared in an MBraun nitrogen glove box. A typical sample contained approximately 60 mg of compounds (~10 mg of Fe) suspended in a plastic cap in heated eicosane, which solidified upon cooling to ambient temperature, in order to immobilize the sample. Another cap with a slightly smaller diameter was squeezed into the previous sample cap to completely encapsulate the solid sample mixture. All spectra were analyzed using the WMOSS Mössbauer Spectral Analysis Software (www.wmoss.org).
Other physical measurements. Infrared spectra were recorded on a Bruker Alpha FTIR spectrometer equipped with an attenuated total reflectance accessory. Solution and solid-state UV-vis-NIR spectra were obtained using an Agilent Cary 5000 spectrophotometer.
Estimation of electron transfer rate by IVCT analysis. 10 The calculation of ambient temperature electron-transfer rate in 2 is based on a method described in a similar mixed-valence Fe2 analog. Location of the IVCT max, extinction coefficient ()and Fe···Fe distance (d) were obtained experimentally as described in the main text. The full width at half maximum (1/2) was determined by fitting IVCT to a Gaussian model in the software OriginPro. The electrontransfer rate (ket) in 2 can be calculated using the following equation: where R is the ideal gas constant and T is temperature. The frequency factor for electron transfer, et, and the thermal free energy, G*, are given by: et = 2π 3/2 abh -1 (kTmax) -1/2 and G* = max(4 -ab) -1 where h is the Planck constant, k is the Boltzmann constant and max is the wavenumber of the IVCT peak maximum. The resonance matrix element, ab, is given by: ab = max, where the extent of electron delocalization  2 = 4.2 × 10 -4 1/2(maxd 2 ) -1 . Here, is the extinction coefficient of IVCT, and d is the Fe···Fe distance determined by X-ray structural analysis.

Viability experiment. Melanoma B16F10 cells (ATCC) were cultured in Dulbecco's Modified
Eagle's Media (Life Technologies) supplemented with 10% fetal bovine serum (Fisher), 1 mM each of sodium pyruvate, non-essential amino acids and L-glycine at 37 °C and 5% CO2. Cells for the experiment were subcultivated for 3 to 4 times after thawing the cell stocks. Cells were incubated with media containing the desired concentration of 3 for 24 h before viability measurements. Cell viability was measured by a Guava EasyCyte Mini Personal Cell Analyzer (EMD Millipore). Each sample subjected for analysis contained 50 L of well-mixed cell suspension and 150 L of Guava ViaCount reagent. Stained samples were vortexed for 20 s and immediately subjected to counting using the ViaCount software module. Viability was measured using the EasyFit software module. Cells not treated by 3 were used as a control to account for normal cell death. Reported %viability was normalized with respect to the control samples.

MRI phantom experiment.
Samples contained 100 mM of NaCl, 100 mM of HEPES buffered at pH 7.4 and overall 10 mM Fe2 concentration with 1:2 ratio ranging from 9:1 to 1:9. ~0.5 mL of each sample was stored in a 0.5 mL Eppendorf tube, which was placed within another scintillation vial filled with H2O solution containing 1 mg/mL CuSO4 and 100 mM NaCl for T1 matching. CEST experiments were carried out on a Bruker Biospec 9.4 T MRI scanner running ParaVision 6.0.1 (Bruker Biospin, Billerica, MA, USA). Temperature was maintained at 37 °C using a warm water circulating system with feedback control from a temperature probe (SA Instruments, Stonybrook, NY, USA). CEST images were acquired using an accelerated spin echo based sequence with a pre-saturation pulse (14 T, 2 s duration) applied at offsets of 83 and 40 ppm (Mz). Other imaging parameters: TR/TE = 2034/14.9 ms, RARE factor 16, matrix = 64 × 64, FOV = 3.2 × 3.2 cm, 2 mm slice thickness, and 2 averages). Matched unsaturated images were acquired using identical parameters except that the pulse amplitude was set to 0 µT (M0). %CEST = (1 -Mz/M0) × 100%. Only regions of the inner Eppendorf tube, where the sample containing the Fe2 probe is shown in Figure 5. Averaged intensities of the same regions were used to calculate CEST83 ppm/CEST40 ppm for fitting. For the fitting, sample A was a significant outlier likely due to weak CEST signal, and therefore was not taken into account for fitting. Trace amounts of precipitation occurred for sample D, E, F during the phantom experiment, likely due to the affected solubility of 1 and/or 2 in the presence of high buffer concentration. Such precipitate did not alter either the phantom experiment or OCP measurement.              Figure S24. CEST spectra for 3.8 mM aqueous solutions of 1 and 2, with ratios of 1:2 from 9:1 (blue) to 1:9 (red) at 35 °C. Each solution contains 100 mM NaCl and 100 mM HEPES buffered at pH 7.5. The legend gives the independently obtained OCP of each sample (mV vs NHE). Inset: Expanded view of the relevant CEST peaks. Figure S25. Open circuit potentials for solutions, containing 100 mM HEPES, 100 mM NaCl and 3.8 mM Fe2 buffered at pH 7.5 at 35 °C, is plotted against both the ratio of CEST effects from application of presaturation at 83 and 40 ppm and the natural log of the ratio (inset). Black circles and the red line represent the experimental data and the fit, respectively. Figure S26. CEST spectra for 3.8 mM aqueous solutions of 1 and 2, with ratios of 1:2 from 9:1 (blue) to 1:9 (red) at 39 °C. Each solution contains 100 mM NaCl and 100 mM HEPES buffered at pH 7.5. The legend gives the independently obtained OCP of each sample (mV vs NHE). Inset: Expanded view of the relevant CEST peaks. Figure S27. Open circuit potentials for solutions, containing 100 mM HEPES, 100 mM NaCl and 3.8 mM Fe2 buffered at pH 7.5 at 39 °C, is plotted against both the ratio of CEST effects from application of presaturation at 83 and 40 ppm and the natural log of the ratio (inset). Black circles and the red line represent the experimental data and the fit, respectively. Figure S28. Comparison of Nernstian fits (from Figures S22, S25 and S27) obtained from data at various temperatures. Figure S29. NMR spectra of 4 mM of 1 in pH 7.4 buffer with (top) and without (bottom) presences of 4 mM of each NaOAc, Na2CO3, NaH2PO4 and Na2SO4. Figure S30. NMR spectra of 4 mM of 1 in D2O with (top) and without (bottom) presences of 4 mM of each NaOAc, Na2CO3, NaH2PO4 and Na2SO4. Figure S31. NMR spectra of 4 mM of 2 in pH 7.4 buffer with (top) and without (bottom) presences of 4 mM of each NaOAc, Na2CO3, NaH2PO4 and Na2SO4. Figure S32. NMR spectra of 4 mM of 2 in D2O with (top) and without (bottom) presences of 4 mM of each NaOAc, Na2CO3, NaH2PO4 and Na2SO4.       Figure S42. Open circuit potentials for solutions for phantom experiments, containing 100 mM of HEPES, 100 mM of NaCl and 10 mM of Fe2 buffered at pH 7.4, is plotted against both the ratio of CEST effects at 37 °C from the averaged phantom image intensity with presaturation at 83 and 40 ppm and the natural log of the ratio (inset). Black circles and the red line represent the experimental data and the fit (equation displayed), respectively. Refer to Experimental Section for fitting details.