Synthesis and characterization of a perchlorotriphenylmethyl (trityl) triester radical: A potential sensor for superoxide and oxygen in biological systems

Article abstract of DOI:10.1016/j.bmcl.2007.04.070

Synthesis and characterization of an inert perchlorotriphenylmethyl triester radical, PTM-TE, are reported. PTM-TE was prepared by a facile 3-step synthesis using Friedel-Crafts reaction of tetrachlorobenzene with chloroform followed by ethoxycarbonylatio

Full text of DOI:10.1016/j.bmcl.2007.04.070

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4062–4065
Synthesis and characterization of a perchlorotriphenylmethyl
(trityl) triester radical: A potential sensor for superoxide
and oxygen in biological systems
Vinh Dang,a Jinhua Wang,b Song Feng,b Christophe Buron,a Frederick A. Villamena,a
Peng George Wangb and Periannan Kuppusamya,*
aCenter for Biomedical EPR Spectroscopy and Imaging, Davis Heart and Lung Research Institute,
Division of Cardiovascular Medicine, Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, USA
bDepartment of Chemistry, The Ohio State University, Columbus, OH 43210, USA
Received 6 March 2007; revised 21 April 2007; accepted 23 April 2007
Available online 27 April 2007
Abstract—Synthesis and characterization of an inert perchlorotriphenylmethyl triester radical, PTM-TE, are reported. PTM-TE
was prepared by a facile 3-step synthesis using Friedel-Crafts reaction of tetrachlorobenzene with chloroform followed by ethoxy-
carbonylation and subsequent oxidation. PTM-TE is paramagnetic and exhibits a single sharp EPR spectrum. In solution, the EPR
linewidth of PTM-TE is highly sensitive to the dissolved oxygen content, thus enabling accurate measurement of oxygen concentra-
tion (oximetry). In addition, the radical also shows high reactivity towards superoxide. The ester radical has the potential for use as a
high-sensitive probe for determination of oxygen concentration and superoxide in biological systems.
ꢀ 2007 Elsevier Ltd. All rights reserved.
In 1900, Moses Gomberg reported the existence of a sta-
ble, trivalent organic free radical which challenged the
prevailing belief that carbon could only have four chem-
ical bonds.1 As this report provided new possibilities to
carbon chemistry, research aimed at understanding and
developing these radicals continued to thrive and perme-
ated various fields of science including medicine and
industrial applications. Most notably, Gomberg’s work
paved the way for future research on using persistent tri-
tyl radicals in clinical applications.2–4 However, it is
known that some radicals and diradicals are short-lived
and very reactive – the half-life of triphenylmethyl rad-
icals in aerated solution may be as low as a fraction of
a second. Studies have also shown that these reactive
radicals become stable and chemically inert upon per-
chlorination.5–8 In light of this endeavor, highly chlori-
nated mono-, di-, and triarylmethanes were developed
and since then they have become the most valuable
chemical precursors of inert free radicals with estimated
half-lives in the order of 100 years.6 Together, these
studies have been regarded as both promising and signif-
icant.5 As a result, the synthesis of perchlorinated trityl
radical has become an immense undertaking with prom-
ising implications.
The role of reactive oxygen species (ROS) such as super-
oxide, hydroxyl, and alkylperoxyl radicals has been
linked to a variety of pathophysiological processes.9
Since an increased production of ROS, especially super-
oxide, leads to oxidative stress, accurate determination
of these free radicals can help us to gain a better under-
standing of oxidative stress-related diseases. Of the
many techniques currently available for the detection
of superoxide in biological systems, electron paramag-
netic resonance (EPR) spectroscopy has the distinct
capability for direct and real-time detection. However,
the EPR technique requires the use of high concentra-
tions (10–100 mM) of spin-trap molecules to maximize
the efficiency of trapping, which may alter the redox
environment of the system.10,11 The poor efficiency of
the spin-trap molecules for superoxide detection is
attributed to small bimolecular reaction rate constants
(typically about 102 Mꢀ1 sꢀ1). Thus, there is a great need
to develop probes with orders of magnitude increased
Keywords: Perchlorotriphenylmethyl radical; Perchlorotriphenylmethyl
triester; Superoxide; Free radicals; EPR; Oximetry; Spectrophotometry.
*
Corresponding author. Tel.: +1 614 292 8998; fax: +1 614 292
8454; e-mail: kuppusamy.1@osu.edu
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.070

