Perchlorotrityl radical-fluorophore conjugates as dual fluorescence and EPR probes for superoxide radical anion

Article abstract of DOI:10.1016/j.bmc.2009.11.034

Perchlorotrityl radicals, mono-substituted with a fluorophore using an amide linker of varying chain length, were synthesized and characterized. Electron paramagnetic resonance (EPR) spectroscopic study indicated free-electron coupling with the aromatic hydrogen nuclei and long-range coupling with the methylene hydrogens of the linker group. Reactivity of the fluorophore-conjugated trityls with superoxide radical anion showed quenching of EPR signal and enhancement of fluorescence emission spectrum. This work presents the first example of a perchlorotrityl-fluorophore conjugate that can potentially be employed as a dual probe for the detection of superoxide under oxidative stress-mediated conditions in biological systems.

Full text of DOI:10.1016/j.bmc.2009.11.034

Bioorganic & Medicinal Chemistry 18 (2010) 922–929
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Perchlorotrityl radical-fluorophore conjugates as dual fluorescence
and EPR probes for superoxide radical anion
Jinhua Wang b, Vinh Dang a, Wei Zhao d, Dongning Lu b, Brian K. Rivera a, Frederick A. Villamena c,
Peng George Wang b, Periannan Kuppusamy a,
*
a Davis Heart and Lung Research Institute, Department of Internal Medicine, 420 West 12th Ave, Room 114, The Ohio State University, Columbus, OH 43210, USA
b Davis Heart and Lung Research Institute, Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA
c Davis Heart and Lung Research Institute, Department of Pharmacology, The Ohio State University, Columbus, OH 43210, USA
d College of Pharmacy, Nankai University, Tianjin 300071, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Perchlorotrityl radicals, mono-substituted with a fluorophore using an amide linker of varying chain
length, were synthesized and characterized. Electron paramagnetic resonance (EPR) spectroscopic study
indicated free-electron coupling with the aromatic hydrogen nuclei and long-range coupling with the
methylene hydrogens of the linker group. Reactivity of the fluorophore-conjugated trityls with superox-
ide radical anion showed quenching of EPR signal and enhancement of fluorescence emission spectrum.
This work presents the first example of a perchlorotrityl-fluorophore conjugate that can potentially be
employed as a dual probe for the detection of superoxide under oxidative stress-mediated conditions
in biological systems.
Received 12 September 2009
Revised 11 November 2009
Accepted 14 November 2009
Available online 4 December 2009
Keywords:
Superoxide
Perchlorotrityl
EPR
Ó 2009 Elsevier Ltd. All rights reserved.
Free radicals
Fluorescence
1. Introduction
tual production of O2ÁÀ, whereas the cytochrome c reduction assay
is affected by the presence of oxidoreductases in cellular sys-
Superoxide radical anion (O2ÁÀ), a reactive free radical species
formed by one-electron reduction of molecular oxygen, has been
implicated in a variety of disease conditions such as ischemia-
reperfusion injury, diabetes, atherosclerosis, chronic inflammation,
viral infection, and malignant diseases.1 Superoxide produced by
mammalian cells serves as a signaling molecule that can modulate
events such as enzyme phosphorylation, cell growth, hypertrophy
tems.8,9 The EPR method requires the use of high concentrations
(10–200 mM) of spin trap that may perturb the redox balance in
the biological system.
Superoxide requires a probe that is sensitive enough to effi-
ciently compete with intracellular components for its detection.
This is due to a relatively short half-life of the molecule and the
presence of endogenous intracellular antioxidant enzymes that
efficiently scavenge O2ÁÀ. Additionally, these probes must have tar-
get specificity to sub-cellular compartments to reflect radical pro-
duction at their site of formation. One of the most promising
approaches to monitor the redox status or the detection of free
radicals in cells is the use of bi-functional fluorogenic spin probes
that can be detected by fluorescence and/or EPR spectroscopy. In
the past, such dual probes containing a fluorescent chromophore
and a paramagnetic nitroxide radical have been reported.10–13 In
these molecules, the paramagnetic nitroxide moiety acts as a
quencher of the fluorescence of the chromophore fragment. Reduc-
tion of the nitroxide by free radicals results in the formation of dia-
magnetic moiety and hence can cause decay of the EPR signal and
in turn, will potentiate fluorescence. Hence, the conjugate allows
all the nitroxide spin probe features to be retained, but also may
gain new advantages by being able to monitor redox and radical
production processes by a very sensitive and simpler fluorescence
technique. However, the chemical property of nitroxide limits its
and apoptosis,2,3 but increased O2 production can lead to cellular
dysfunction. Therefore, there is a need for improved techniques for
the detection and quantification of O2 in order to gain knowledge
on its role in the progression of oxidative stress-related diseases
and pathophysiological conditions.
ÁÀ
ÁÀ
A variety of analytical techniques have been developed to detect
and quantify O2 radicals in biological systems. These include
ÁÀ
spectrophotometric methods that use reduction of ferricytochrome
c or nitroblue tetrazolium (NBT) by O2ÁÀ, chemiluminescence, fluo-
rescence-based techniques and electron paramagnetic resonance
(EPR) spectroscopy using spin-trapping techniques.4–7 However,
these methods have certain limitations for measuring O2ÁÀ. In the
lucigenin-based luminescence detection of O2ÁÀ, it has been dem-
onstrated that the probe itself can redox-cycle, resulting in artifac-
* Corresponding author.
