Toward stable electron paramagnetic resonance oximetry probes: Synthesis, characterization, and metabolic evaluation of new ester derivatives of a tris-(para-carboxyltetrathiaaryl)methyl (TAM) radical

Article abstract of DOI:10.1021/tx400250a

Tris(p-carboxyltetrathiaaryl)methyl (TAM) radicals, such as 1a ("Finland" radical), are useful EPR probes for oximetry. However, they are rapidly metabolized by liver microsomes in the presence of NADPH, with the formation of diamagnetic quinone-methide metabolites resulting from an oxidative decarboxylation of one of their carboxylate substituents. In an effort to obtain TAM derivatives potentially more metabolically stable in vivo, we have synthesized four new TAM radicals in which the carboxylate substituents of 1a have been replaced with esters groups bearing various alkyl chains designed to render them water-soluble. The new compounds were completely characterized by UV-vis and EPR spectroscopies, high resolution mass spectrometry (HRMS), and electrochemistry. Two of them were water-soluble enough to undergo detailed microsomal metabolic studies in comparison with 1a. They were found to be stable in the presence of the esterases present in rat liver microsomes and cytosol, and, contrary to 1a, stable to oxidation in the presence of NADPH-supplemented microsomes. A careful study of their possible microsomal reduction under anaerobic or aerobic conditions showed that they were more easily reduced than 1a, in agreement with their higher reduction potentials. They were reduced into the corresponding anions not only under anaerobic conditions but also in the presence of dioxygen. These anions were much more stable than that of 1a and could be characterized by UV-vis spectroscopy, MS, and at the level of their protonated product. However, they were oxidized by O₂, giving back to the starting ester radicals and catalyzing a futile cycle of O₂ reduction. Such reactions should be considered in the design of future stable EPR probes for oximetry in vivo.

Full text of DOI:10.1021/tx400250a

Article
pubs.acs.org/crt
Toward Stable Electron Paramagnetic Resonance Oximetry Probes:
Synthesis, Characterization, and Metabolic Evaluation of New Ester
Derivatives of a Tris‑(para-carboxyltetrathiaaryl)methyl (TAM)
Radical
Christophe Decroos,† Veronique Balland,‡ Jean-Luc Boucher,*,† Gildas Bertho,† Yun Xu-Li,†,§
́
and Daniel Mansuy†
†Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite
Paris Descartes, Sorbonne
Paris Cite, 45 rue des Saints-Peres, 75270 Paris, France
‡Laboratoire d’Electrochimie Molec
́ ́
ulaire, UMR CNRS 7591, Universite Paris Diderot, Sorbonne Paris Cite, 15 rue J.-A. de Baïf,
́
́
̀
́
75205 Paris, France
S
* Supporting Information
ABSTRACT: Tris(p-carboxyltetrathiaaryl)methyl (TAM)
radicals, such as 1a (“Finland” radical), are useful EPR probes
for oximetry. However, they are rapidly metabolized by liver
microsomes in the presence of NADPH, with the formation of
diamagnetic quinone-methide metabolites resulting from an
oxidative decarboxylation of one of their carboxylate
substituents. In an effort to obtain TAM derivatives potentially
more metabolically stable in vivo, we have synthesized four new TAM radicals in which the carboxylate substituents of 1a have
been replaced with esters groups bearing various alkyl chains designed to render them water-soluble. The new compounds were
completely characterized by UV−vis and EPR spectroscopies, high resolution mass spectrometry (HRMS), and electrochemistry.
Two of them were water-soluble enough to undergo detailed microsomal metabolic studies in comparison with 1a. They were
found to be stable in the presence of the esterases present in rat liver microsomes and cytosol, and, contrary to 1a, stable to
oxidation in the presence of NADPH-supplemented microsomes. A careful study of their possible microsomal reduction under
anaerobic or aerobic conditions showed that they were more easily reduced than 1a, in agreement with their higher reduction
potentials. They were reduced into the corresponding anions not only under anaerobic conditions but also in the presence of
dioxygen. These anions were much more stable than that of 1a and could be characterized by UV−vis spectroscopy, MS, and at
the level of their protonated product. However, they were oxidized by O2, giving back to the starting ester radicals and catalyzing
a futile cycle of O2 reduction. Such reactions should be considered in the design of future stable EPR probes for oximetry in vivo.
stability in the presence of biological systems, low toxicity, and
good water solubility.
INTRODUCTION
■
The dioxygen partial pressure (pO2) is an important parameter
in the metabolic processes of living organisms. Measurement of
dioxygen concentration in vitro and in vivo is of crucial
importance in the study of both physiological and pathological
situations including cancers and ischemia-reperfusion.1,2
Various methods have been developed to detect and monitor
pO2.3−10 Among them, electron paramagnetic resonance
spectroscopy measures the O2-dependent linewidths of
exogenous paramagnetic contrast agents such as nitroxides
and triarylmethyl radicals, as well as particulate-based probes
such as chars and lithium phthalocyanines.11−13 Spin−spin
interaction between the paramagnetic spin probes and
dioxygen, also a paramagnetic molecule, modifies the relaxation
time of the probe and its peak-to-peak linewidth.3,14 For these
spectroscopic and imaging modalities, useful spin probes should
display one single sharp line in their EPR spectra with a high
O2-dependent line width. Furthermore, it should possess good
Triarylmethyl (TAM) radicals 1a and 1b (called Oxo63)
(Figure 1) are stable carbon-centered radicals that have been
developed for use as contrast agents in NMR imaging and as
probes for EPR spectroscopy and EPR imaging (EPRI).9,14−18
They have been demonstrated to be very effective spin probes
for measuring pO2 in vitro and in vivo through line-broadening
analysis because their EPR spectrum displays one narrow single
line whose linewidth linearly depends upon the O2 concen-
tration.3,14−19 Oxo63, 1b, is highly soluble in water and weakly
toxic, and its pharmacokinetics in mice has been followed by
EPR spectroscopy and imaging.19 In vitro, 1b is stable in the
presence of oxidoreductants such as ascorbate, glutathione,
NADPH, hydrogen peroxide, and hydroxyl radical.20−22
However, several studies have shown that TAMs 1a−b readily
Received: July 8, 2013
© XXXX American Chemical Society
A
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Figure 1. Structure and metabolism of TAM 1a and 1b by liver microsomes. Adapted from ref 24. Copyright 2009 American Chemical Society.