V. Dang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4062–4065
4063
reactivity with superoxide. To this end, we have devel-
oped perchlorotriphenylmethyl triester radical
a
Solid
(PTM-TE) that can overcome this limitation. PTM-TE
has a high detection-sensitivity due to sharp single-line
EPR spectrum. In addition, PTM-TE is an attractive
candidate for measurement of oxygen concentration
(oximetry). Furthermore, the ester nature of the PTM-
TE radical is expected to facilitate its penetration into
cells, followed by hydrolysis by cellular enzymes such
as esterases into carboxylate radical and thus can be
trapped in the cells. This will enable the detection of
superoxide radicals produced inside the cells. As an oxy-
gen probe, PTM-TE can be utilized for measurement as
well as mapping (imaging) of oxygen distribution in tis-
sues.12 In this communication, we report the synthesis,
characterization, and preliminary evaluation of PTM-
TE for oximetry and detection of superoxide.
*
*
Solution
3479
3487
3496
Magnetic Field (G)
Figure 1. EPR spectra of PTM-TE. The spectra were measured from a
sample of crystalline powder and in a solution of PTM-TE (100 lM) in
DMSO at ambient conditions using an X-band EPR spectrometer. The
EPR acquisition settings were: modulation amplitude, 160 mG;
microwave power, 1 mW; time constant, 40 ms; scan time, 15 s. The
first derivative peak-to-peak widths were 5.650 G for solid and 0.540 G
The synthesis of PTM-TE radical 4 (Scheme 1) used
tris(2,3,5,6-tetrachlorophenyl)methane generated from
the Friedel-Crafts reaction of 1,2,4,5-tetrachlorobenzene
and chloroform.13 Reaction of compound 2 with slight
excess of butyllithium and TMEDA in THF at low tem-
perature yielded the corresponding tri-anion. This tri-
anion was reacted with ethylchloroformate in situ to
yield compound 3.14 Compound 3 was then converted
into 4 by a two-step process: conversion into the triphe-
nylcarbanion with NaOH in a mixture solution of
DMSO and Et2O, followed by oxidation with I2.15 Since
compound 3 has the ethoxycarbonyl functional group, it
can be easily modified to afford more variants for future
research.
for solution. The small satellite peaks indicated with on both sides of
*
the main peak are due to hyperfine coupling with 13C nuclei (natural
abundance).
dependent on the concentration of dissolved molecular
oxygen in solution. We measured the linewidth of
PTM-TE (100 lM) in DMSO as well as in hexafluoro-
benzene (HFB) as a function of partial pressure of equil-
ibrating oxygen (pO2). As shown in Figure 2, the
linewidth increased proportionally to pO2. The oxy-
gen-induced broadening is attributed to the paramag-
netic nature of molecular oxygen, which upon collision
with PTM-TE molecule can undergo Heisenberg spin
exchange interaction resulting in broadening of the ob-
served EPR signal. It should be noted that this exchange
interaction does not chemically alter the two molecules
involved. Furthermore, the oxygen-induced broadening
has no effect on the EPR signal intensity (double-inte-
PTM-TE was characterized by EPR spectroscopy
(Fig. 1). The solid showed a broad EPR signal with a
peak-to-peak width (linewidth, DBpp) of 5.650 G in room
air. However, a solution of PTM-TE (100 lM) in
dimethylsulfoxide (DMSO) at ambient conditions
showed a single sharp peak with a linewidth of
0.540 G. No self-induced spin-spin broadening was ob-
served for up to 12 mM concentration of PTM-TE.
The linewidth of PTM-TE in DMSO, however, was
7
6
Cl
Cl
in HFB
Cl
Cl
5
Cl
Cl
Cl
Cl
Cl
Cl
4
3
a
b
Cl
Cl
Cl
Cl
Cl
Cl
1
2
2
in DMSO
Cl
Cl
Cl
Cl
Cl
Cl
EtOOC
Cl
Cl
Cl
COOEt
Cl
1
0
EtOOC
Cl
Cl
Cl
COOEt
Cl
c
Cl
Cl
Cl
Cl
0
50
100
150
pO2 (mmHg)
200
250
300
350
Cl
Cl
Cl
Cl
COOEt
Cl
Cl
COOEt
Figure 2. Effect of oxygen on the EPR linewidth of PTM-TE in
solution. Measurements were made by equilibrating a solution of
PTM-TE (100 lM) in DMSO or HFB with pre-calibrated gas mixtures
of known O2 concentration. The data show a linear dependence of
linewidth with pO2. The oxygen-sensitivity was 17.71 mG/mmHg for
HFB and 1.6 mG/mmHg for DMSO.
3
4
Scheme 1. Synthesis of PTM-TE radical. Reagents and conditions: (a)
CHCl3, AlCl3; (b) n-BuLi, TMEDA, ethyl chloroformate, 77%; (c) 1—
NaOH, DMSO/Et2O, 2—I2, Et2O, 93%.

