Dual-Function pH and Oxygen Phosphonated Trityl Probe - Analytical

Jun 15, 2012 - Andrey A. Bobko, Ilirian Dhimitruka, Denis A. Komarov, and Valery V. .... Vladimir G. Vasiliev , Leonid A. Shundrin , Howard J. Halpern...
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Dual-Function pH and Oxygen Phosphonated Trityl Probe Andrey A. Bobko, Ilirian Dhimitruka, Denis A. Komarov, and Valery V. Khramtsov* Dorothy M. Davis Heart & Lung Research Institute and Division of Pulmonary, Allergy, Critical Care & Sleep Medicine, Department of Internal Medicine, The Ohio State University, Columbus, Ohio 43210, United States S Supporting Information *

ABSTRACT: Triarylmethyl radicals (TAMs) are used as persistent paramagnetic probes for electron paramagnetic resonance (EPR) spectroscopic and imaging applications and as hyperpolarizing and contrast agents for magnetic resonance imaging (MRI) and proton−electron double-resonance imaging (PEDRI). Recently we proposed the concept of dual-function pH and oxygen TAM probes based on the incorporation of ionizable groups into the TAM structure (J. Am. Chem. Soc. 2007, 129, 7240−7241). In this paper we report the synthesis of a deuterated derivative of phosphonated trityl radical, pTAM. The presence of phosphono substitutes in the structure of TAM provides pH sensitivity of its EPR spectrum in the physiological range from 6 to 8, the phosphorus hyperfine splitting acting as a convenient and highly sensitive pH marker (spectral sensitivity, 3ΔaP/ΔpH ≈ 0.5 G/pH unit; accuracy of pH measurements, ±0.05). In addition, substitution of 36 methyl protons with deuterons significantly decreased the individual line width of pTAM down to 40 mG and, as consequence, provided high sensitivity of the line-width broadening to pO2 (ΔH/ΔpO2 ≈ 0.4 mG/mmHg; accuracy of pO2 measurements, ≈1 mmHg). The independent character of pH and [O2] effects on the EPR spectra of pTAM provides dual functionality to this probe, allowing extraction of both parameters from a single EPR spectrum.

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derivatives1,4,6 display a very narrow single line of about 100 mG or less, which makes them attractive for continuous wave (CW) electron paramagnetic resonance imaging (EPRI) applications.7 The long relaxation time of TAMs8 provides an advantage over nitroxides for pulsed EPRI,9,10 proton−electron double-resonance imaging (PEDRI),1,11,12 and hyperpolarized nuclear magnetic resonance (NMR)13 and magnetic resonance imaging (MRI).14 Applications of TAM radicals as functional probes include EPR oximetry1,11,12 and recently reported sensitivity to the superoxide anion15,16 and pH.17−19 The oxygen-dependent broadening of the TAM line of about 500 mG/mM oxygen1 provides the basis for its oximetric applications. Importantly, the concentration broadening of the TAMs is only about 10−30 mG/mM,1 which is 1 order of magnitude less than that for the nitroxides.20 These properties make TAM radicals superior oximetric probes for in vivo EPR/ EPRI and PEDRI applications.1,11 The applications of other

itroxides and triarylmethyl radicals, TAMs, represent two main classes of soluble paramagnetic probes used for electron paramagnetic resonance (EPR) spectroscopy and imaging applications. TAMs have advantages over nitroxides in extraordinary stability toward tissue redox processes, longer relaxation time, and narrower line width, making them particularly attractive for imaging applications.1 In spite of the fact that the synthesis of triphenylmethyl radical was reported by Gomberg more than a century ago,2 TAM structure with sterically protected trivalent carbon only recently regained attention as the basic fragment for synthesis of stable organic radicals after innovative development by Nycomed.1,3 The placement of heteroatoms in TAM structure eliminated the numerous hyperfine splittings and broadenings of the EPR signal caused by coupling of the unpaired electron with the hydrogen nuclear spins. Scheme 1 shows the most popular TAM derivatives developed by Nycomed that contain carboxyl groups, namely, cTAM and its more hydrophilic derivative, Oxo63. General synthesis of TAMs, including large-scale production of cTAM and its deuterated derivatives, was recently described.4−6 The EPR spectra of these TAM © 2012 American Chemical Society