according to a previously described method.35 Cytochrome c was
purchased from Sigma-Aldrich (St. Quentin Fallavier, France). All
other chemicals and solvents were of the highest grade commercially
available.
react with superoxide20−22 with the formation of the
corresponding diamagnetic quinone-methides QMa-b22 (Fig-
ure 1), and some TAM radicals may be used for the specific
measurement of superoxide, O2•−, in cell-free and cellular
systems.20,21,23
Spectroscopic Methods. All NMR experiments (1H, 13C,
heteronuclear single quantum correlation, and heteronuclear multiple
bond correlation) were carried out at room temperature on a Bruker
Biospin Advance II 500 MHz spectrometer. Chemical shifts (δ) are
reported in ppm relative to tetramethylsilane and coupling constants
(J) in Hz. UV−vis spectra were recorded on a Cary 300 spectrometer
(Varian, Les Ulis, France). Mass spectra were obtained on an LCQ
Advantage ion trap spectrometer (Thermo Scientific, Courtaboeuf,
France) 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). Data were recorded and analyzed with
the XCalibur acquisition system. High-resolution mass spectra
(HRMS) were obtained by ESI in time-of-flight detection mode on
a LCT (Waters-Micromass, Guyancourt, France) spectrometer at
ICSN (Gif sur Yvette, France). EPR spectra were recorded at 21 °C
using a Bruker Elexsys 500 EPR spectrometer (Bruker, Wissenheim,
France) operating at X-band (9.85 GHz) and an AquaX quartz cell
(Bruker) fitted in a TM 110 cavity 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
Xepr software.
We have recently demonstrated that 1a−b undergo several
metabolic transformations.24,25 Their aerobic incubations in the
presence of rat, human, and pig liver microsomes and NADPH
led to the quinone-methides QMa-b that result from the
oxidative decarboxylation of 1a−b by superoxide24 that is
known to be generated by cytochromes P450 and P450
reductase under decoupling conditions.26,27 Under anaerobic
conditions, 1a−b are reduced by NADPH to the diamagnetic
triarylmethane derivatives 1a-b-H (Figure 1) in reactions also
catalyzed by P450s and P450 reductase.24 Finally, we have also
shown that hemeproteins (hemoglobin and myoglobin) and
peroxidases (lactoperoxidase, prostaglandin hydroperoxide
synthase, and horseradish peroxidase) efficiently catalyze the
oxidative decarboxylation of 1a−b by hydrogen peroxide or
hydroperoxides, leading eventually to the quinone-methides
QMa-b, via the corresponding cations 1a−b+.25
These data indicated that decarboxylation of 1a−b was
mainly at the origin of the metabolic instability of these
compounds. In an effort to improve the metabolic stability of
these EPR probes, we have synthesized new compounds in
which the carboxylate groups of 1a−b were replaced with ester
functions. In order to obtain probes with sharp EPR signals, we
excluded amide functions since some TAM radicals bearing an
amide group have been synthesized and found to display large
linewidths due to hyperfine coupling with the N-atoms.28,29
Some ester derivatives of 1a−b have been synthesized, and EPR
studies have shown that they display intermediate linewidths
since the H-atoms of the ester alkyl chain also induce hyperfine
coupling, but this was smaller than that observed with amide
groups.30−34
Synthesis of Esters 2−5. Tris(8-[3-trimethylammonium]-
propyloxycarboxyl-2,2,6,6-tetramethylbenzo[1,2-d;4,5-d′]bis[1,3]-
dithiol-4-yl)methyl Radical, Bromide, 2. Cesium carbonate (65.0 mg,
200 μmol) was added to a solution of 1a (50.0 mg, 50.0 μmol) in 1
mL of freshly distilled N,N-dimethylformamide (DMF). After 30 min
at room temperature, (3-bromopropyl)trimethylammonium bromide
(196.0 mg, 750 μmol) and KI (2.0 mg, 12.0 μmol) were added, and
the mixture was stirred under argon for 15 h. The solvent was
evaporated under high vacuum, and the crude product was purified by
chromatography on neutral alumina with CH2Cl2/EtOH gradients to
afford compound 2 as a green-orange solid (70.0 mg, 91%). Rf = 0.11
(Al2O3, CH2Cl2/ethanol/water 50:45:5 v/v/v). HRMS (ESI+) calcd
for C58H81N3O6S12, 1299.2774; found, 1299.2757.
Tris(8-[3-sulfonic acid]propyloxycarboxyl-2,2,6,6-
tetramethylbenzo[1,2-d;4,5-d′]bis[1,3]dithiol-4-yl)methyl Radical,
Sodium Salt, 3. Cesium carbonate (32.6 mg, 100 μmol) was added
to a solution of 1a (25.0 mg, 25.0 μmol) in 1.5 mL of freshly distilled
DMF. After 30 min at room temperature, sodium 3-bromo-
propanesulfonate (84.4 mg, 375 μmol) and KI (1.0 mg, 6.0 μmol)
were added. The mixture was stirred under argon for 24 h, and the
solvent was evaporated under high vacuum. Five milliliters of ethanol
were added, and the solution was filtered. Ethanol was evaporated, and
the crude product was purified by flash-chromatography on a
This article describes the synthesis and UV−vis, HRMS,
EPR, and electrochemical characteristics of four new TAM
esters bearing either positive or negative charges or polyether
chains that should make them water-soluble enough. It also
compares the metabolic stability of the two most water-soluble
new TAM esters in the presence of liver microsomes, with or
without NADPH, with that previously reported for 1a−b.