E-mail address: Kuppusamy.1@osu.edu (P. Kuppusamy).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.034

J. Wang et al. / Bioorg. Med. Chem. 18 (2010) 922–929
923
application for intracellular radical detection due to its susceptibil-
ity to be metabolically reduced in the cell. Thus, a highly EPR sen-
sitive (narrow/single line) probe with good biostability against
cellular oxidoreductants and high specificity for O2 is desirable
for radical detection in biological systems.
ethylene oxide, afforded compound 2 with spacer(s) incorporated
(Scheme 1). By controlling the molar ratios of 1, ethylene oxide
and base, compounds 2a or 2b can be isolated as major products.
However, only small amounts of compound 2c were formed even
when six equivalents of ethylene oxide and n-butyl lithium were
added. It was found that compounds 2a and 2b have good solubility
in THF and dichloromethane, while compound 2c was insoluble in
any commonly-used solvents. The solubility differences in com-
pounds 2a–2c can control the equilibrium concentrations of these
three compoundsin solution. The primary amine, 4, was synthesized
viaMitsunobureactionusing2aandphthalimidefollowedbyhydra-
zinolysis, with sufficiently high purity for use in the succeeding step
according to previously establish procedure.20 Two different linker
distances were introduced to compounds 4 in order to shed insights
into how these distances can affect the chromophore-radical inter-
action. Amidation reaction of 4 with both 1-pyrenecarboxylic acid
N-hydroxysuccinimide ester and pyrenebutyric acid N-hydroxy-
succinimide ester was carried on to form fluorescence chromo-
phore-conjugated compounds 5 and 6, respectively.21 Finally,
radicals7 and 8 were obtained by treatingcompounds5 or 6 with ex-
cess of nBu4N+OHÀ and subsequent oxidation of the resulting anions
with p-chloranil in a one-pot reaction with near quantitative yield.22
Two methods were employed for the synthesis of 10 (Scheme 2).
In Method A, treatment of 1 with 2.4 equiv of n-BuLi and TMEDA fol-
lowed by 2.4 equiv of ethyl chloroformate afforded di-ethoxycar-
bonyl trityl 9 in 46.7% yield as well as mono-ethoxycarbonyl trityl,
which can be reused.19 However, reaction of 9 with ethylene oxide
gave poor yield and a complex mixture that was difficult to charac-
terize. The unsuccessful synthesis of 10 using Method A could be
due to the susceptibility of ethoxycarbonyl functionality to nucleo-
philic addition by ethylene oxide under strong basic condition.
ÁÀ
Recently, triarylmethyl (trityl) radicals have been reported as
probes for detecting O2ÁÀ in biological systems.14,15 Perchlorotriphe-
nylmethyl radicals are a novel class of trityls whose structures are
based on perchlorotriphenyl methyl radical (PTM). Due to the full
steric blockage of the central carbon, the PTM radical and its deriva-
tives are highly stable against a variety of reactive chemical agents,
and hence are called ‘inert free radicals’.16,17 The ability of two
derivatives, tris(2,3,5,6-tetrachloro-4-carboxylate-phenyl) methyl
radical (PTM-TC) and tris(2,3,5,6-tetrachloro-4-ethyloxycarbonyl-
phenyl) methyl radical (PTM-TE), to trap O2ÁÀ have been tested.18,19
These species demonstrated enhanced stability in biological sys-
tems, excellent water solubility (in the cases of carboxylic acid),
and a narrow, single EPR signal. Therefore, the development of tri-
tyl-fluorophore conjugate is proposed which may offer improved
ÁÀ
O2 detection by using EPR and/or fluorescence techniques.
2. Results and discussion
2.1. Synthesis
Due to the steric hindrance and high stability of tris(2,3,5,6-tetra-
chlorophenyl)methane, 1, it is difficult to directly derivatize the
compound with a chromophore. Instead, our initial efforts involved
adding a linker with appropriate functional group between the trityl
core structure and the chromophore. Treatment of 1 with n-butyl
lithium at À78 °C followed by nucleophilic addition of the trityl to
Cl
Cl
Cl
Cl
Cl
Cl
R2
Cl
Cl
Cl
R1
Cl
n
-BuLi , TMEDA
Cl
Cl
ethylene oxide
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
R3
1
2a R1 = CH2CH2OH, R2, R3 = H;
2b R1, R2 = CH2CH2OH, R3 = H;
2c R1, R2, R3 = CH2CH2OH;
O
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H2N
N
O
Cl
phthalimide, PPh3,
DIAD, THF
NH2-NH2
90.5%
2a
Cl
Cl
Cl
Cl
97.7%
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
4
3
O
N
O
n
Cl
Cl
Cl
Cl
Cl
Cl
H
N
O
H
N
O
Cl
Cl
Cl
Cl
n = 0 or 3
Et3N, THF
n
O
n
O
1)
n
BuNOH, THF
Cl
Cl
Cl
Cl
2) p-chloranil
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
7 n = 0, 99.3%
8 n = 3, 95.4%
5 n = 0, 59.3%
6 n = 3, 67.0%
Scheme 1. Synthesis of compounds 7 and 8.

924
J. Wang et al. / Bioorg. Med. Chem. 18 (2010) 922–929
Therefore, an alternative method (Method B) was carried out in
which1 wasreactedwith ethyleneoxide firstfollowedbyethylchlo-
roformate. Although one more hydrolysis step was required, the
overall yield increased twofold. Following the earlier procedure,
radical 15 was synthesized from intermediate 10 (Scheme 3) which
was slightly soluble in water, making radical 15 possible to be char-
acterized in aqueous solution.
group, suggesting that the unpaired electron is delocalized over the
whole PTM moiety (see Table 1 for the simulated EPR parameters).