Received: April 2, 2012 Accepted: June 15, 2012 Published: June 15, 2012 6054

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mG, in the physiological pH range from 6.8 to 9.0.18 Note that changes in oxygen concentration do not influence spectral shifts but result in line broadening (≈6 mG/% [O2]). The independent character of pH and [O2] effects on the EPR spectra of aTAM1 provides dual functionality to this probe, allowing for an extraction of both parameters from a single EPR spectrum.18 However, a complex spectral pattern of aTAM1 decreases its value for oxygen and pH mapping. Further simplification of spectral properties of dual-function TAM probes is desired, ideally providing pH-sensitive doublet hyperfine splitting. The development of TAM derivatives based on more hydrophilic structures is also important for elaboration of nontoxic dual function probes. Recently Driesschaert et al.19 reported the synthesis of a very promising phosphonated TAM probe, phTAM (Scheme 1). This probe combines excellent aqueous solubility and stability with spectral pH sensitivity in the physiological pH range. The presence of three phosphorus atoms with a nuclear spin of 1/2 should split the EPR signal into a simple quartet pattern, allowing the assessment of pH by measuring the value of aP hyperfine splitting. In practice, the three phosphonic acids are ionized consecutively, which results in superposition of EPR spectra of four ionic species with different aP values and gfactors. Due to partial overlap of these four forms present together at physiological pH, the authors reported only averaged pKa of phosphonated groups of phTAM being equal to 7.1. This partial overlap also complicates the extraction of individual line shapes and analysis of oxygen-induced line broadening; therefore oxygen sensitivity of phTAM spectra was not reported. In our opinion, for phosphonated TAMs to achieve dual functionality, further derivatization to simplify the EPR spectral pattern is required. As a first step in this direction, in this work we synthesized a deuterated derivative of the phosphonated TAM, pTAM (Scheme 1). Significant narrowing of individual spectral lines of pTAM compared with phTAM allowed us to resolve spectral patterns of all four ionic species, as well as analyzing oxygen-induced line broadening. The independent character of pH and [O2] effects on the EPR spectra of pTAM provides dual functionality to this probe, allowing for concurrent monitoring of both parameters.

Scheme 1. Chemical Structures of cTAM, Oxo63, cTAM1, aTAM1, phTAM, and pTAMa

a

cTAM, Oxo63, and cTAM1 contain carboxyl groups; aTAM1 contains an amino group; and phTAM and pTAM contain phosphono groups.

oxygen-sensitive paramagnetic materials include particulate probes such as lithium phthalocyanine particles21,22 and carbonaceous materials (chars, coals, carbon blacks).23 It should be noted that particulate probes measure oxygen partial pressure in the place of probe implantation, whereas soluble TAM probes are more suitable for imaging applications.22,24 We proposed a concept of dual-function pH and oxygen probes17 based on TAM structures containing ionizable groups and reported the first observation of pH effect on EPR spectra of TAM derivatives containing carboxyl17 and amino18 groups. The pH-dependent shift of a single EPR line of cTAM and Oxo63 was observed in acidic medium, reflecting changes in gfactor value upon protonation of carboxyl groups.17 Low pKa value of the carboxyl group in TAM derivatives limits their application to the pH range from 2 to 4, which still could be useful, for example, for studies of stomach acidity.25 Another limitation of TAM pH probes with a single EPR line is a linear proportionality of the value of pH-dependent line shift to the EPR frequency, which makes this shift impractically small at low magnetic fields used for in vivo applications (about 30 mG at L-band). Note that nitroxide pH probes are successfully used for in vivo pH mapping at low-field EPR due to a frequencyindependent pH effect on the hyperfine splitting, aN.26,27 Accordingly, cTAM derivatives with hydrogen atom attached to the aryl groups (e.g., cTAM1, Scheme 1) demonstrated reversible changes in hydrogen hyperfine splitting, aH, in acidic medium owing to reversible protonation of carboxyl groups.17 In turn, TAMs containing amino groups18 (e.g., aTAM1, Scheme 1) demonstrated strong pH-induced changes of the hyperfine splittings from nitrogen and hydrogen atoms in direct proximity to protonatable amino groups, Δhfs ≈ 300−1000