EXPERIMENTAL PROCEDURES
Chemicals. Tris-(8-carboxyl-2,2,6,6-tetramethylbenzo-[1,2-d;4,5-
d′]bis[1,3]dithiol-4-yl)methyl sodium salt (1a) was synthesized
■
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preconditionned C18 column (AIT, Houilles, France) using water/
CH3CN gradients to afford compound 3 as a green-orange solid (32.0
mg, 89%). Rf = 0.37 (SiO2, CH2Cl2/methanol 2:1 v/v + 0.1% acetic
acid). HRMS (ESI−) calcd for C49H54O15S15, 1361.9273; found,
1361.9226.
and in methanol containing 0.05 M tetrabutylammonium hexafluor-
oborate for radicals 1a-5. Solutions were carefully degassed by the
bubbling of nitrogen before each measurement. The redox potentials
were calculated according to the equation E = (Ep,a + Ep,c)/2.
HPLC Analysis. Analysis of products was performed at room
temperature on a Super ODS (TSK gel reverse-phase, 50 mm × 4.6
Tris(8-[2-[2-(2-methoxyethoxy)ethoxy]ethoxycarboxyl-2,2,6,6-
tetramethylbenzo[1,2-d;4,5-d′]bis[1,3]dithiol-4-yl)methyl Radical, 4.
Cesium carbonate (19.5 mg, 60.0 μmol) was added to a solution of 1a
(15.0 mg, 15.0 μmol) in 1.0 mL of freshly distilled DMF. After 30 min
at room temperature, 1-bromo-2-[2-(2-methoxyethoxy)ethoxy]ethane
(obtained from 2-[2-(2-methoxyethoxy) ethoxy]ethanol using CBr4
and triphenylphosphine in THF, following a described protocol)36
(52.0 mg, 229 μmol) and KI (1.0 mg, 6.0 μmol) were added. The
mixture was stirred under argon for 36 h, and the solvent was
evaporated under high vacuum. The crude product was purified by
chromatography on SiO2 with diethyl ether/cyclohexane gradients to
afford compound 4 as a brown solid (18.0 mg, 82%). Rf = 0.85 (SiO2,
CH2Cl2/methanol 9:1 v/v + 0.1% acetic acid). HRMS for 4 chelating
Na+(ESI+): calcd for C61H81NaO15S12, 1460.2122 ; found, 1460.2122.
Tris(8-[4-triphenylphosphonium]butyloxycarboxyl-2,2,6,6-
tetramethylbenzo[1,2-d;4,5-d′]bis[1,3]dithiol-4-yl)methyl Radical,
Bromide, 5. Cesium carbonate (78.0 mg, 240 μmol) was added to a
solution of 1a (60.0 mg, 60.0 μmol) in 3 mL of freshly distilled DMF.
After 30 min at room temperature, (4-bromobutyl)-
triphenylphosphonium bromide (431.0 mg, 900 μmol) and KI (2.5
mg, 15.0 μmol) were added, and the mixture was stirred under argon
for 24 h. The solvent was evaporated under high vacuum, and the
crude product was purified by chromatography on neutral alumina
with CH2Cl2/ethanol gradients to afford compound 5 as a green-
orange solid (115.0 mg, 87%). Rf = 0.69 (Al2O3, CH2Cl2/ethanol/
water 50:45:5 v/v/v). HRMS (ESI+) calcd for C106H105O6P3S12,
1950.3773; found, 1950.3771.
Synthesis of Triarylmethanes 2-H and 3-H. Tris(8-[3-
trimethylammonium]propyloxycarboxyl-2,2,6,6-tetramethylbenzo-
[1,2-d;4,5-d′]bis[1,3]dithiol-4-yl)methane, Bromide, 2-H. Radical 2
(10.0 mg, 6.5 μmol) was dissolved in 10 mL of 0.01 M deoxygenated
HCl, and Na2S2O4 (2.3 mg, 13.2 μmol) was added. After 15 min at
room temperature under argon, water was evaporated under vacuum,
and the crude product was dissolved in 1 mL of CH3CN. The solution
was filtered and concentrated under vacuum to afford triarylmethane
2-H (10.0 mg, 100%) as a yellow solid. 1H NMR (CD3OD) δ 5.52 (s,
1H), 4.44−4.52 (m, 6H), 3.60 (t, 6H, J = 8.5), 3.21 (s, 27 H), 2.34−
2.40 (m, 6H), 1.80 (s, 9H), 1.79 (s, 9H), 1.75 (s, 9H), 1.68 (s, 9H).
13C NMR (CD3OD) δ 23.95, 28.82, 28.94, 33.87, 34.99, 54.01, 63.48,
mm, 2 μm, Interchim, Montluco̧ n, France) using a Spectra Physics
HPLC system. The mobile phase was a mixture of solvent A (10 mM
ammonium acetate, pH 6.5) and solvent B (acetonitrile) with the
following gradient: 0−2 min, isocratic elution with 5% B; 2−22 min,
linear increase from 5 to 90% B; 22−25 min, isocratic elution with
95% B; 25−27 min, linear decrease to 5% B; and 27−35 min, re-
equilibration at 5% B. The flow rate was 1 mL·min−1, and the
absorbance was monitored at 270 nm using a Borwin data acquisition
software. Under these conditions, the retention times for 1a and 1a-H,
QMa, 2, and 2-H, and 3 and 3-H were 7.3, 10.7, 16.5, and 9.6 min,
respectively. HPLC-MS studies were performed on a Surveyor HPLC
instrument coupled to the LCQ Advantage ion trap spectrometer
(Thermo Scientific, Courtaboeuf, France) under the above-described
conditions, except that the flow rate was 500 μL.min−1.