However, trityl 15 exhibited a triplet spectrum with 1:2:1 ratio due
to the methylene protons from the linker group. The observed dif-
ferences in hyperfine coupling constants of the two aromatic and
methylene protons are due to the asymmetry of the molecule as
shown in Figure 3.
2.3. Reactivity with superoxide radical anion
2.2. EPR Studies
2.3.1. EPR Spectroscopy
Figure 1 shows the solid state EPR spectra of fluorophore-trityls
The addition of KO2 DMSO solution to trityls 7, 8 or 15 resulted
in the disappearance of their EPR signal (Fig. 4) suggesting the for-
mation of a diamagnetic product. Using the analogue perchorotri-
tylmethyl-tricarboxylate, PTM-TC, we postulated that the nature of
7, 8 and 15. The peak-to-peak linewidth (DBpp) for 15 is the broad-
est compared to 7 and 8. In spite of the absence of aromatic pro-
tons in 15, this suggests that the line-broadening mechanism is
caused mostly by spin–spin exchange interaction. It can be as-
sumed that the packing interaction for 15 is more efficient due,
perhaps, to the short linker group that allows a more ordered lat-
tice structure. Figure 2 shows the EPR of spectra 7, 8 and 15 in
DMSO under anaerobic conditions. Both 7 and 8 exhibited a multi-
plet (7-line) EPR spectrum arising from the hyperfine coupling of
two aromatic and two methylene hydrogen atoms from the linker
ÁÀ
PTM reactivity to O2 is a redox reaction resulting in the formation
of the reduced product, perchlorotriphenylmethane and oxidation
ÁÀ
of O2 to molecular oxygen as analyzed by electrochemical and
mass spectrometric methods.18 Competitive kinetics experiments
revealed that PTM-TC reacts with O2ÁÀ with an apparent second-or-
der rate constant of 8.3 Â 108 MÀ1 sÀ1. The high reactivity of PTM-
Method A
Cl
Cl
Cl
Cl
O
O
Cl
Cl
Cl
Cl
HO
OEt
OEt
n
-BuLi , TMEDA
n
-BuLi , TMEDA
ethylene oxide
ClCOOEt
Cl
Cl
Cl
Cl
1
Cl
Cl
Cl
Cl
Cl
Cl
9.9%
46.7%
Cl
Cl
Cl
Cl
Cl
Cl
O
O
EtO
EtO
9
10
Method B
Cl
Cl
Cl
Cl
Cl
O
Cl
Cl
HO
Cl
Cl
EtO
O
OEt
n
-BuLi , TMEDA
n
-BuLi , TMEDA
O
Cl
Cl
LiOH, THF
61.5%
ClCOOEt
ethylene oxide
Cl
Cl
10
1
Cl
Cl
Cl
Cl
Cl
Cl
Cl
70.2%
33.7%
Cl
Cl
Cl
Cl
2a
O
EtO
11
Scheme 2. Methodologies for the synthesis of compound 10.
O
Cl
Cl
Cl
Cl
Cl
Cl
Cl
O
Cl
O
O
Cl
Cl
Cl
Cl
N
O
HO
Cl
Cl
H2N
OEt
OEt
OEt
phthalimide, PPh3,
DIAD, THF
Cl
Cl
Cl
NH2-NH2
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
O
12
O
10
O
13
EtO
EtO
EtO
O
Cl
Cl
Cl
Cl
Cl
O
O
H
H
N
O N
O
N
Cl
Cl
Cl
Cl
OEt
OEt
O
O
O
Et3N, THF
1)
nBuNOH, THF
Cl
Cl
Cl
Cl
2) p-chloranil
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
O
O
15
EtO
EtO
14
Scheme 3. Synthesis of compound 15.

J. Wang et al. / Bioorg. Med. Chem. 18 (2010) 922–929
925
8
7
15
A
10 G
Figure 1. X-band EPR spectra of fluorophore-trityls 7, 8 and 15 as crystalline
powders in air as measured at room temperature. The acquisition settings were:
microwave frequency, 9.786 GHz (X-band), modulation amplitude, 1 G; microwave
power, 2 mW; time constant, 40 ms; scan time, 15 s. The first derivative peak-to-
peak widths of 7, 8, and 15 were 3.26 G, 2.76 G, and 5.14 G, respectively.
B
C
ÁÀ
TC with O2 may offer a potential opportunity for measurement of
superoxide in biological systems. It can, therefore, be assumed that
ÁÀ
based on these preliminary evidences that O2 reactivity with 7, 8
or 15 can parallel that of PTM-TC. Further, we have previously
shown that the perchloro-class of trityl radicals, for example
PTM-TC, is not reactive or has no specificity of reaction toward hy-
droxyl, peroxyl, or nitric oxide radicals in aqueous solutions.18
2.3.2. Fluorescence spectroscopy
The excitation and emission profiles for compounds 7, 8 and 15
are shown in Figure 5. The spectrum showed two emission bands,
at 377 nm and 397 nm, corresponding to excitations at 330 and
346 nm, respectively. Enhanced fluorescence spectra can be ob-
served for all compounds upon addition of O2ÁÀ, however, this ef-
fect is more pronounced for compounds 7 and 15. This enhanced
ÁÀ
effect of O2 on the emission spectra is consistent with the trityl
radical quenching as demonstrated by the EPR studies mentioned
above. This study further demonstrates the potential use of fluoro-
3503
3508
3513
Magnetic Field (G)
3518
3523
3528
phore-trityl conjugate as both an EPR and fluorescent probe for the
ÁÀ
detection of O2
.