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MATERIALS AND METHODS Synthesis. The synthesis of pTAM was based on the recently published procedure for the nondeuterated analogue phTAM.19 Compound 1 (Scheme 2) was synthesized according to ref 5. Tris{(2,2,6,6-Tetramethylbenzo[1,2-d;4,5-d′]bis[1,3]dithiol-4-yl)phosphonic acid diethyl ester}methanol (2). Compound 1 (260 mg, 0.29 mmol) was dissolved in 5 mL of benzene. N,N,N′,N′-Tetramethylethylenediamine (348 mg, 0.45 mL, 3 mmol) was added, followed by butyllithium 2.5 M solution in hexanes (1.2 mL, 3 mmol). The solution was stirred for 30 min at room temperature and then was added slowly via syringe to a solution of diethyl chlorophosphate (517 mg, 0.43 mL, 3 mmol) in 3 mL of benzene placed on an acetone−dry ice bath, and the mixture was stirred over a weekend. The reaction was quenched with aqueous ammonium chloride solution and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and evaporated. The crude residue was purified by flash chromatography on silica gel eluted with hexanes/ethyl acetate 3:1. Product 2 (100 mg) , mixed with smaller amounts of other compounds, was obtained. 1H NMR 6055

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40% yield was obtained. MS (ESI, (M)+] 1143.0939 (obsd), 1143.0950 (calcd). Electron Paramagnetic Resonance Characterization of pTAM. EPR measurements were performed on an X-band EMX EPR spectrometer (Bruker, Germany). Temperature and gas composition during EPR measurements were controlled by a temperature and gas controller (Noxygen, Germany). The spectra of pTAM radical were recorded in solutions of various buffer compositions, oxygen partial pressures, pH values, and temperatures. To control gas composition, Teflon tubes with a diameter of 1.14 mm and wall thickness of 60 μm (Zeus, Inc.) were used throughout experiments. To optimize spectral intensity and minimize power saturation effects on line width, the EPR spectra were measured at 0.13 mW microwave power, which did not result in significant broadening (less than 10%). Titration of Solutions. A solution of radical (4 mL, 200 μM) was titrated by addition of a small volume of NaOH or HCl, with the final dilution of sample less than 1%. pH was controlled by an electrode calibrated at 22 or 37 °C by use of pH values for reference solution recommended by the U.S. National Bureau of Standards. Temperature of reference and titrated solutions during pH measurements was controlled by use of a jacketed reaction beaker attached to a Lauda circulator E100. Calculation of pKa Values. Analysis of EPR Spectra Measured at pH > 4 (Slow Proton Exchange). EPR spectra measured in 1.125 mM sodium phosphate buffer solutions at pH > 4 are represented by superposition of the EPR spectra of four ionization states of pTAM (pTAM3−, pTAM4−, pTAM5−, and pTAM6−; see Scheme 3) characteristic for slow proton exchange between the states. The spectra were simulated by use of P.E.S.T. WINSIM software (NIEHS), which yielded the fraction, f, of each ionization state of pTAM. The obtained data were fitted by standard titration equations, yielding corresponding values of pK21,2,3 (see Supporting Information). Analysis of EPR Spectra Measured at pH < 4 (Fast Proton Exchange). The EPR spectra of pTAM in acidic conditions are represented by simple quartet spectral pattern because of fast proton exchange between ionization states of pTAM. The distance between low- and high-field components (3 × aP) of

