Reactions of Amino Acids, Peptides, and Proteins with Oxidized

Article pubs.acs.org/crt

Reactions of Amino Acids, Peptides, and Proteins with Oxidized Metabolites of Tris(p‑carboxyltetrathiaaryl)methyl Radical EPR Probes Christophe Decroos,† Jean-Luc Boucher,*,† Daniel Mansuy,† and Yun Xu-Li†,∥ †

Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Université Paris Descartes, Sorbonne Paris Cité, 45 rue des Saints-Pères, 75270 Paris, France ABSTRACT: Oxidation of the tris(p-carboxyltetrathiaaryl)methyl (TAM) EPR radical probe, TAMa•, by rat liver microsomes (RLM) + NADPH, or horseradish peroxidase (HRP) + H2O2, or K2IrCl6, led to an intermediate cation, TAMa+, which was treated with glutathione (GSH), with formation of an adduct, TAMa-SG•, resulting from the substitution of a TAMa• carboxylate group with the SG group. L-α-Amino acids containing a strong nucleophilic residue (NuH), such as L-cysteine or L-histidine, also reacted with TAMa+, with formation of radical adducts TAMa-Nu• in which a carboxylate group of TAMa• was replaced with Nu. Other less nucleophilic L-α-amino acids, such as L-arginine, Lserine, L-threonine, L-tyrosine, or L-aspartate, as well as the tetrapeptide H−(Gly)4−OH, reacted with TAMa+ via their α-NH2 group, with formation of an iminoquinone methide, IQMa, deriving from an oxidative decarboxylation and amination of TAMa•. Upon reaction of TAMa+ with L-proline and L-lysine, Nsubstituted iminoquinone methide adducts, IQMa-Pro and IQMa-Lys, were formed. Finally, preliminary results showed that oxidation of TAMa• in the presence of bovine serum albumin (BSA), led to the covalent binding of TAMa-derived metabolites to BSA. Oxidation of another frequently used TAM probe, TAMb• (Oxo63), in the presence of GSH, N-acetyl-cysteine methyl ester, or histidine also led to TAMb-Nu• adducts equivalent to the corresponding TAMa-Nu• adducts, suggesting that the oxidative metabolism of such TAM• probes could lead to protein covalent binding. Moreover, the above data describe an easy access to new TAM radical EPR probes coupled to amino acids, peptides or proteins that could be useful for addressing various biological targets.

■

INTRODUCTION Triarylmethyl radicals (TAM•) are stable carbon-centered radicals discovered over 100 years ago by Gomberg.1 Much attention has been recently paid to the tris-(pcarboxyltetrathiaaryl)methyl radicals, such as TAMa• and TAMb• (known as Oxo63) (Scheme 1), as they offer a large number of potential applications. Thus, they are used as contrast agents in NMR imaging, particularly in Overhauserenhanced magnetic resonance imaging, dynamic nuclear polarization, and as probes for in vivo and in vitro oximetry by electron paramagnetic resonance imaging (EPRI).2−7 These paramagnetic compounds exhibit several interesting properties, including their EPR narrow single line, whose line width linearly depends upon the O2 concentration. Moreover, they are water-soluble and weakly toxic.8 Among these, TAMa• (Scheme 1) remains the most accessible derivative because its large-scale synthesis has been recently described.9,10 As part of a research program directed toward the application of EPRI to in vivo biological studies, we have evaluated the chemical and biological reactivity of TAMa• in various redox situations. It was found that three-electron oxidants such as O2•− and ROO• efficiently oxidize TAMa• via a selective © 2014 American Chemical Society

oxidative decarboxylation reaction leading to the corresponding diamagnetic quinone methide QMa (Scheme 1).11 It was also shown that the metabolism of TAMa• by human, pig, and rat liver microsomes led to two major metabolites, QMa and the triarylmethane TAMa-H, resulting from the oxidation or the reduction of TAMa•, respectively.12 Moreover, we have shown that peroxidases, such as horseradish peroxidase, lactoperoxidase, and prostaglandin synthase, and other hemeproteins, such as methemoglobin, metmyoglobin, and catalase, also efficiently catalyze the oxidation of TAMa• to QMa by H2O2 or alkylhydroperoxides.13 These reactions involve the intermediate formation of the corresponding cation TAMa+ (Scheme 1) that has also been cleanly generated by oxidation of TAMa• by K2IrIVCl6 and characterized by UV−visible spectroscopy and mass spectrometry.13 Very similar reactions have been found to occur on the TAMb• radical.12,13 Cation TAMa+ readily reacts with various kinds of nucleophiles (NuH), such as phosphines, secondary amines, imidazoles, and thiols, leading eventually to new TAM• radicals in which a carboxylate substituent of Received: December 16, 2013 Published: February 24, 2014 627