Figure 2. X-band EPR spectra of fluorophore-trityls in anaerobic solution. The EPR
spectra of compounds (A) 8; (B) 7; and (C) 15 in DMSO (100 M) measured at room
l
temperature (see Section 4 for instrument settings). The broken red-colored trace
superimposed on each spectrum was simulated using the parameters shown in
Table 1.
3. Summary and conclusions
Perchlorotrityl-fluorophore conjugates were synthesized and
characterized, and EPR spectra show long-range electronic coupling
with the methylene protons of the linker group. Reactivity with O2
ÁÀ
was investigated using EPR and fluorescence spectroscopy and the
results show disappearance of the EPR signal of the trityl with a
concomitant increase in the emission intensity, respectively, sug-
gesting a strong, perhaps a through-space interaction, between the
unpaired spin of the trityl and the excited electronic state of the fluo-
rophore. This work suggests potential application of perchlorotrityl-
Table 1
Experimental EPR hyperfine splitting constants (aHx) and anaerobic linewidths (
in G for trityls 7, 8 and 15 in DMSO at 298 K
DBpp)
ÁÀ
Cl
Cl
flurophore conjugates as dual EPR and fluorescence probe for O2
detection.
Cl
Cl
Hd
Ha
Cl
Hc
Cl Cl
The new class of bimodal probes, with higher sensitivity when
compared to existing probes, may enable accurate and reliable
quantitative determination of superoxide concentration in biolog-
ical systems. Superoxide determination in cells in vitro would re-
quire cellular internalization of the probe, while in vivo
measurements need further considerations including mode of
administration of the probe, detection sensitivity, and other com-
plexities in the living system. A further difficulty may be the limi-
tation of the methods for deep tissue measurements. In the case of
experimental animal models, one can use excised tissue biopsies
following intravenous administration of the probe. The tissue sam-
ples could then be immediately flash frozen, preserved, and sec-
tioned for EPR or fluorescence microscopy imaging. However, in
Cl
Cl
Cl
Cl
Cl
Hb
Compound
Hyperfine splitting constants aH (G)
DBpp (G)
Aromatic H’s
(Ha and Hb)
Methylene H’s
(Hc and Hd)
7
8
15
2.09, 1.91
2.05, 1.96
n/a
1.24, 0.95
1.14, 1.02
1.26, 0.94
0.70
0.62
0.66

926
J. Wang et al. / Bioorg. Med. Chem. 18 (2010) 922–929
A
B
C
D
E
F
Figure 3. Preferred conformations for trityls 7 (top) and 8 (bottom) using MMFF
*
MonteCarlo search and geometry optimization at the B3LYP/6-31G level of theory
showing the intramolecular distance between the trityl and fluorophore moieties.
3514
3519
3524
3529
3534
Magnetic Field (G)
the case of clinical situations this may not be readily feasible. Thus
the near-term usefulness of the probes is in non-clinical research
applications both in vitro using cell culture models and in vivo
using animal models.
Figure 4. EPR spectra of (A) 8, (C) 7 and (D) 15; and (B), (D) and (F), respectively, are
the resulting spectra upon addition of KO2 in DMSO. The acquisition settings were:
microwave frequency, 9.786 GHz, modulation amplitude, 1 G; microwave power,
5 mW; time constant, 40 ms; scan time, 21 s. The broken red-colored trace
superimposed on each spectrum was simulated using the parameters shown in
*
Table 1. The small satellite peaks indicated with on both sides of the main peaks
are due to hyperfine coupling with 13C nuclei (natural abundance).
4. Experimental section
4.1. Bis[2,3,5,6-tetrachlorophenyl]-[4-(2-hydroxyetheyl)-2,3,5,6-
tetrachlorophenyl] methane (2a),bis[4-(2-hydroxyetheyl)-2,3,5,6-
tetrachlorophenyl]-[2,3,5,6-tetrachloro-phenyl] methane (2b),
tri[4-(2-hydroxyetheyl)-2,3,5,6-tetrachlorophenyl] methane (2c)
134.8, 134.8, 134.6 (2 carbons), 134.4, 133.8, 133.7, 133.5, 133.4,
133.4, 133.3, 132.3, 130.2, 60.2, 56.4, 37.4. HRMS (M+Na)+ calcd for
C23H12Cl12O2Na 768.6909, found 768.6929. Compound 2c: 1H NMR
(400 MHz, CDCl3) d 7.02 (s, 1H), 3.90 (m, 6H), 3.40 (t, J = 7.2 Hz,
6H); 13C NMR (100 MHz, CDCl3) d 136.8 (6 carbons), 134.8 (3 car-
bons), 134.5 (3 carbons), 133.8 (3 carbons), 133.4 (3 carbons),
60.3(3 carbons), 56.7, 37.4 (3 carbons). HRMS (M+Na)+ calcd for
C25H16Cl12O3Na 812.7171, found 812.7204.