Scheme 2. Synthesis of pTAM Trityl Radical

(500 MHz, CDCl3) δ = 6.5 (s, 1 H), 4.2 (m, 12 H), 1.3 (m, 18H). Tris{(2,2,6,6-Tetramethylbenzo[1,2-d;4,5-d′]bis[1,3]dithiol-4-yl)phosphonic acid}methane Radical (pTAM). Compound 2 (25 mg, 0.018 mmol) was dissolved in 5 mL of methylene chloride, and bromotrimethylsilane (0.1 mL, 0.116 g, 0.75 mmol) was added. The solution was stirred at room temperature overnight. Methanol (4 mL) was added, and the resulting solution was refluxed for 4 h. The reaction was evaporated until dry, dissolved in 6 mL of methanol, and passed through a Hypersep C18 cartridge. The compound was collected and eluted with 2 × 5 mL of methanol. The methanol was evaporated, giving an impure solid that contains the desired radical pTAM. The product was dissolved in diluted aqueous NaOH and eluted with water through a Hypersep C18 cartridge. A green solution of the product was eluted first, followed by impurities. The green solution was acidified with concentrated hydrochloric acid (final concentration 1 M) and eluted with 50% methanol/water mixture through a Hypersep C18 cartridge for further purification. The eluent was collected, and water was evaporated with methanol. Overall 10 mg of pure product in Scheme 3. Acid−Base Equilibrium for pTAM Trityl Radicala

K1i and K2i denote the first and second ionization constants for each (i = 1, 2, 3) phosphonic acid residue of the pTAM molecule. The dotted arrows reflect the situation of fast proton exchange between two experimentally distinguishable ionization states. K1* and K2* denote the corresponding first and second cumulative ionization constants for phosphono groups of pTAM molecule (see Supporting Information for details of pKa value measurements). a

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us to distinguish the EPR spectra of all these forms, with the spectral parameters summarized in Table 1.

the EPR spectrum at each pH value was measured, and the hyperfine splitting constant (aP) was calculated. The obtained ap data were fitted by standard titration equations, yielding the corresponding value of pK1 (see Supporting Information). Analysis of Oxygen-Induced EPR Spectral Line Broadening. Line-shape analysis of the EPR spectra was performed in order to calculate oxygen-induced line broadening as previously described.28 At each oxygen concentration, the experimental spectra were fitted by rational approximation of the Voigt function given by Hui et al.29 The nonlinear leastsquares fitting were performed by Levenberg−Marquardt algorithm by use of a home-designed MATLAB code. The obtained dependence of the Lorentz component of EPR spectrum line width on oxygen concentration can be used as a calibration curve in further oximetric measurements.

Table 1. EPR Spectral Parameters for Different Ionization States of pTAM ionization state

aP1 (PO32−), ±0.01 G

6−

a

pTAM pTAM5− pTAM4− pTAM3− pTAM0 a

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3.40 (3) 3.43 (2)a 3.45 (1)a na na

aP2 (HPO31−), ±0.01 G

aP3 (H2PO3), ±0.01 G

ΔHpp, mG

Δg, mG

na 3.58 (1)a 3.63 (2)a 3.66 (3)a na

na na na na 3.80 (3)a

35 38 42 47 38

± ± ± ± ±

0 15 ± 2 42 ± 2 74 ± 2 nd

2 2 2 2 2

Numbers in parentheses denote the number of phosphono groups.

RESULTS Synthesis of the deuterated pTAM derivative was achieved in a two-step sequence (Scheme 2) from the starting material, deuterated trityl alcohol 1, synthesized as we recently described.5 Figure 1 shows the pH-dependent variation of

Figure 2A shows the pH dependence of the fraction of ionized forms of pTAM, calculated from the corresponding

Figure 1. EPR spectra of 200 μM pTAM solution in 1.125 mM sodium phosphate buffer with 150 mM NaCl, measured at various pH values under nitrogen atmosphere and temperature 37 °C. Spectral parameters were as follows: microwave power, 0.13 mW; time constant, 20.48 ms; conversion time, 10 ms; sweep time, 81.92 s; modulation amplitude, 0.02 G; modulation frequency, 10 kHz; sweep width, 14 G; number of points, 8192. Left panel represents enlarged spectra of low-field spectral components.