Preparation of Rat Liver Microsomes and Cytosols. Male
Sprague−Dawley rats (200−250 g) were provided laboratory chow
and water ad libitum. After 7 days of adaptation, animals were treated
with phenobarbital (80 mg·kg−1, in 0.9% saline, i.p. for 4 days). Liver
cytosols and microsomes were prepared by differential centrifugation
as previously reported and stored at −80 °C until use.37 Protein
concentrations were determined by the Bradford assay with bovine
serum albumin as standard.38 Cytochrome P450 contents were
determined by the method of Omura and Sato.39
Hydrolytic Stability of Radicals 2 and 3 in the Presence of Rat
Liver Cytosol or Microsomes. The hydrolytic stability of esters 2 and 3
(100 μM) was tested at 37 °C in the presence of 0.10 mg·mL−1 rat
liver cytosolic or 1.0 mg·mL−1 microsomal proteins in 0.1 M
phosphate buffer containing 0.1 mM EDTA. The reactions were
stopped by the addition of cold acetonitrile followed by centrifugation
for 15 min at 13,000 rpm, and the supernatants were analyzed by UV−
vis spectroscopy. The final concentrations of radicals were estimated
using ε491 nm = 14,500 M−1·cm−1 and 15,000 M−1·cm−1 for 2 and 3,
respectively. The supernatants were also analyzed by HPLC as
described above. The esterase activity of rat liver cytosol and
microsomes toward para-nitrophenol acetate was determined
following a previously described colorimetric method.40 The reactions
were performed at 37 °C in 0.1 M phosphate buffer at pH 7.4
containing 0.1 mM EDTA, 0.10 mg·mL−1 cytosolic, or 1.0 mg·mL−1
microsomal proteins and were initiated by the addition of 1 mM para-
nitrophenol acetate (from a fresh 0.1 M solution in Me2SO). The
formation of para-nitrophenol was determined by following changes in
63.92, 64.16, 64.52, 65.49, 121.26, 131.43, 140.73, 141.46, 142.05,
142.97, 167.02. HRMS (ESI+) calcd for C58H82N3O6S12, 1300.2852;
found, 1300.2816.
Tris(8-[3-sulfonic acid]propyloxycarboxyl-2,2,6,6-
tetramethylbenzo[1,2-d;4,5-d′]bis[1,3]dithiol-4-yl)methane, Sodium
Salt, 3-H. Radical 3 (10.0 mg, 7.0 μmol) was dissolved in 10 mL of
0.01 M deoxygenated HCl, and Na2S2O4 (2.4 mg, 13.8 μmol) was
added. After 15 min at room temperature under an argon atmosphere,
water was evaporated under vacuum, and the crude product was
dissolved in 1 mL of methanol. The solution was filtered and
concentrated under vacuum to afford triarylmethane 3-H (10.0 mg,
absorbance at 405 nm for 3 min and quantitated using Δε405 nm
=
13,000 M−1·cm−1.
Incubations of Radicals 2 and 3 in the Presence of Rat Liver
Microsomes and NADPH under Aerobic Conditions. Incubations
were performed at 37 °C in Eppendorf tubes. Typical aerobic
incubation mixtures (final volume 150 μL) contained 100 μM radicals
2 or 3 in 0.1 M phosphate buffer at pH 7.4, 0.1 mM EDTA and 0.5−
0.8 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 mM, final concentration). After 30 min, the reaction
mixtures were quenched by the addition of cold acetonitrile (same
volume) and centrifuged for 10 min at 13,000 rpm, and aliquots were
analyzed by HPLC as described above. The final concentrations of
radicals were estimated using ε491 nm = 14,500 M−1·cm−1 and 15,000
M−1·cm−1 for 2 and 3, respectively.
Metabolism of 2 and 3 by Rat Liver Microsomes in the Presence
of NADPH under Anaerobic Conditions. Anaerobic incubations were
performed at 37 °C in 1-cm path length quartz cuvettes previously
purged with argon and stopped with a rubber septum. Liver
microsomes were gently degassed by argon flowing at the surface of
the sample for 5 min at 4 °C. Meanwhile, the buffer was also degassed
by argon bubbling for at least 30 min at 4 °C before being added to
degassed liver microsomes. Typical anaerobic incubation mixtures
1
100%) as a yellow solid. H NMR (CD3OD) δ 5.46 (s, 1H), 4.44−
4.55 (m, 6H), 3.07 (t, 6H, J = 7.5), 2.27−2.33 (m, 6H), 1.80 (s, 9H),
1.77 (s, 9H), 1.74 (s, 9H), 1.67 (s, 9H). 13C NMR (CD3OD) δ 25.93,
29.14, 29.36, 33.51, 34.62, 49.75, 63.42, 63.64, 64.26, 66.51, 121.44,
131.29, 140.57, 141.40, 141.91, 142.96, 167.38. HRMS (ESI−) calcd
for C49H55O15S15, 1362.9352; found, 1362.9318.