dx.doi.org/10.1021/tx400467p | Chem. Res. Toxicol. 2014, 27, 627−636

Chemical Research in Toxicology

Article

Scheme 1. Structure and Biological and Chemical Oxidations of Radicals TAMa• and TAMb•a

a

(1) Reaction with O2•−, ROO•, or liver microsomes + NADPH + O2. (2) Reaction with peroxidase + H2O2 or with K2IrCl6.

TAMa• has been replaced with the nucleophile via an ipsoaromatic nucleophilic substitution (SNAr) reaction that is coupled to an oxidative decarboxylation of TAMa• (Scheme 2).14

synthesized according to a previously described method.9 Oxo63 (TAMb•) was a generous gift from Dr M.C. Krishna (NIH, Bethesda, MD, U. S. A.). NADPH, HRP, H2O2, K2IrCl6, α-amino acids used in this study, H(Gly)4OH, BSA, and sinapinic acid (used as the matrix for matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) experiments) were purchased from Sigma-Aldrich (St. Quentin Fallavier, France). All chemicals and solvents were of the highest grade commercially available and used without further purification. Spectroscopic Methods. UV−visible spectra were recorded on a Uvikon 942 spectrometer (Kontron Biotech). HPLC−MS studies were performed on a Surveyor HPLC instrument coupled to a LCQ Advantage ion trap spectrometer (Thermo, Les Ulis, France). Mass spectra were obtained by electrospray ionization in both positive (ESI+) and negative (ESI−) ionization detection modes under the following conditions: sheath gas, 40; auxiliary gas, 10; spray voltage, 5 kV; capillary temperature, 300 °C; capillary voltage, 7 V; and scanning in full scan mode (m/z from 200 to 1500 for experiments with TAMa• and from 200 to 2000 for experiments with TAMb•). Data were recorded and analyzed with the XCalibur acquisition system. MALDI-TOF mass spectrometry experiments were performed using a Voyager DE-STR MALDI-TOF mass spectrometer (ABSciex, Les Ulis, France), equipped with a 337-nm pulsed nitrogen laser (20 Hz) and an Acqiris 2 GHz digitizer board. Mass spectra were obtained in linear positive ion mode with the following settings: accelerating voltage 25 kV, grid voltage 90% of accelerating voltage, extraction delay time of 300 ns. The laser intensity was set just above the ion generation threshold to obtain peaks with the highest possible signal-to-noise ratio without significant peak broadening. The mass spectrometer was externally calibrated using BSA. All data were processed using the Data Explorer software package (ABSciex). EPR spectra were recorded at 20 °C using a Bruker Elexsys 500 EPR spectrometer (Bruker, Wissenheim, France) operating at X-band (9.85 GHz) with a TM 110 cavity and an AquaX quartz cell, under the following conditions: modulation frequency, 100 kHz; modulation amplitude, 0.03 G; time constant, 40.96 ms; conversion time, 40.96 ms; and microwave power, 1 mW. Data acquisition and processing were performed using Bruker software. Preparation of Rat Liver Microsomes. Male Sprague− Dawley rats (200−250 g, Charles River, L’Arbresle, France) were provided laboratory chow and water ad libitum. After 7 days of adaptation, animals were treated with phenobarbital (50

Scheme 2. Ipso-Aromatic Nucleophilic Substitution of TAMa+ by Nucleophilesa

a NuH = thiols, imidazoles, secondary amines; (1) = K2IrCl6 or peroxidase + H2O2.