To a solution of 1 (1.6 g, 2.46 mmol) and TMEDA (0.44 ml,
2.95 mmol) in anhydrous THF (30 ml) at À78 °C, n-BuLi (1.84 ml,
1.6 M solution in THF) was added and stirred for 1 h. Ethylene oxide
was added (2.95 ml, 1.0 M solution in Et2O) and the reaction mixture
was warmed to room temperature and stirred overnight. The reac-
tion was quenched by addition of 1.0 ml water. The solvent was re-
moved and the residue was dissolved with CH2Cl2, washed with
water and brine, and dried with Na2SO4. Removal of the solvent fol-
lowed by column chromatography (hexanes/EtOAc = 5:1 to 1: 1.5)
afforded the product 2a (0.58 g, 0.48 mmol, 33.7%) 2b (0.36 g,
0.48 mmol, 19.6%), and 2c (0.12 g, 0.15 mmol, 6.2%). Compound
2a: 1H NMR (400 MHz, CDCl3) d 7.61 (s, 2H), 6.97 (s, 1H), 3.87 (t,
J = 7.2 Hz, 2H), 3.37 (t, J = 7.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) d
138.8, 138.7, 137.0, 136.4, 134.8, 134.6, 134.4, 134.4, 133.8, 133.6
(2 carbons), 133.5, 133.3 (2 carbons), 132.4, 132.4, 130.3 (2 carbons),
60.2, 56.3, 37.4; HRMS (M+Na)+ calcd for C21H8Cll2ONa 724.6644,
found 724.6649. Compound 2b: 1H NMR (400 MHz, CDCl3) d 7.61
(s, 1H), 6.97 (s, 1H), 3.87 (t, J = 7.2 Hz, 4H), 3.37 (t, J = 7.2 Hz, 4H);
13C NMR (100 MHz, CDCl3) d 138.9, 136.9, 136.9, 136.6, 136.5,
4.2. Bis(2,3,5,6-tetrachlorophenyl)-[4-(2-phthalimideetheyl)-
2,3,5,6-tetrachlorophenyl] methane (3)
To a solution of 2a (0.32 g, 0.46 mmol), phthalimide (0.13 g,
0.91 mmol), triphenylphosphine (0.24 g, 0.91 mmol) in anhydrous
THF (10 ml), DIAD (0.18 L, 0.91 mmol) was added. The reaction
mixture was stirred overnight at room temperature. The solvent
was removed in vacuo and the residue was dissolved in EtOAc,
washed with water and brine, and dried with Na2SO4. Removal of
the solvent followed by column chromatography (hexanes/
EtOAc = 1:0 to 10:1) afforded the product 3 (0.37 g, 0.45 mmol,
97.7%). 1H NMR (500 MHz, CDCl3) d 7.81 (dd, J = 5.4 Hz, 3.0 Hz,
2H), 7.68 (dd, J = 5.4 Hz, 3.0 Hz, 2H), 7.62 (s, 1H), 7.61 (s, 1H),
6.93 (s, 1H), 4.10 (m, 1H), 4.01 (m, 1H), 3.52 (m, 1H), 3.44 (m,
1H); 13C NMR (125 MHz, CDCl3) d 167.9 (2 carbons), 138.8, 138.6,

J. Wang et al. / Bioorg. Med. Chem. 18 (2010) 922–929
927
133.9, 133.6 (2 carbons), 133.4, 133.3, 132.5, 132.45 (2 carbons),
131.1, 130.6, 130.5, 130.3 (2 carbons), 128.62 (2 carbons), 128.56,
127.0, 126.3, 125.8, 125.7, 124.6, 124.5, 124.35, 124.28, 124.17,
56.5, 38.2, 34.3; HRMS (M+Na)+ calcd for C38H17Cll2NONa
951.7384, found 951.7368.
8 + KO2
10
8
8
6
4
4.5. Bis(2,3,5,6-tetrachlorophenyl)-{4-[2-(1-
pyrenebutylcarboxy)aminoethyl]-2,3,5,6-tetrachlorophenyl}
methane (6)
2
0
To a solution of 4 (0.14 g, 0.20 mmol), 1-pyrenebutyric acid N-
hydroxysuccinimide ester (0.09 g, 0.24 mmol) in anhydrous THF
(10 ml), Et3N (0.10 ml, 0.72 mmol) was added. The reaction mix-
ture was stirred at 50 °C for 2 h. The solvent was removed and
the residue was purified by column chromatography (hex-
anes = 100% to hexanes/EtOAc/CH2Cl2 = 1:0.33:1) afforded the
product 6 (0.13 g, 0.13 mmol, 67.0%). 1H NMR (400 MHz, CDCl3) d
8.25 (d, J = 10.0 Hz, 1H), 8.13 (d, J = 7.5 Hz, 2H), 8.0–8.1 (m, 2H),
7.9–8.0 (m, 3H), 7.79 (d, J = 7.8 Hz, 1H), 7.59 (s, 1H), 7.58 (s, 1H),
6.94 (s, 1H), 5.58 (t, J = 5.9 Hz, 1H), 3.4–3.6 (m, 2H), 3.33 (t,
J = 7.0 Hz, 2H), 3.23 (t, J = 7.0 Hz, 2H), 2.1–2.2 (m, 4H); 13C NMR
(125 MHz, CDCl3) d 174.7, 140.67, 140.65, 139.6, 138.5, 137.8,
136.8, 136.5, 136.4, 135.8, 135.5, 135.4, 135.3, 134.4, 133.4,
132.9, 132.3, 132.0, 130.8, 129.5, 129.4, 128.8, 127.9, 127.2,
127.0, 126.96, 126.8, 125.4, 58.3, 39.3, 37.9, 36.2, 34.8, 29.2; HRMS
(M+Na)+ calcd for C41H23Cll2NONa 993.7851, found 993.7851.