Cyclic Voltammetry. Cyclic voltammetry was carried out with a
PST20 Autolab potensiostat (Metrohm Autolab BV, Utrecht, The
Netherlands) interfaced with a PC computer. Experiments were
performed in a water-jacketed electrochemical cell maintained at 20 °C
with a circulating water bath, using a platinum-wire auxiliary electrode,
and a saturated calomel reference electrode (+ 0.242 V vs standard
hydrogen electrode). The working electrode was a glassy carbon
electrode (0.071 cm2). Measurements were carried out in 0.1 M
phosphate buffer at pH 7.4 containing 0.1 mM EDTA for radicals 1a-3
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Scheme 1. Synthesis of Radicals 2−5 from TAM 1a
(final volume 150 μL) contained 100 μM radicals 2 or 3 in 0.1 M
phosphate buffer at pH 7.4 containing 100 μM EDTA and 0.005−
0.008 mg·mL−1 microsomal proteins (∼10 nM P450). After
equilibration for 5 min at 37 °C, the reactions were started by the
addition of NADPH (1 mM final concentration) and monitored by
UV−vis at 37 °C either by repetitive scanning between 380 and 880
nm or by following the increase in absorbance at 644 nm to estimate
the rate of formation of anions 2− and 3− with ε644 nm values of 31,800
and 36,300 M−1·cm−1, respectively. Protonation of 2− and 3− was
neglected on the time scale of the experiments (3 min).
their metabolic stability in phosphate buffer at pH 7.4. By
contrast, esters 4 and 5, bearing a neutral group or a
triphenylphosphonium moiety, were not soluble enough in
water to permit such metabolic studies. However, their
spectroscopic and electrochemical characteristics were deter-
mined in organic solvents such as methanol or acetonitrile.
In acetonitrile, all radicals 2−5 displayed two intense UV−vis
absorption bands around 410 and 490 nm, and a broad band
around 680 nm. In phosphate buffer at pH 7.4, radicals 2 and 3
exhibited very similar spectra with bands that were about 30 nm
red-shifted when compared to those of 1a (Table 1). In
oxygenated phosphate buffer, radicals 2 and 3 displayed almost
identical EPR spectra that were characterized by a single signal
centered at g = 2.0050 with a linewidth of 380 2 mG (Figure
2A). This linewidth was sensitive to the concentration of
Reaction of 2− with O2. Reactions were performed at room
temperature in 1-cm path length quartz cuvettes previously purged
with argon and stopped with a rubber septum. A solution of 100 μM
radical 2 in degassed 0.1 M phosphate buffer at pH 7.4 containing 0.1
mM EDTA was treated with a substoichiometric amount of sodium
dithionite (65 μM) to generate the anion 2−. The effects of successive
additions of aerated buffer solution (containing ∼260 μM O2) were
monitored by UV−vis spectroscopy either by repetitive scanning
between 380 and 880 nm or by following the decrease in absorbance at
644 nm (maximal absorption of 2−).
dioxygen and decreased to 290
2 mG in anaerobic buffer
(Table 1). Because of the presence of six protons in their ester
chains, the EPR signal of radicals 2 and 3 consisted of a
septuplet with a hyperfine coupling constant aH = 0.11 G
(Figure 2B−C).32 In order to compare the EPR spectra of 2−5
under identical conditions, further experiments were performed
in a solvent where all compounds were soluble. The effects of
spin exchange between radicals 2−5 and O2 on the linewidth
broadening of the signal were thus measured in acetonitrile. All
new radicals displayed an identical behavior with a 4-fold
increase in their signal linewidth when passing from anaerobic
to aerobic conditions (Table 1).
The redox behavior of esters 2−5 was investigated by cyclic
voltammetry in methanol, a solvent where all compounds are
soluble, and compared to that of 1a. The cyclic voltammograms
recorded under argon at 0.1 V·s−1 are given as Supporting
Information (Figure S1). For all compounds, two electro-
chemical processes were identified corresponding either to the
reduction of the radical to the corresponding anion or to the
oxidation of the radical to the corresponding cation. For
compound 1a, the electrochemical reduction process showed a
nonideal, only quasi-reversible behavior, which most likely
arose from the presence of water traces in methanol that led to
a partial protonation of the anion of 1a to triarylmethane 1a-H
(see below). For compounds 3 and 4, both electrochemical
processes were fully reversible, allowing the unambiguous
determination of the corresponding standard potentials. At the
same scan rate, the waves associated with the oxidation of 2 and
5 were poorly defined because of their overlap with the very
intense oxidation wave of the associated bromide counterions.
This interpretation was confirmed by the electrochemical study
of tetrabutylammonium bromide that showed an oxidation
wave at the same potential (data not shown). Accordingly, the
standard potential of the oxidation process was better estimated
RESULTS AND DISCUSSION
■
Synthesis and Characterization of TAM Esters 2−5.
Esters 2−5 were obtained in good yields (80−90%) by the
reaction of 1a with an excess of the corresponding bromides in
the presence of Cs2CO3 and catalytic amounts of KI (see
Experimental Procedures) (Scheme 1). They were charac-
terized by UV−vis, MS, HRMS, and EPR spectroscopy (see
Experimental Procedures and Table 1). Esters 2 and 3 that bear
a trimethylammonium or sulfonate group at the end of their
alkyl-chains were soluble in water, which allowed us to study
Table 1. UV−Vis and EPR Characteristics of Radicals 1a and
2−5 in Phosphate Buffer or Acetonitrile and in the Absence
or Presence of O2
linewidth
the EPR
a
signal
c
of
b
radical
solvent
λmax (nm)
−O2
+O2
1a
2
phosphate buffer
phosphate buffer
phosphate buffer
acetonitrile
374, 469, 641
408, 491, 682
410, 491, 684
408, 490, 677
406, 487, 673
405, 487, 676
407, 489, 664
90
2
180
2
2
2
290
290
350
370
350
340
2
2
5
5
5
5
381
380
3
2
1430 20
1470 20
1430 20
1450 20
3
acetonitrile
4
acetonitrile
5
acetonitrile
a
Linewidths (in mG) measured as the differences between the two
b
highest lines of the septuplet. Phosphate buffer (0.1 M, pH 7.4) and
acetonitrile were deoxygenated by bubbling with argon for 1 h.
c
Phosphate buffer (0.1 M, pH 7.4) and acetonitrile were exposed to
normal atmosphere (260 μM O2 at 25 °C for buffer).