These data strongly suggested that, during the oxidative metabolism of TAMa•, biological nucleophiles from proteins or nucleic acids could react with TAMa+ and lead to irreversible modifications of these cell components. This article reports first data in that direction and shows that oxidation of TAMa• and TAMb•, in the presence of the main natural amino acids or of small peptides, leads to two kinds of irreversible modification of those compounds. It also describes preliminary results on the consequences of the oxidation of TAMa• in the presence of bovine serum albumin.

■

EXPERIMENTAL PROCEDURES Chemicals. Tris-(8-carboxyl-2,2,6,6-tetramethylbenzo-[1,2d;4,5-d′]bis[1,3]dithiol-4-yl)methyl sodium salt, TAMa•, was 628



Article

mg.kg−1, ip in saline, for 5 days). Liver microsomes were prepared by differential centrifugation as previously reported and stored at −80 °C until use.15 Protein concentrations were determined by the Bradford assay with BSA as standard.16 Cytochrome P450 contents were determined by the method of Omura and Sato.17 Incubation Conditions for the Oxidations of TAM• in the Presence of Various Nucleophiles. Oxidation of TAMa• by RLM in the Presence of GSH. Incubations were performed at 37 °C in 1 mL Eppendorf tubes. Typical aerobic incubation mixtures (final volume 200 μL) contained 100 μM TAMa• in 0.1 M phosphate buffer pH = 7.4, 0.1 mM EDTA, 5.0 mM GSH, 1000 U mL−1 superoxide dismutase, and 0.5−1.0 mg mL−1 microsomal proteins (∼1.0 μM P450). After equilibration for 5 min at 37 °C, the reactions were started by the addition of NADPH (1.0 mM, final concentration). After 30 min, the reaction mixtures were quenched by the addition of cold acetonitrile (same volume), centrifuged for 10 min at 10000g, and aliquots were analyzed by HPLC−MS method A1 as described below. Oxidation of TAMa• by H2O2 and HRP in the Presence of GSH. Incubations were performed at 25 °C in 1 mL Eppendorf tubes (200 μL final volume) containing 100 μM TAMa•, 1.0 μM HRP, 0.25 mM H2O2, and 5.0 mM GSH in 0.1 M phosphate buffer (pH = 7.4) containing 0.1 mM EDTA. After 5 min, the reactions were stopped by the addition of 250 U mL−1 catalase. After 5 min, cold acetonitrile (same volume) was added and the tubes were centrifuged for 10 min at 10000g. Aliquots were analyzed by HPLC−MS method A1 as described above. Oxidation of TAMa• by K2IrCl6 in the Presence of Various Nucleophiles. Incubations were performed at 25 °C in 1 mL Eppendorf tubes (200 μL final volume). TAMa+ was generated by adding, under stirring, 4 μL of a 10 mM solution of K2IrCl6 (freshly prepared in distilled water) to a solution containing 100 μM TAMa• in 0.1 M phosphate buffer (pH = 7.4). After 1 min, 20 μL of a 50 mM solution of nucleophile (GSH, amino acids, or tetrapeptide H−(Gly)4−OH) in phosphate buffer were added. If not soluble in water, the nucleophile was dissolved in a minimum amount of an organic solvent such as THF or methanol. When thiols were used as nucleophiles, they were first deprotonated with NaOH (1 equiv) before reacting with TAM a+. After 15 min, the reaction mixtures were analyzed by HPLC−MS method A1. In the case of GSH, a preparative experiment was also performed on 2.0 mg TAMa•; after removing of water, the crude adduct was purified by HPLC method A2. Transformations of TAMb• were performed under the same conditions except that aliquots were analyzed by HPLC−MS method A3. HPLC−MS Method A1. Analyses of TAMa• metabolites were performed at room temperature on a Super-ODS column (reverse phase TSK gel, 50 × 4.6 mm, Interchim, Montluçon, France) using a Surveyor HPLC system (Thermo) coupled to an ion trap LCQ Advantage (Thermo) mass spectrometer with electrospray ionization in positive (ESI+) and negative (ESI−) modes. The mobile phase was a mixture of solvent A (water + 0.1% formic acid) and solvent B (acetonitrile + 0.1% formic acid) with the following gradient: 0−2 min, isocratic elution with 95% A; 2−42 min, linear increase from 5 to 95% B; 42−45 min, isocratic elution with 95% B; 45−47 min, linear decrease from 95 to 5% B; 47−55 min, re-equilibration at 5% B. The flow rate was 0.5 mL min−1. Retention times for TAMa•, QMa,