Figure 2. (A) Dependence of the fraction of pTAM ionization forms pTAM3− (○), pTAM4− (●), pTAM5− (□), and pTAM6− (■) on pH value calculated from the EPR spectra (see Figure 1). Solid lines represent the best fits from eqs 1−4 (see Supporting Information), yielding pK21 = 6.42 ± 0.05, pK22 = 6.89 ± 0.05, and pK23 = 7.73 ± 0.05. (B) Dependence of hyperfine splitting, 3 × aP, of pTAM on pH value calculated from the EPR spectra (see Figure 1). Sample composition and acquisition parameters are the same as in a Figure 1 caption. Solid line represents the best fit using eq 5 (see Supporting Information), yielding pK1* = 1.32 ± 0.05.

EPR spectra of pTAM measured in 1.125 mM phosphate buffer. A simple quartet spectral pattern (1:3:3:1), arising from three equivalent phosphorus hyperfine splittings (S = 1/2), was observed for symmetrical pTAM ionization states, namely, for completely deprotonated pTAM6− (pH 10.2), completely protonated pTAM0 (pH 0.0), and pTAM3− (pH 4.1) with all three phosphonic acids being only partially deprotonated (see Scheme 3 for ionization states of pTAM). At neutral pH, four ionization states, pTAM6−, pTAM5−, pTAM4−, and pTAM3−, that correspond to consecutive first protonations of three phosphono groups were simultaneously present in solution. The corresponding EPR spectra show superposition of all four signals (see Figure 1, pH 6.9), in agreement with slow proton exchange between these forms on the EPR time scale.30 The narrow lines of the deuterated pTAM (ΔHpp ≈ 40 mG) allow

EPR spectra. The experimental data are described by standard titration curves, with the ionization constants for first protonations of three phosphono groups being equal to 7.73, 6.89, and 6.42. For the second protonation of phosphono groups of pTAM in strong acidic conditions, we did not observe superposition of the EPR spectra of different ionization states, pTAM3−, pTAM2−, pTAM1−, and pTAM0, but only a smooth increase of aP splitting from 3.66 G (Figure 1, pH 4.1) to 3.80 G (Figure 1, pH 0.0) in according to standard titration curve with a single pK1* being equal to 1.32 (see Figure 2B). This is in agreement with fast proton exchange between corresponding ionization states on the EPR time scale due to high proton concentration in acidic solutions. The condition of 6057

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fast proton exchange on the EPR time scale requires that inverse protonation time (1/τ) was larger than the difference in the EPR frequencies (Δω) between the forms participating in exchange, or 1/τ > Δω.30 In case of exchange between pTAM3− and pTAM0, the difference in the peak positions in frequency units, Δω ≈ 5 × 106 s−1 (Table 1), and 1/τ ≈ kd × [H+] ≈ 1010 M−1·s−1 × 10−2 M ≈ 108 s−1 at pH ≈ 2 (assuming kd ≈ 1010 M−1·s−1 for diffusion-controlled protonation31), which is in agreement with the fast exchange requirement. The pKa values of acid−base equilibria between different ionization states of pTAM were found to vary with ionic strength and temperature (see Supporting Information); therefore, calibration of the probe at specific experimental conditions is required. The fast proton exchange between four ionization states of pTAM and corresponding EPR lines coalescence at pH close to pK1* is a consequence of high proton concentration in acidic solution. A similar effect can be achieved at neutral pH close to pK2* by increasing concentration of the proton-donating molecules that participate in proton exchange. In the case of proton transfer from H2PO4− buffer molecule to nonprotonated phosphono group of pTAM, the line coalescence is observed at buffer concentration about 20 mM (see Supporting Information). For the most biologically relevant applications, localization of membrane-impermeable pTAM probe is extracellular where concentration of phosphate buffer is about 1 mM; therefore slow exchange between ionization states of pTAM is expected. Figure 3A demonstrates the effect of oxygen-induced line broadening measured at pH 7.2. The narrow individual line width allow for accurate line-shape analysis based on rational approximation of Voigt function (see Materials and Methods) and extraction of Lorentz component of EPR spectrum line width. Figure 3B shows the corresponding dependence of Lorenz line width on oxygen concentration, which can be used as calibration curves in further oximetric measurements. The independent character of the two effects, pH on phosphorus hyperfine splitting and [O2] on line broadening, provides dual functionality to the pTAM probe, allowing extraction of both parameters from a single EPR spectrum.