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Figure 2. EPR spectrum of radical 3 in phosphate buffer (0.1 M, pH 7.4) in the presence of O2 (A) or in the absence of O2 (B). Enlargement of the
spectrum recorded under anaerobic conditions to observe the hyperfine structure (C).
from the cyclic voltammograms recorded at higher scan rates (5
V·s−1, Figure S1, Supporting Information). For compound 2,
the shape of the reduction wave associated with the oxidation
process indicated some adsorption of the product on the
electrode, leading to a less precise estimation of the standard
potential associated with the reduction process. A similar
adsorption process also occurred for the reduction of
compound 5.
Electrochemical characterization of the water-soluble esters 2
and 3 was also performed in phosphate buffer (100 mM, pH 7)
and compared to that of 1a. The corresponding cyclic
voltammograms are given Figure 3. For compound 1a, the
The standard potentials obtained for compounds 2−5 are
given in Table 2 and compared to those of 1a. Replacement of
the carboxylate substituents with esters resulted in a large
increase (about 500 mV) of the oxidation and reduction
potentials.31 Easier reduction and more difficult oxidation of the
esters should be related to the greater electron-withdrawing
effect of the ester substituents relative to carboxylates. Within
this ester series, one must note the effects of their global charge
on their redox potentials. Thus, the negatively charged
compound 3 was the most difficult to reduce and the easiest
to oxidize.
Table 2. Redox Potentials of TAM Radicals 1a and 2−5
Determined by Cyclic Voltammetry in Phosphate Buffer or
Methanol
a
a
radicals
solvent
Ered
Eox
b
c
c
1a
2
phosphate buffer
phosphate buffer
phosphate buffer
methanol
−660 (−70)
−230
−325
−920
−350
415 (−410)
d
d
3
Figure 3. Cyclic voltammograms of radicals 1a, 2, and 3 (400 μM) in
phosphate buffer (0.1 M, pH 7.4, containing 0.1 mM EDTA) at 0.02
V·s−1 (solid line) and 0.5 V·s−1 (dashed line). Intensities (ordinate, in
μA) were divided by the square root of the scan rate to allow direct
comparisons.
1a
2
390
e
methanol
890
3
methanol
−435
−415
−350
820
850
890
4
methanol
e
5
methanol
a
Redox potentials (mV/saturated calomel electrode) (SCE) of TAM
reduction process was irreversible at a scan rate of 0.02 V·s−1
because of the fast protonation of the anion formed at the
electrode in aqueous solvent. Full reversibility could be
recovered by increasing the scan rate to 0.5 V·s−1. Simulation
of the cyclic voltammograms recorded at various scan rates
according to an EC mechanism, where the electrochemical
radicals 1a and 2−5 calculated as the half sum of the anodic and the
cathodic potentials for each redox process. 0.1 M phosphate buffer at
pH 7.4 containing 0.1 mM EDTA. Data from ref 29 recorded in 0.1
M phosphate buffer at pH 7.4 containing 0.1 M NaCl; redox potentials
were recalculated vs SCE. Not determined. Estimated value due to
adsorption at the electrode.
b
c
d
e
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process is coupled to a quasi-irreversible chemical reaction,
allowed us to determine the pseudo-first-order kinetic rate of
the protonation process that was estimated to be 1.2 s−1 under
our experimental conditions (see Figure S2, Supporting
Information). Assuming that the proton arises from the
phosphate buffer, the second order kinetic rate was estimated
to be 24 mol−1·L·s−1. For compounds 2 and 3, the reduction
processes were fully reversible at all scan rates. The standard
potentials associated with the reduction process are given in
Table 2. As in methanol, one observed a large increase of the
values reported for 2 and 3 as compared to those of 1a.
Moreover, the lack of reactivity of anions 2− and 3− is most
likely related to their pKa value, which should be lower than
that of 1a−, if one takes into account the greater electron-
withdrawing effect of the ester substituents relative to
carboxylates.
Stability of TAM Ester Radicals 2 and 3 in the
Presence of Rat Liver Microsomes and Cytosol. In a
first series of experiments, radicals 2 and 3 were incubated for
30 min at 37 °C in the presence of rat liver cytosol or rat liver
microsomes, in the absence of NADPH. UV−vis and HPLC
analyses of the incubation mixtures did not show any
transformation of 2 and 3 that were fully recovered unchanged.
Similar incubations performed in the presence of para-
nitrophenyl acetate, a common substrate of microsomal and
cytosolic esterases,40 showed that these esterases were active,
375 30 and 5500 155 nmol·min−1·mg protein−1 of para-
nitrophenol being formed with microsomes and cytosol,
respectively. These data showed that compounds 2 and 3
were stable toward microsomal and cytosolic esterases under
our conditions. A similar resistance to pig liver esterase was
previously reported for the methyl- and tert-butyl esters of
1a.32,33 This should be due to the great steric hindrance around
the ester group directly bound to the aryl substituents of these
helical TAM radicals.32
In a second series of experiments, similar incubations of 2
and 3 were performed in the presence of rat liver microsomes
and NADPH to study their possible reductase- and/or P450-
dependent metabolism. After 30 min of incubation under these
conditions, a UV−vis study of the reaction mixture showed that
2 and 3 were mostly recovered unchanged (96 3 and 94
3%, respectively) and that no new product could be detected
using HPLC. So, as expected for compounds having no para-
carboxyl aromatic substituents, 2 and 3 were much more stable
toward microsomal oxidation than 1a.