and TAMa-SG• were 25.8, 30.0, and 21.6 and 21.9 min (two TAMa-SG• diastereoisomers), respectively. HPLC Method A2. Purification of TAMa-SG• adduct by semipreparative HPLC was performed on a Super ODS column (reverse phase TSK gel, 50 × 4.6 mm, Interchim, Montluçon, France) using a Spectra Physics HPLC system. A gradient of water + 0.1% formic acid (solvent A) and acetonitrile + 0.1% formic acid (solvent B) was used under the following conditions: 0−2 min, isocratic elution with 95% A; 2−22 min, linear increase from 5 to 95% B; 22−25 min, isocratic elution with 95% B; 25−27 min, linear decrease from 95 to 5% B; 27−32 min, re-equilibration at 5% B. The flow rate was 1.0 mL min−1 and the absorbance was monitored at 270 and 240 nm using the Borwin data acquisition system. Retention times for TAMa•, QMa, and TAMa-SG• were 14.4, 16.4, and 10.2 and 10.5 min (2 TAMa-SG• diastereoisomers), respectively. HPLC−MS Method A3. Separation and identification of TAMb• metabolites were performed at room temperature on a Super-ODS column (reverse phase TSK gel, 50 × 4.6 mm, Interchim, Montluçon, France) using a Spectra-Physics HPLC system. The mobile phases were identical to those described in method A1 with the following gradient: 0−27 min, linear increase from 0 to 10% B; 27−42 min, linear increase from 10 to 95% B; 42−45 min, isocratic elution with 95% B; 45−47 min, linear decrease from 95 to 0% B; 47−55 min, reequilibration at 100% A. The flow rate was 0.5 mL min−1. Retention times for TAMb•, QMb, and TAMb-SG• were 22.2, 28.5, and 17.5 min, respectively (the 2 TAMb-SG• diastereoisomers were not resolved under these conditions). Oxidation of TAMa• in the Presence of BSA. BSA (100 μM in 0.1 M phosphate buffer, pH = 7.4, final volume 200 μL) was reacted with 20 μM, 100 μM, 500 μM, or 1 mM TAMa+ (formed by the stoichiometric reaction of TAMa• and K2IrCl6, before addition on the BSA solution). After 10 min reaction, samples were concentrated on a centrifugal filter device (Centricon, 30 kDa molecular cutoff, Merck, Darmstadt, Germany), washed twice with distilled water, and redissolved in 200 μL of distilled water before analysis by MALDI-TOF MS. Control experiments consisting of BSA alone, and BSA + TAMa• (TAMa•/BSA ratio of 1 and 5) were done using the same workup before analysis by MALDI-TOF MS (mass resolution of 0.05% leading to a molecular ion at m/z = 66 430 ± 33 for BSA, result from four determinations, expected molecular mass: 66 430).

■

RESULTS Oxidation of TAMa• by Either HRP and H2O2 or K2IrCl6 in the Presence of Glutathione. Incubation of 0.1 mM TAMa• with 1 μM HRP, 0.25 mM H2O2, and 5 mM GSH in 0.1 M phosphate buffer pH 7.4 at 25 °C for 5 min led to three products different from starting TAMa•. Almost identical results were obtained after oxidation of TAMa• with 2 equiv of K2IrCl6 and further addition of 50 equiv of GSH. HPLC−MS and HPLC−UV−vis analyses of the reaction mixtures showed the formation of quinone methide QMa as a minor metabolite (∼5% yield, based on starting TAMa). QMa was previously described as the predominant metabolite of the oxidation of TAMa• by H2O2 in the presence of HRP (without GSH).13 The major products (20% yield based on starting TAMa•; 80% yield based on consumed TAMa•, as about 75% TAMa• was recovered at the end of the reaction) corresponded to two HPLC peaks of equal intensity with very close retention times and identical UV−vis and mass spectra. Their mass spectra 629