Figure 3. (A) Low-field component of EPR spectra of 200 μM pTAM solution in 1.125 mM sodium phosphate buffer, pH 7.2, measured at various oxygen partial pressures at 37 °C. Spectral parameters were as follows: microwave power, 0.13 mW; time constant, 167.84 ms; conversion time, 20 ms; sweep time, 40.96 s; modulation amplitude, 0.02 G; modulation frequency, 10 kHz; sweep width, 1.5 G; number of points, 2048. (B) Dependence of Lorentz line width of EPR spectra of pTAM on oxygen partial pressure. Data were calculated for the most intense spectral component, pTAM5−, of the EPR spectra shown in panel A. Solid line is the linear fit with a slope of 0.42 ± 0.04 mG/ mmHg (or about 300 mG/mM).

and ischemia, and the dependence of chemical shifts on ionic strength.36 EPR oximetry is one of the most promising and rapidly developing techniques for measurement of oxygen in living tissues. 24 EPR oximetry is based on a pure physical phenomenon, Heisenberg exchange between the diradical molecule of oxygen and a radical probe. As a consequence, EPR oximetry does not interfere with oxygen metabolism, therefore providing a basis for noninvasive oxygen measurements in biological systems, including those in vivo. Spectral− spatial EPRI allows for extraction of an oxygen concentration map and spin probe distribution. To date, to obtain the highest sensitivity and best quality images, minutes to hours of time are required with common CW EPRI. Due to technical restrictions, pulsed EPR approaches are limited to paramagnetic probes with long relaxation times.37 An alternative imaging modality that employs unpaired electrons is proton−electron doubleresonance imaging (PEDRI), which also strongly relies on probes with long relaxation times.12 Up to now, in vivo EPR measurements of pH exclusively rely on application of pHsensitive nitroxides.26−28,38,39 Recently we reported tumor pH monitoring using L-band EPR and tumor pH mapping using PEDRI in living mice based on specially designed nitroxide probe.27 In other L-band EPR applications, we used an oxygensensitive trityl probe to monitor ischemia-induced myocardial oxygen depletion in isolated perfused rat hearts, while in separate experiments we measured related myocardial acidosis using a nitroxide pH probe.28 The development of a dual-

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DISCUSSION Tissue pH and oxygen concentration are among the most important parameters in physiology and pathophysiology of living organisms. In certain stress conditions, for example, high exercise levels, interruption of normal blood supply, or biochemical shock, the oxygen supply and the body’s ability to regulate pH, at least locally, may be compromised. A reasonable depth of penetration of microwaves in living tissues makes NMR and low-field EPR-based techniques the most appropriate approaches for noninvasive in vivo oxygen and pH assessment. After the invention of MRI, oxygen was the primary candidate to be considered as potential contrast agent, but this possibility was ruled out due to the small effect of oxygen on the relaxation times of water protons. Several NMR oximetric approaches were developed that utilize blood oxygen leveldependent effects32 or use exogenous probes such as perfluorocarbon emulsions33,34 and fluorinated nitroimidazoles.35 However, these methods still suffer from low intrinsic NMR sensitivity and difficulties with quantitative data interpretation. For in vivo pH measurements, 31P NMR using endogenous inorganic phosphate has proven to be the most suitable noninvasive approach but still suffers from lack of resolution, the fact that Pi concentrations vary with metabolism 6058