Figure 4. UV−vis spectra of radical 2 (solid line), its anion 2− (dotted
line), and triarylmethane 2-H (dashed line). Trityl anion 2− was
formed by the reduction of radical 2 (50 μM 2 in 0.1 M phosphate
buffer at pH 7.4 containing 0.1 mM EDTA) with an excess of sodium
dithionite.
corresponded to the anion 2− resulting from a one-electron
2+
reduction of 2 (m/z calcd for C58H81N3O6S12 = 649.6).
Protonation of these anions derived from 2 and 3 in anaerobic
phosphate buffer at pH 7.4 was very slow (6 and 10% after 30
min for 2− and 3−, respectively). It was much faster at lower
pHs and, at pH 2, one observed a complete disappearance of
the 644 nm absorbing anion in less than 5 min with the
formation of a new species absorbing at 410 nm (Figure 4).
Further HPLC-MS analyses of the resulting solution showed
that only one compound exhibiting MS characteristics expected
for the triarylmethane 2-H or 3-H was formed in these
experiments. After extraction and purification, triarylmethanes
2-H and 3-H were completely identified by HRMS, and 1H and
13C NMR spectroscopy (see Experimental Procedures and
1
Tables S1 and S2 of Supporting Information). Their H and
13C NMR spectra are very similar to those previously described
for TAM 1a-H and indicate that these three reduced
metabolites have a C-3 symmetry axis and noncoplanar
aromatic rings.24
Thus, anaerobic reduction of radicals 1a, 2, and 3 either by
NADPH in the presence of rat liver microsomes or by
dithionite led to the same final TAM-H metabolites, via the
same TAM− anions. However, the stabilities of the intermediate
anions were quite different, 1a− being hardly detectable during
the formation of 1a-H,24 whereas 2− and 3− were stable for at
least 30 min in phosphate buffer at pH 7.4. Another main
difference between 1a and 2 or 3 was the much greater rate of
anaerobic microsomal reduction of 2 and 3 in the presence of
NADPH, when compared to 1a. Table 3 shows that the initial
rates of microsomal reduction of 100 μM 2 and 3 by 1 mM
NADPH were comparable to the reduction rate of cytochrome
c by these microsomes under identical conditions (586 53
nmol·min−1·mg protein −1). By contrast, the initial rate of 1a
reduction into 1a-H under these conditions was very much
smaller (29 2 nmol 1a-H·min−1·mg protein−1). These greater
rates of reduction of 2 and 3 and greater stability of their anions
when compared to those of 1a were in agreement with their
greater reduction potentials (Table 2).
From these data, it appeared that esters 2 and 3 were stable
toward esterases and much more resistant toward microsomal
metabolism than 1a. However, as the small part of 2 and 3
(about 5%) consumed upon microsomal incubation in the
presence of NADPH could have come from their reduction
into triarylmethanes whose retention times were identical to
those of 2 and 3, we have more deeply investigated their
possible reductive metabolism.
Microsomal Reduction of 2 and 3 under Anaerobic
Conditions. UV−vis studies of incubations of 2 or 3 with rat
liver microsomes in the presence of NADPH under anaerobic
conditions showed a fast disappearance of the bands of the
starting compounds and the appearance of an intense band at
644 nm (Figure 4). The corresponding species have been
prepared by a reaction of 2 or 3 with sodium dithionite and
characterized by MS. Thus, the direct injection into the mass
spectrometer of a solution of 2 just after its reduction by
dithionite led to a molecular ion at m/z = 649.7 that well
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Table 3. Comparison of the Anaerobic Reduction Rates of 2
and 3 to the Anions 2− and 3− Catalyzed by Rat Liver
Microsomes
a
radicals
reduction rates
2
3
510 40
450 30
a
The initial rates of anaerobic reduction of radicals 2 and 3 were
determined by UV−vis spectroscopy by monitoring the absorbance at
644 nm and quantified using Δε values 31,800 and 36,300 M−1·cm−1
for 2− and 3−, respectively. The protonation of the anions has been
neglected on the time scale of the experiments (3 min). Incubations
were performed at 37 °C in deoxygenated 0.1 M phosphate buffer at
pH 7.4 containing 0.1 mM EDTA, 100 μM 2 or 3, 0.005−0.008 mg
microsomal protein·mL−1, and 1 mM NADPH. Rates are expressed in
nmol radical reduced·min−1·mg prot−1 and are the means SD from 3
experiments.
Microsomal Reduction of 2 and 3 under Aerobic
Conditions. Careful examination of the UV−vis spectra of
aerobic incubations of 2 and 3 with rat liver microsomes in the
presence of NADPH revealed the formation of the 644 nm
absorbing species corresponding to anions 2− and 3−. However,
as indicated above, 2 and 3 were found in great part unchanged
after 30 min of reaction. A possible explanation for these results
could be that 2− and 3− would be reoxidized by O2 into 2 and
3, respectively. Figure 5A shows that progressive additions of
phosphate buffer at pH 7.4 containing 260 μM O2 to an
anaerobic solution of 2− previously prepared by reduction of
100 μM 2 with substoichiometric amounts of dithionite (65
μM) led to the progressive disappearance of the 644 nm peak
of 2− and appearance of the characteristic peak of 2 at 491 nm.
Figure 5B shows that 0.25 mol of O2 was sufficient to reoxidize
1 mol of 2− into 2, which means that 1 mol of O2 was able to
oxidize 4 mol of 2−.