Article

(ESI+) were characterized by a molecular ion at m/z = 1260.9 corresponding to [TAMa• + GS − CO2], in agreement with a structure deriving from the substitution of a carboxylate group of TAMa• with the GS moiety (TAMa-Nu• with Nu = SG in Scheme 2; calc. m/z = 1261.0, see Table 1). Such a formation

They are in complete agreement with the indicated structure TAMa-SG•. Actually, TAMa-SG• existed as a 50:50 mixture of two diastereoisomers because of the chirality of TAMa• itself associated with its helicoidal structure and the chirality of the SG moiety.18,19 Oxidation of TAMa• by Rat Liver Microsomes in the Presence of GSH. The formation of adducts TAMa-SG• was also observed upon incubation of TAMa• with RLM in the presence of NADPH and GSH (data not shown). In the latter experiment, the presence of superoxide dismutase was required to limit the formation of QMa because of a fast reaction between TAMa• and O2•−.11,12,20−22 Oxidation of TAMa• in the Presence of L-Cysteine, LCysteine Derivatives, and L-Histidine. In the following experiments, L-α-amino acids (5 mM) were added to reaction media in which oxidation of 0.1 mM TAMa• was performed by K2IrCl6 (2 equiv), as these oxidative conditions led us to the best yields of adducts formation with GSH and were without effects on the ratios of the observed end products of the reactions. L-Cysteine led to results very similar to those described above for GSH, with the major formation of two diastereomer metabolites (50:50 mixture) whose spectral characteristics (λmax = 478 nm ; MS molecular ion at m/z = 1074.9) (Table 1) were in agreement with a structure deriving from the replacement of a TAMa• carboxylate substituent with the SCH2CH(COOH)NH2 group. The Cys derivative, Nacetyl-Cys-methyl ester, or a close analog, D-penicillamine, led to the same kind of adducts (Table 1). Minor amounts of QMa (∼2%) were also detected by HPLC−MS in all these reactions. It is noteworthy that GSH and Cys contain two potential nucleophilic groups, the SH and α-amino acid NH2 groups. A priori, two adducts with either a C−S or a C−NH bond could have resulted from the reaction of these nucleophilic groups with TAM+. The EPR spectrum of the obtained adducts that exhibited a sharp signal (see, for instance, Figure 1) is only compatible with the C−S adducts shown in Figure 1 and Table 1, as a C−N adduct would have led to a more complex EPR spectrum because of hyperfine coupling with the N atom (for which I = 1).14 HPLC−MS and HPLC−UV−vis analyses of similar oxidations of TAMa• with K2IrCl6 performed in the presence of L-histidine showed the formation of QMa in small amounts (∼5%) and of two new products. The major one (20% yield based on the starting amount of TAMa•) exhibited UV− vis (λmax = 472 nm) and MS (molecular ion at m/z = 1108.9) characteristics expected for a TAMa-His• adduct resulting from the replacement of a carboxylate substituent of TAMa• with

Table 1. Characteristics of the Adducts TAMa-Nu• Formed upon Reaction of TAMa+ with GSH and Some L-α-Amino Acids or Their Derivatives

Yields based on consumed TAMa• and determined by HPLC. bMS (ESI+) molecular ion experimentally found for the TAMa-Nu• adducts and corresponding to [M + 3H]+; calculated value for [M + 3H]+ in parentheses (M is the molecular mass of the TAM-Nu• compounds bearing two para-COO− substituents). cMajor peak of the UV−vis spectrum of the compounds in a mixture of CH3CN and H2O containing 0.1% formic acid (HPLC solvent). a

of a GSH adduct of TAMa• was a further example of the nucleophilic substitutions previously described upon oxidative decarboxylation of TAMa• in the presence of nucleophiles (Scheme 2).14 These major metabolites were characterized by UV−vis spectra with a major band at 478 nm (Table 1) and a minor band at 660 nm and by EPR spectra with a sharp signal at g = 2.0030 (Figure 1). These spectral data were very similar to those of TAMa• and of its previously reported derivatives in which a CO2 substituent has been replaced with an SR group.14

Figure 1. Structure and EPR spectrum of the adduct formed upon oxidation of TAMa• by liver microsomes + NADPH, HRP + H2O2, or K2IrCl6 in the presence of GSH. 630