10
8
7 + KO2
6
7
4
2
0
10
8
15 + KO2
6
15
4
2
0
4.6. Bis(2,3,5,6-tetrachlorophenyl)-{4-[2-(1-
pyrenecarbonylamino)etheyl]-2,3,5,6-tetrachlorophenyl}
methyl radical (PTMH2-PyC) (7)
360
390
420
450
Wavelength (nm)
Figure 5. Excitation and emission profiles of 8, 7, and 15 in the presence and
absence of KO2 in DMSO. Data show two emission bands, at 377 nm and 397 nm,
corresponding to excitations at 330 and 346 nm, respectively.
An aqueous solution of tetra n-butyl ammonium hydroxide
(84.0 mg, 0.105 mmol) was added to a solution of 5 (65.0 mg,
0.070 mmol) in dry tetrahydrofuran (5 ml) at room temperature.
The resulting red solution was stirred overnight, and then p-chlora-
nil (51.7 mg, 0.21 mmol) was added. The resulting intensely brown
solution was stirred for 1 h. After this time, the solution was evapo-
rated to dryness and the resulting product was purified by chroma-
tography (hexanes = 100% to hexanes/EtOAc/CH2Cl2 = 9:1:2) and
136.9, 136.7, 134.8, 134.6, 134.5, 134.3, 134.0 (2 carbons), 133.7,
133.6, 133.5, 133.5, 133.4, 133.2, 132.4, 132.3, 131.9 (2 carbons),
130.2, 130.2, 123.3 (2 carbons), 56.3, 34.7, 33.1; HRMS (M+Na)+
calcd for C29H11Cll2NO2Na 851.6888, found 851.6903.
afforded the product
7 (64.6 mg, 0.070 mmol, 99.3%). HRMS
4.3. Bis(2,3,5,6-tetrachlorophenyl)-[4-(2-aminoetheyl)-2,3,5,6-
tetrachlorophenyl] methane (4)
(M+Na)+ calcd for C38H16Cll2NONa 950.7305, found 950.7291.
4.7. Bis(2,3,5,6-tetrachlorophenyl)-{4-[2-(1-
pyrenebutylcarboxy)aminoetheyl]-2,3,5,6-tetrachlorophenyl}
methyl radical (PTMH2-PyB) (8)
To a solution of 3 (0.25 g, 0.30 mmol) in absolute EtOH (5 ml)
was added hydrazine hydrate. The reaction mixture was refluxed
for 4 h. The mixture was then cooled, diluted with water, and ex-
tracted with CH2Cl2 (2Â). The organic extracts were washed with
brine and dried over MgSO4. Removal of the solvent afforded the
crude product 4 (0.14 g, 0.20 mmol), which was used in the next
Aqueous solution of tetra n-butyl ammonium hydroxide
(73.6 mg, 0.092 mmol) was added to a solution of 6 (60.0 mg,
0.062 mmol) in dry tetrahydrofuran (5 ml) at room temperature.
The resulting red solution was stirred overnight and then p-chloranil
(46.0 mg, 0.186 mmol) was added. The resulting intensely brown
solution was stirred for 1 h. After this time, the solution was evapo-
rated to dryness and the resulting product was purified by chroma-
tography (hexanes = 100% to hexanes/EtOAc/CH2Cl2 = 1:0.33:1)
step without purification. HRMS (M+H)+ calcd for C21H10Cl12
701.6987, found 701.6931.
N
4.4. Bis(2,3,5,6-tetrachlorophenyl)-{4-[2-(1-
pyrenecarbonyl)aminoetheyl]-2,3,5,6-tetrachlorophenyl}
methane (5)
afforded the product
8 (57.2 mg, 0.059 mmol, 95.4%). HRMS
(M+Na)+ calcd for C41H22Cll2NONa 992.7776, found 992.7775.
To
a solution of 4 (0.14 g, 0.20 mmol), 1-pyrenecarboxyl
N-hydroxysuccinimide ester (0.08 g, 0.24 mmol) in anhydrous THF
(10 ml), Et3N (0.10 ml, 0.72 mmol) was added. The reaction mixture
was stirred at 50 °C for 2 h. The solvent was removed and the residue
was purified by column chromatography (hexanes = 100% to hex-
4.8. Bis[2,3,5,6-tetrachloro-4-ethoxycarbonyl-phenyl]-(2,3,5,6-
tetrachlorophenyl) methane (9)
To a solution of 1 (1.15 g, 1.76 mmol) and TMEDA (0.64 ml,
4.22 mmol) in anhydrous THF (80 ml) at À78 °C, n-BuLi (2.65 ml,
1.6 M solution in THF) was added and stirred for 1 h. Ethyl chloro-
formate was added (0.41 ml, 4.2 mmol) and the reaction mixture
was allowed to warm up to room temperature and stirred over-
night. The reaction was quenched by addition of 1 M HCl to pH
2. The solvent was removed and the residue was dissolved with
anes/EtOAc/CH2Cl2 = 15:4:10) afforded the product
5 (0.11 g,
0.12 mmol, 59.3%).1H NMR (500 MHz, CDCl3) d 8.44 (d, J = 10.0 Hz,
1H), 8.16 (d, J = 7.5 Hz, 1H), 8.13 (d, J = 7.5 Hz, 1H), 7.9–8.1 (m, 6H),
7.64 (s, 1H), 7.63 (s, 1H), 7.05 (s, 1H), 6.48 (t, J = 5.9 Hz, 1H), 3.8–
4.0 (m, 2H), 3.54 (t, J = 6.9 Hz, 2H); 13C NMR (125 MHz, CDCl3) d
170.1, 138.8, 138.7, 137.8, 136.7, 135.0, 134.7, 134.44, 134.39,

928
J. Wang et al. / Bioorg. Med. Chem. 18 (2010) 922–929
CH2Cl2, washed with brine, and dried with Na2SO4. Removal of the
solvent followed by column chromatography (hexanes/CH2Cl2/
drous THF (5 ml), DIAD (51 lL, 0.26 mmol) was added. The reaction
mixture was stirred overnight at room temperature. The solvent was
removed and the residue was dissolved with EtOAc, washed with
water and brine, and dried with Na2SO4. Removal of the solvent
was followed by column chromatography (hexanes/EtOAc = 8:1 to
3:1) which afforded the product 12 (66.7 mg, 0.068 mmol, 52.3%)
with minimal impurity. 1H NMR (400 MHz, CDCl3) d 7.78 (d,
J = 5.4 Hz, 3.1 Hz, 2H), 7.67 (d, J = 5.4 Hz, 3.1 Hz, 2H), 6.94 (s, 1H),
4.46 (q, J = 6.9 Hz, 4H), 4.0–4.1 (m, 2H), 3.4–3.6 (m, 2H), 1.40 (t,
J = 6.9 Hz, 6H); 13C NMR (100 MHz, CDCl3) d 167.9, 163.2, 139.0,
138.8, 137.3, 136.1, 135.30, 135.27, 135.1, 134.94, 134.89, 134.6,
134.0, 133.96, 133.85, 133.76, 133.67, 131.9, 130.4, 130.3, 129.34,
129.26, 123.3, 63.0, 56.5, 35.2, 33.1, 14.0; HRMS (M+Na)+ calcd for
C35H19Cll2NO6Na 997.7286, found 997.7291.