4064
V. Dang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4062–4065
gral of the EPR spectrum). Hence, the width of the EPR
signal can be used to measure the concentration of O2 in
the system. The calibration curves of EPR linewidth ver-
sus pO2 can be generated for any solvent by equilibrat-
ing it with known concentrations of oxygen.
PTM-TE was specifically designed with the ethoxycar-
bonyl substituent attached to each of the phenyl rings
for a specific purpose, that is, cellular internalization.
The hydrophobic character of the molecule permits it
to easily diffuse across the membrane lipid bilayers.
Then, by the nonspecific action of intracellular esterases,
the molecule is expected to be cleaved to yield the corre-
sponding tricarboxylate radical, PTM-TC. The resulting
PTM-TC is another stable ‘inert’ radical which also
gives a single sharp EPR peak in aqueous solutions.17
Additionally, PTM-TC gives a characteristic UV–vis
absorption at 380 nm. Our recent study has shown that
PTM-TC, on reaction with superoxide, induces a de-
crease in EPR signal or optical absorption at 380 nm
with high specificity and sensitivity.18 Thus, PTM-TE
can be used to monitor and quantify the production of
intracellular superoxide.
The results shown in Figure 2 indicate a significant con-
trast between the slopes of the calibration curves of
PTM-TE in DMSO (1.6 mG/mmHg) and HFB
(17.71 mG/mmHg). The higher sensitivity in HFB is lar-
gely attributed to higher solubility of oxygen in HFB.
For example, the solubility of oxygen at atmospheric
pressure (21% of oxygen) at 25 ꢁC is 4400 lM in HFB
as compared to 478 lM in DMSO.16 In addition, viscos-
ity and polarity of solvent molecule may also contribute
to the observed difference.
The reactivity of PTM-TE to superoxide was deter-
mined. PTM-TE in DMSO (0.8 mM) was treated with
a solution of potassium superoxide (KO2) in DMSO
(2.4 mM). As shown in Figure 3, the EPR spectrum of
PTM-TE was completely quenched by superoxide sug-
gesting that PTM-TE radical reacts with superoxide to
form a diamagnetic product. Although the mechanism
by which PTM-TE reacts with superoxide is unclear,
previous reports suggested that the neutral protonated
In summary, we have developed an efficient synthetic
route for the synthesis of PTM-TE. Additionally, we
have demonstrated the effect of molecular oxygen and
superoxide on the EPR spectrum of PTM-TE in solu-
tion. Unlike molecular oxygen, superoxide induces a sig-
nal loss. Thus, PTM-TE potentially can be used to
determine oxygen and/or superoxide generation in bio-
logical applications.
form of superoxide, OOH, rather than OÅ2ꢀ is involved
Å
in the reaction, as illustrated in the following equation:
½R3CÅꢁ þ ÅOOH ! ½R3C–OOHꢁ
It is likely that the superoxide anion or OOH radical
Acknowledgment
Å
This study was supported by NIH Grant EB004031.
interacts with PTM-TE to form a covalent bond be-
tween the methyl radical epicenter and the oxygen atom
Å
of OOH.10 The resulting adduct is diamagnetic
and hence, EPR silent. This observation is confirmed
References and notes
1
by H NMR spectroscopy where only the triplet and
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quartet splittings for the ethoxycarbonyl substituent
were identified. The absence of a singlet peak at d 7
(ppm) corresponding to the epicenter methine proton
(data not shown) indicates that the resulting compound
appears most likely to be the proposed adduct,
[R3C–OOH].
5. Ballester, M.; Pascual, I.; Carreras, C.; Vidal-Gancedo, J.
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3484
3489
3494
Magnetic Field (G)
14. Tris(2,3,5,6-tetrachlorophenyl)methane (486 mg, 0.74 mmol)
and TMEDA (377 ll, 2.4 mmol) were dissolved in dry
THF (50 ml) under dry nitrogen atmosphere and cooled to
ꢀ78 ꢁC. A solution of 1.6 M butyllithium in hexanes
(1.6 ml, 2.5 mmol) was added in one portion and the
Figure 3. The effect of superoxide on the EPR spectrum of PTM-TE
radical in DMSO. (A) EPR spectrum of PTM-TE in DMSO (0.8 mM).
(B) EPR spectrum of PTM-TE in DMSO following addition of solid
KO2 (2.4 mM). The PTM-TE signal is quenched by O2Åꢀ
.