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function pH and oxygen probe will advance these applications, allowing for simultaneous pH and oxygen monitoring and dualfunction tissue mapping. This is of particular importance when one takes into account the complex relationship between tissue hypoxia and acidosis and the high heterogeneity of these parameters in various pathological states, for example, in tumors. A concept of dual-function pH and oxygen probes based on TAM structures containing ionizable groups was proposed several years ago,17 and observations of pH effects on EPR spectra of TAM derivatives containing carboxyl,17 amino,18 and phosphono19 groups were reported. The phosphonated trityl probe phTAM (Scheme 1), synthesized by Driesschaert et al.,19 combines excellent aqueous solubility and stability with spectral pH sensitivity in the physiological pH range. Oxygen sensitivity of phTAM spectra was not reported, probably due to complex spectral analysis required. In this work, we synthesized a deuterated derivative of the phosphonated TAM, pTAM (Scheme 1). Significant narrowing of individual spectral lines of pTAM compared with phTAM allowed us to avoid spectral line overlaps and accurately analyze both pH-dependent shifts of the hyperfine splittings and oxygen-dependent line broadening. The phosphorus hyperfine splitting and line width broadening were found to be sensitive pH and pO2 markers (3ΔaP/ΔpH ≈ 0.5 G/pH unit, accuracy of pH measurements, ±0.05; ΔHL/ΔpO2 ≈ 0.4 mG/mmHg, accuracy of pO2 measurements, ≈1 mmHg). The obtained data demonstrate the potential of the pTAM probe for dual-function applications in biological systems. Note that, for the most biologically relevant applications (neutral pH and phosphate buffer concentration of about 1 mM for extracellular medium), slow exchange between ionization states of pTAM is expected. The observed effects of high proton (pH < 4, Figure 1) or phosphate buffer (≥10 mM; Figure S1, Supporting Information) concentrations on the EPR line shape of the pTAM probe is explained by increased rates of proton exchange between different ionization states of pTAM, resulting in frequency exchange between corresponding EPR lines. The manifestation of exchange phenomena was first theoretically described in NMR40 and explored for measurements of the rates of proton exchange reactions.41,42 Similar studies of proton exchange in EPR spectra include short-lived radicals43−45 as well as stable nitroxyl radicals.30,46,47 The proton exchange/transfer reactions are among the fastest in solution, and methods allowing direct measurements of the rate constants are valuable.31 The EPR line-shape sensitivity to the rate of proton exchange might be useful for studying the influence of various factors such as steric factors, solvent, and electron-donating/electron-accepting substituents on the kinetics of proton transfer.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; phone (614) 6883664; fax (614) 293-4799. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partly supported by NIH Grant EB01454201A1.

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ABBREVIATIONS TAM triarylmethyl radicals PEDRI proton−electron double-resonance imaging EPRI electron paramagnetic resonance imaging CW EPR continuous-wave electron paramagnetic resonance cTAM trityl radical containing carboxyl groups aTAM trityl radical containing amino group phTAM phosphonated trityl radical pTAM deuterated derivative of phosphonated trityl radical

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Additional text describing pH dependence of pTAM ionization state fractions and hyperfine splitting; equations for calculating pKa values; and two figures and one table showing EPR spectral pH dependence on phosphate buffer concentration; pKa values measured at different temperature, buffer concentration, and ionic strength; and estimates for the rate of proton exchange between different ionization states of pTAM. This material is available free of charge via the Internet at http://pubs.acs.org. 6059

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dx.doi.org/10.1021/ac3008994 | Anal. Chem. 2012, 84, 6054−6060