EtOAc = 100:1:1) afforded the product
9 (0.66 g, 0.82 mmol,
46.7%) and mono-substituted compound (0.43 g, 0.59 mmol,
33.4%). 1H NMR (400 MHz, CDCl3) d 7.64 (s, 1H), 6.98 (s, 1H),
4.46 (q, J = 7.1 Hz, 4H), 1.40 (t, J = 7.1 Hz, 6H); 13C NMR
(100 MHz, CDCl3) d 163.2, 138.8 (2 carbons), 138.0, 135.4 (2 car-
bons), 135.02, 134.99, 134.4, 134.0, 133.9, 133.7, 133.3, 132.6,
130.6, 130.5, 130.4, 129.43, 129.40, 63.1, 56.3, 14.0; HRMS
(M+Na)+ calcd for C25H21Cll2O4Na 824.6805, found 824.6819.
4.9. Bis[2,3,5,6-tetrachloro-4-ethoxycarbonyl-phenyl]-[4-(2-
hydroxyetheyl)-2,3,5,6-tetrachloro-phenyl] methane (10)
Method A: To a solution of 9 (0.48 g, 0.60 mmol) and TMEDA
(0.23 ml, 1.50 mmol) in anhydrous THF (30 ml) at À78 °C, n-BuLi
(0.94 ml, 1.6 M solution in THF) was added and stirred for 1 h. Eth-
ylene oxide was added (1.5 ml, 1.0 M solution in Et2O) and the
reaction mixture was stirred allowing the reaction to warm to
room temperature. The reaction was quenched by addition of
1 M HCl to pH 2. The solvent was removed and the residue was dis-
solved with CH2Cl2, washed with brine, and dried with Na2SO4. Re-
moval of the solvent followed by column chromatography
(hexanes/EtOAc/CH2Cl2 = 6:1:1) afforded the product 10 (0.05 g,
4.12. Bis[2,3,5,6-tetrachloro-4-ethoxycarbonyl-phenyl]-[4-(2-
aminoetheyl)-2,3,5,6-tetrachlorophenyl] methane (13)
To a solution of 12 (50 mg, 0.0513 mmol) in absolute EtOH (5 ml)
was added hydrazine hydrate. The reaction mixture was heated and
refluxed for 2 h. The mixture was cooled while precipitate appeared.
The reaction mixture was diluted with water, and extracted with
CH2Cl2 (2Â). The organic extracts were washed with brine, dried over
MgSO4. Removal of the solvent afforded the crude product 13 (45 mg,
0.053 mmol), which was used in the next step without purification.
HRMS (M+H)+ calcd for C27H18Cl12NO4 845.7410, found 845.7401.
0.059 mmol, 9.9%). Method B: To
a solution of 11 (0.67 g,
0.73 mmol) in THF/H2O (1:1, 10 ml) at room temperature, LiOH
(0.07 g, 2.92 mmol) was added and stirred overnight. After the
reaction completed, the pH of the reaction mixture was adjusted
to 2 by addition of 1 M HCl. The reaction mixture was extracted
by dichloromethane three times. Removal of the solvent followed
by column chromatography (hexanes/EtOAc/CH2Cl2 = 6:1:1) affor-
ded the product 10 (0.38 g, 0.45 mmol, 61.5%). 1H NMR (400 MHz,
CDCl3) d 6.98 (s, 1H), 4.46 (q, J = 7.1 Hz, 4H), 3.87 (t, J = 7.2 Hz, 2H),
3.37 (t, J = 7.2 Hz, 2H), 1.40 (t, J = 7.1 Hz, 6H); 13C NMR (100 MHz,
CDCl3) d 163.3, 139.0, 138.9, 137.4, 135.7, 135.34, 135.31, 135.02,
134.99, 134.75, 134.69, 133.98, 133.92, 133.74, 133.64, 130.43,
130.39, 129.38, 129.36, 63.1, 60.2, 56.5, 37.4, 14.0; HRMS
(M+Na)+ calcd for C27H16Cll2O5Na 868.7068, found 868.7065.