V. Dang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4062–4065
4065
mixture was stirred at low temperature for 1 h. The
reaction was quenched with ethyl chloroformate and
allowed to reach room temperature overnight. Solvent
was evaporated and the residue dissolved in dichloro-
methane. The organic layer was washed with water and
dried over sodium sulfate. Solvent was evaporated under
vacuum. Silica gel chromatography (hexanes/AcOEt:12/1)
yielded 500 mg (77%) of tris(2,3,5,6-tetrachloro-4-ethoxy-
The mixture was filtered through a dry funnel into a
solution of iodine (1.9 g) in Et2O (100 ml). The solution
was left undisturbed in the dark for 24 h. The resulting
solution was then washed with concentrated NaHCO3
(39%, 50 ml), brine, and then water (50 ml · 2). Solvent
was dried over Na2SO4 and evaporated. The residue was
purified by silica gel chromatography (Hexane/AcOEt: 80/
20) to give a red solid (1.378 g, 92.5%). EPR (solid, 298 K):
single peak, DBpp = 5.650 G. UV–vis (hexane): kmax(e) 289
(5010), 378 (8830). IR: m 2938, 1740, 1536, 1445, 1380,
1329, 1284, 1222, 1136, 1007, 858, 720 cmꢀ1. HRMS
[M+Na]+ calculated for C28H15Cl12O6Na 895.694, found
895.692.
1
carbonyl-phenyl)methane 3 as a colorless solid. H NMR
(400 MHz, CDCl3) d 7.00 (s, 1H); 4.47 (q, J = 7.1 Hz, 6H);
1.41(t, J = 7.1 Hz, 9H); 13C NMR (100 MHz, CDCl3) d
163.2, 138.5, 135.5, 135.0, 134.0, 130.5, 129.5, 63.1, 56.3,
14.0. UV–vis (hexanes): kmax(e) 292 (2220), 302 (2480). IR:
m 2982, 2936, 1743, 1556, 1465, 1446, 1371, 1341, 1329,
16. Murov, S. L.; Hug, G. L.; Carmichael, I. Hand Book of
Photochemistry; M. Dekker: New York, 1993.
17. Maspoch, D.; Gerbier, P.; Catala, L.; Ruiz-Molina, D.;
Vidal-Gancedo, J.; Wurst, K.; Rovira, C.; Veciana, J.
Chem. Eur. J. 2002, 8, 3635.
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D.; Roques, N.; Veciana, J.; Rovira, C.; Kuppusamy, P.
Biochem. J. (in review).
1299, 1263, 1225, 1208, 1121, 1096, 1019, 859 cmꢀ1
.
HRMS [M+Na]+ calcd for C28H16Cl12O6Na 896.7018,
found 896.7041.
15. Tris(2,3,5,6-tetrachloro-4-ethoxycarbonyl-phenyl)methane
(1.49 g, 1.78 mmol) was dissolved in the mixture of
DMSO/Et2O (75/425). To the solution, powdered NaOH
was added and the mixture was stirred in the dark for 24 h.