Article

Scheme 3. Adducts Formed upon Oxidation of TAMa• in the Presence of Lys or Nα-AcLys

Scheme 4. Possible Mechanisms for the Formation of IQMa upon Reaction of TAMa+ with Either the α-NH2 Group of α-Amino Acids or with NaN3

similar to those of IQMa-Lys except for its MS molecular ion that appeared at m/z = 1141.0. This increase of m/z of 42 amu well corresponds to the presence of an α-NHCOCH3 group in Nα-AcLys and in the IQMa-Nα-AcLys adduct instead of an αNH2 group in Lys and in IQMa-Lys. Reaction with Nα-AcLys also led to QMa in low yield but did not lead to the 580 nmabsorbing product. Attempts to purify IQMa-Nα-AcLys showed us that this species could be reversibly transformed (Scheme 3) into a species absorbing at 470 nm and exhibiting an EPR signal at g = 2.0030 that should correspond to TAMa-Nα-AcLys•, the radical expected from the nucleophilic attack of the Nω-atom of NαAcLys on TAMa+ according to the general mechanism of Scheme 2. The IQMa-Nα-AcLys species was favored under the acidic conditions used in HPLC−MS studies (CH3CN−H2O containing formic acid), whereas basic and presumably reducing conditions used for purification (CH3CN−H2O containing NH3) favored the TAMa-Nα-AcLys• species. Oxidation of TAMa• in the Presence of Other L-αAmino Acids. With all the other L-α-amino acids that were tested under identical conditions, Arg, Ser, Thr, Tyr, Trp, and Asp, an HPLC−MS analysis of the reaction mixture showed the formation of QMa and of the 580 nm-absorbing product already found with His and Lys. The yields in the latter product varied between 10 and 15% (based on starting TAMa). Its UV− vis spectrum showed a blue-shifted peak at 580 nm as the UV− vis spectra of QMa and IQMa-Lys that were characterized by maxima at 542 and 595 nm, respectively. Its mass spectrum

deprotonated His bound by one of its imidazole nitrogen atom (Table 1). Previous data have shown the formation and complete characterization of a very similar adduct TAMabenzimidazole• upon reaction of TAMa•, oxidized under identical conditions, with benzimidazole.14 The second, minor product (8% yield based on starting TAMa•) showed UV−vis characteristics (λmax = 580 nm) quite different from those of the previously obtained TAMa-Nu• adducts (λmax around 470 nm). Its structure will be discussed below. Oxidation of TAMa• in the Presence of L-Lysine. HPLC−MS and HPLC−UV−vis analyses of oxidations of TAMa• performed in the presence of Lys under identical conditions showed the formation of QMa in low amounts ( imidazole > ω-NH2 (of Lys) > (HOOCCH)NH2 > OH. They suggest that covalent binding of TAMa• metabolites such as TAMa+ could result from its reactions with Cys, His, and Lys protein residues. Our preliminary results with BSA indicate that one or two TAMa moieties are covalently bound to the protein presumably through Cys, His, or Lys residues. Our data also suggest that another reaction, the deamination of the terminal α-NH2 group of peptides or proteins, could occur during metabolic oxidation of TAM• radicals. The detection of IQMa upon reaction of BSA with TAMa+ is in favor of this proposition. An analogous deamination of the terminal NH2 group of proteins has been recently reported after the oxidative bioactivation of another class of EPR probes, the nitroxide radicals.25 One-electron oxidation of these nitroxides leads to the corresponding oxoammonium cations whose reaction with protein terminal NH2 group leads to the corresponding imines. Hydrolysis of the terminal imine function occurs with loss of NH3 and leads to a deaminated protein bearing a terminal carbonyl moiety.25 What is the possible relevance of the above data in toxicology? TAM radicals such as TAMa• and TAMb• are widely used for in vivo EPR spectroscopy and imaging.2−8 Our data show that their oxidative metabolism by liver microsomes or peroxidases leads to a new class of electrophilic metabolites able to bind covalently to proteins, to deaminate proteins (with formation of IQMa), and to irreversibly react with GSH. The knowledge of these reactions should be useful for understanding and preventing possible secondary toxic effects of these EPR probes. Finally, the above data describe an easy access to new TAM• radical EPR probes coupled to amino acids, peptides, or proteins that could be useful for addressing various biological targets.