These data indicated that radicals 1a, 2, and 3 underwent a
one-electron reduction into the corresponding anions by
NADPH-containing liver microsomes under anaerobic con-
ditions. Anions 2− and 3− were stable in the incubation
medium (phosphate buffer at pH 7.4), whereas 1a− would be
quite rapidly protonated with the formation of 1a-H. It has
been reported that such microsomal reduction of 1a was
catalyzed by the P450/P450 reductase system,24 and it is likely
that this system was also involved in the reduction of 2 and 3.
Under aerobic conditions, 1a cannot compete with dioxygen
for the electrons coming from the P450/P450 reductase
system, and it was not reduced under these conditions.24 By
contrast, 2 and 3, which have higher reduction potentials
(Table 2), successively compete with O2 to receive electrons
with the formation of 2− and 3−. However, these anions are
rapidly oxidized by O2 leading back to the starting radicals 2
and 3. The latter radicals thus catalyzed a futile cycle of O2
reduction by NADPH-containing microsomes (Scheme 2) and
were almost not consumed in those reactions. This could
explain why they appear globally stable after aerobic micro-
somal incubation (see above). The small amounts of 2 and 3
(about 5%) lost after 30 min of aerobic incubation in the
presence of rat liver microsomes and NADPH could be due to
the formation of 2-H and 3-H upon protonation of 2− and 3−,
which is slow at pH 7.4.
Figure 5. Reaction of anion 2− with O2. (A) UV−vis spectra showing
the effects of the stepwise additions of a buffer solution containing 260
μM O2 to a solution of the anion formed upon the reduction of radical
2 (100 μM in 0.1 M phosphate buffer at pH 7.4 containing 0.1 mM
EDTA) by substoichiometric amounts of sodium dithionite (65 μM).
(B) Plot of the absorbance at 644 nm as a function of the O2
equivalents added to 2−.
the presence of NADPH with the formation of diamagnetic
QM metabolites resulting from oxidative decarboxylation. In an
effort to obtain TAM derivatives potentially more metabolically
stable in vivo, we have synthesized four new TAM radicals, 2−5,
in which the carboxylate sustituents of 1a were replaced with
esters bearing various alkyl chains that were designed to render
them water-soluble. They were characterized by UV−vis, EPR
spectroscopy, HRMS, and electrochemistry. Two of them, 2
and 3, were water soluble enough to undergo detailed
microsomal metabolic studies in comparison with 1a. They
were found to be metabolically stable toward the esterases
present in rat liver microsomes and cytosol. Radicals 2 and 3
were also found to be oxidation-resistant and globally stable in
the presence of aerobic NADPH-supplemented microsomes,
contrary to 1a. However, a careful study of their possible
microsomal reduction under anaerobic or aerobic conditions
CONCLUSIONS
■
TAM radicals 1a and 1b are useful EPR probes for oximetry;
however, they are rapidly metabolized by liver microsomes in
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a
Scheme 2. Fates of Radical 2 in the Presence of Rat Liver Microsomes and NADPH
a
Radical 2 was reduced to the corresponding anion 2− that was either protonated to triarylmethane derivative 2-H in the absence of O2 or oxidized
by O2 to starting radical 2 under aerobic conditions.
the trinity of normoxia, hypoxia, and hyperoxia in disease and therapy.
Antioxid. Redox Signaling 9, 1717−1730.
showed that they were more easily reduced than 1a, in
agreement with their higher reduction potentials. They were
reduced into the corresponding anions not only under
anaerobic conditions but also in the presence of dioxygen.
These anions were much more stable than that of 1a and have
been characterized by UV−vis spectroscopy, MS, and at the
level of their protonated diamagnetic products. However, they
were oxidized by O2, giving back the starting radicals 2 or 3 and
catalyzing a futile cycle of O2 reduction. Such reactions should
be considered in the design of future metabolically stable TAM
EPR probes for oximetry in vivo.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Assignment of 1H and 13C NMR signals of triarylmethanes 2-H
and 3-H; cyclic voltammograms of 1a and 2−5 recorded in
methanol; and experimental cyclic voltammograms of 1a in
phosphate buffer and simulations obtained with the DigiElch
program. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 33 1 42 86 21 91. Fax: 33 1 42 86 83 87. E-mail:jean-luc.
boucher@parisdescartes.fr.
Present Address
§Y.X.-L.: Laboratoire de Chimie des Processus Biologiques,
̀
College de France, 11 Place M. Berthelot, 75005 Paris, France.
Funding
This work was supported by a fellowship to C.D. from the
̀
French “Ministere de l’Education et de la Recherche.”
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Y. Frapart (UMR 8601 CNRS, Paris) for his help
in EPR experiments.
(11) Burks, S. R., Bakhshai, J., Makowsky, M. A., Muralidharan, S.,
Tsai, P., Rosen, G. M., and Kao, J. P. (2010) 2H,15N-Substituted
nitroxides as sensitive probes for electron paramagnetic resonance
imaging. J. Org. Chem. 75, 6463−6467.
ABBREVIATIONS
■
(12) Jordan, B. F., Baudelet, C., and Gallez, B. (1998) Carbon-
centered radicals as oxygen sensors for in vivo electron paramagnetic
resonance: screening for an optimal probe among commercially
available charcoals. Magn. Reson. Mater. Phys. Biol. Med. 7, 121−129.
(13) Pandian, R. P., Parinandi, N. L., Ilangovan, G., Zweier, J. L., and
Kuppusamy, P. (2003) Novel particulate spin probe for targeted
determination of oxygen in cells and tissues. Free Radical Biol. Med. 35,
1138−1148.
DMF, N,N-dimethylformamide; EPRI, electron paramagnetic
resonance imaging; ESI, electrospray ionization; HRMS, high-
resolution mass spectrometry; pO2, dioxygen partial pressure;
QM, quinone-methide; SCE, saturated calomel electrode;
TAM, triarylmethyl radical
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