4.13. Pyrene-1-carboxylic acid (2-{4-[bis-(2,3,5,6-tetrachloro-4-
ethoxycarbonyl-phenyl)- methyl]-2,3,5,6-tetrachloro-phenyl} -
ethyl)-amide (14)
To a solution of 13 (45 mg, 0.20 mmol), 1-pyrenecarboxyl N-
hydroxysuccinimide ester (0.08 g, 0.053 mmol) in anhydrous THF
(5 ml), Et3N (0.033 ml, 0.24 mmol) was added. The reaction mixture
was stirred at 50 °C for 2 h. The solvent was removed and the residue
was purified by column chromatography (hexanes = 100% to hex-
anes/EtOAc/CH2Cl2 = 4.5:0.8:2) afforded the product 14 (30 mg,
0.03 mmol, 52.5%). 1H NMR (400 MHz, CDCl3) d 8.46 (d, J = 10.0 Hz,
1H), 8.19 (d, J = 8.5 Hz, 2H), 7.9–8.1 (m, 6H), 7.05 (s, 1H), 6.37 (m,
1H), 4.48 (m, 4H), 3.8–4.1 (m, 2H), 3.56 (m, 2H), 1.41 (m, 6H); 13C
NMR (100 MHz, CDCl3) d 170.1, 163.3, 139.0, 138.9, 138.2, 136.1,
135.39, 135.37, 135.1, 135.0, 134.9, 134.8, 134.97 (2 carbons),
133.92, 133.7, 132.6, 131.1, 130.7, 130.6, 130.5, 130.4, 129.42,
129.40, 128.7 (2 carbons), 128.6, 127.1, 126.3, 125.8 (2 carbons),
124.8, 124.44, 124.39, 124.35, 124.26, 63.0, 56.5, 38.2, 34.3, 14.0;
HRMS (M+Na)+ calcd for C44H25Cll2NO5Na 1095.7809, found
1095.7797.
4.10. Bis[2,3,5,6-tetrachloro-4-ethoxycarbonyl-phenyl]-[4-(2-
ethoxycarbonyloxy-ethyl)-2,3,5,6-tetrachloro-phenyl] methane
(11)
To a solution of 2a (0.73 g, 1.04 mmol) and TMEDA (0.73 ml,
4.84 mmol) in anhydrous THF (60 ml) at À78 °C, n-BuLi (3.03 ml,
1.6 MsolutioninTHF)wasadded andstirred, for 1 h. Ethylchlorofor-
mate (0.86 ml, 8.96 mmol) was added and the reaction mixture was
stirred, allowing thereaction to warmup to À20 °C. The reaction was
quenched by addition of 1 M HCl to pH 2. The solvent was removed
and the residue was dissolved with CH2Cl2, washed with brine, and
dried with Na2SO4. Removal of the solvent followed by column chro-
matography (hexanes/CH2Cl2/EtOAc = 20:1:1) afforded the product
11 (0.67 g, 0.73 mmol, 70.2%). 1H NMR (500 MHz, CDCl3) d 7.00 (s,
1H), 4.46 (q, J = 7.1 Hz, 4H), 4.35 (m, 2H), 4.18 (q, J = 7.1 Hz, 2H),
3.48 (t, J = 7.0 Hz, 2H), 1.40 (t, J = 7.1 Hz, 6H), 1.28 (t, J = 7.1 Hz,
3H); 13C NMR (100 MHz, CDCl3) d 163.2, 155.0, 138.92, 138.85,
136.3, 136.2, 135.38, 135.35, 135.01, 134.98, 134.85, 134.83,
133.96, 133.92, 133.81, 133.77, 130.44, 130.40, 129.39, 129.37,
64.2, 63.1, 56.5, 36.6, 33.6, 14.3, 14.0; HRMS (M+Na)+ calcd for
C30H20Cll2O7Na 940.7281, found 940.7281.
4.14. Bis(2,3,5,6-tetrachlorophenyl)-{4-[2-(1-
pyrenecarbonyl)aminoetheyl]-2,3,5,6-tetrach-lorophenyl}
methyl radical (15)
An aqueous solution of tetra n-butyl ammonium hydroxide
(22.6
18.6
l
L, 0.028 mmol) was added to a solution of 14 (20.0 mg,
l
mol) in dry tetrahydrofuran (5 ml) at room temperature. The
resulting red solution was stirred overnight, and then p-chloranil
(13.7 mg, 0.056 mmol) was added. The resulting intensely brown
solution was stirred for 1 h. After this time, the solution was evapo-
rated to dryness and the resulting product was purified by chroma-
tography (hexanes = 100% to hexanes/EtOAc/CH2Cl2 = 6:2:1)
afforded the product 15 (19.8 mg, 18.5 lmol, 99.0%); HRMS
(M+Na)+ calcd for C44H24Cll2NO5Na 1094.7731, found 1094.7732.
4.11. Bis[2,3,5,6-tetrachloro-4-ethoxycarbonyl-phenyl]-[4-(2-
phthalimide-etheyl]-2,3,5,6-tetrachlorophenyl] methane (12)
4.15. EPR measurements
EPR measurements were performed at room temperature using
a Bruker EMX spectrometer equipped with a high-sensitivity
To a solution of 10 (110 mg, 0.13 mmol), phthalimide (38 mg,
0.26 mmol), triphenylphosphine (68.4 mg, 0.26 mmol) in anhy-

J. Wang et al. / Bioorg. Med. Chem. 18 (2010) 922–929
929
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4.16. Fluorescence measurements
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Acknowledgment
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This work was supported by NIH grant EB006363.
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