■

Article

REFERENCES

(1) Gomberg, M. (1900) An instance of trivalent carbon: Triphenylmethyl. J. Am. Chem. Soc. 22, 757−771. (2) Joergensen, M., Rise, F., Andersson, S., Almen, T., Aabye, A., Wistrand, L. G., Wikstroem, H., Golman, K., Servin, R., and Michelsen, P. (1991) Triarylmethyl radicals and the use of inert carbon free radicals in MRI. PCT Int. Appl. WO Patent 91/12024. (3) Thaning, M. (2005) Triarylmethyl free radicals as image enhancing agents. PCT Int. Appl. WO Patent 98/39277. (4) Krishna, M. C., English, S., Yamada, K., Yoo, J., Murugesan, R., Devasahayam, N., Cook, J. A., Golman, K., Ardenkjaer-Larsen, J. H., Subramanian, S., and Mitchell, J. B. (2002) Overhauser enhanced magnetic resonance imaging for tumor oximetry: coregistration of tumor anatomy and tissue oxygen concentration. Proc. Natl. Acad. Sci. U. S. A. 99, 2216−2221. (5) Krishna, M. C., Matsumoto, S., Yasui, H., Saito, K., Devasahayam, N., Subramanian, S., and Mitchell, J. B. (2012) Electron paramagnetic resonance imaging of tumor pO2. Radiat. Res. 177, 376−386. (6) Ardenkjaer-Larsen, J. H., Laursen, I., Leunbach, I., Ehnholm, G., Wistrand, L. G., Petersson, J. S., and Golman, K. (1998) EPR and DNP properties of certain novel single electron contrast agents intended for oximetric imaging. J. Magn. Reson. 133, 1−12. (7) Gallez, B., Baudelet, C., and Jordan, B. F. (2004) Assessment of tumor oxygenation by electron paramagnetic resonance: principles and applications. NMR Biomed. 17, 240−262. (8) Matsumoto, K., English, S., Yoo, J., Yamada, K., Devasahayam, N., Cook, J. A., Mitchell, J. B., Subramanian, S., and Krishna, M. C. (2004) Pharmacokinetics of a triarylmethyl-type paramagnetic spin probe used in EPR oximetry. Magn. Reson. Med. 52, 885−892. (9) Reddy, T. J., Iwama, T., Halpern, H. J., and Rawal, V. H. (2002) General synthesis of persistent trityl radicals for EPR imaging of biological systems. J. Org. Chem. 67, 4635−4639. (10) Dhimitruka, I., Velayutham, M., Bobko, A. A., Khramtsov, V. V., Villamena, F. A., Hadad, C. M., and Zweier, J. L. (2007) Large scale synthesis of a persistent trityl radical for use in biomedical EPR applications and imaging. Bioorg. Med. Chem. Lett. 17, 6801−6805. (11) Decroos, C., Li, Y., Bertho, G., Frapart, Y., Mansuy, D., and Boucher, J.-L. (2009) Oxidation of tris-(p-carboxyltetrathiaaryl)methyl radical EPR probes: evidence for their oxidative decarboxylation and molecular origin of their specific ability to react with O2•−. Chem. Commun., 1416−1418. (12) Decroos, C., Li, Y., Bertho, G., Frapart, Y., Mansuy, D., and Boucher, J.-L. (2009) Oxidative and reductive metabolism of tris(pcarboxyltetrathiaaryl)methyl radicals by liver microsomes. Chem. Res. Toxicol. 22, 1342−1350. (13) Decroos, C., Li, Y., Soltani, A., Frapart, Y., Mansuy, D., and Boucher, J.-L. (2010) Oxidative decarboxylation of tris-(pcarboxyltetrathiaaryl)methyl radical EPR probes by peroxidases and related hemeproteins: intermediate formation and characterization of the corresponding cations. Arch. Biochem. Biophys. 502, 74−80. (14) Decroos, C., Prangé, T., Mansuy, D., Boucher, J.-L., and Li, Y. (2011) Unprecedented ipso aromatic nucleophilic substitution upon oxidative decarboxylation of tris(p-carboxyltetrathiaaryl)methyl (TAM) radicals: A new access to diversely substituted TAM radicals. Chem. Commun. 47, 4805−4807. (15) Kremers, P., Beaune, P., Cresteil, T., de Graeve, J., Columelli, S., Leroux, J. P., and Gielen, J. E. (1981) Cytochrome P450 monooxygenase activities in human and rat liver microsomes. Eur. J. Biochem. 118, 599−606. (16) Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248−254. (17) Omura, T., and Sato, R. (1964) The Carbon Monoxide-Binding Pigment of Liver Microsomes. II. Solubilization, Purification, and Properties. J. Biol. Chem. 239, 2379−2385. (18) Driesschaert, B., Robiette, R., Lucaccioni, F., Gallez, B., and Marchand-Brynaert, J. (2011) Chiral properties of tetrathiatriarylmethyl spin probes. Chem. Commun. 47, 4793−4795.

AUTHOR INFORMATION

Corresponding Author

*J.-L. Boucher. Tel.: + 33 1 42 86 21 91. Fax: + 33 1 42 86 83 87. E-mail: [email protected]. Present Address ∥

Laboratoire de Chimie des Processus Biologiques, Collège de France, 11 Place M. Berthelot, 75005 Paris, France. Funding

This work was supported by a fellowship to C.D. from the French Ministère de l’Education et de la Recherche. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank Dr. Y. Frapart (UMR 8601 CNRS, Paris) for his help in EPR experiments, Dr. M. Krishna (NIH, Bethesda, MD, U. S. A.) for a generous gift of Oxo63 (TAMb•), and Dr V. Guerineau (ImaGif, ICSN, Gif sur Yvette, France) for MALDI-TOF MS experiments.

■

ABBREVIATIONS Nα-AcLys: Nα-acetyl-lysine; BSA: bovine serum albumin; EPRI: electron paramagnetic resonance imaging; ESI: electron spray ionization; HRP: horseradish peroxidase; IQM: iminoquinone methide; MALDI-TOF MS: matrice assisted laser desorption ionization-time-of-flight mass spectroscopy; NuH: nucleophile; RLM: rat liver microsomes; QM: quinone methide; TAM•: triarylmethyl radicals 635



Article

(19) Driesschaert, B., Robiette, R., LeDuff, C. S., Collard, L., Robeyns, K., Gallez, B., and Marchand-Brynaert, J. (2012) Configurationnaly stable tris(tetrathioaryl)methyl molecular propellers. Eur. J. Org. Chem., 6517−6525. (20) Liu, Y., Song, Y., De Pascali, F., Liu, X., Villamena, F. A., and Zweier, J. L. (2012) Tetrathiatriarylmethyl radical with a single aromatic hydrogen as a highly sensitive and specific superoxide probe. Free Radical Biol. Med. 53, 2081−2091. (21) Rizzi, C., Samouilov, A., Kutala, V. K., Parinandi, N. L., Zweier, J. L., and Kuppusamy, P. (2003) Application of a trityl-based radical probe for measuring superoxide. Free Radical Biol. Med. 35, 1608− 1618. (22) Kutala, V. K., Parinandi, N. L., Zweier, J. L., and Kuppusamy, P. (2004) Reaction of superoxide with trityl radical: implications for the determination of superoxide by spectrophotometry. Arch. Biochem. Biophys. 424, 81−88. (23) Guengerich, F. P. (2001) Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity. Chem. Res. Toxicol. 14, 611−650. (24) Makris, T. M., Denisov, I., Schlichting, I., and Sligar, S. G. (2005) in Cytochrome P450: Structure, Mechanism, and Biochemistry, (Ortiz de Montellano, P. R., Ed.) pp 149−170, Kluwer Academic/ Plenum Publishers, New York. (25) Lardinois, O. M., Maltby, D. A., Medzihradszky, K. F., Ortiz de Montellano, P. R., Tomer, K. B., Mason, R. P., and Deterding, L. J. (2009) Spin scavenging analysis of myoglobin protein-centered radicals using stable nitroxide radicals: Characterization of oxoammonium cation-induced modifications. Chem. Res. Toxicol. 22, 1034− 1049.

636


Reactions of Amino Acids, Peptides, and Proteins with Oxidized

Recommend Documents