Reactivity of molecular oxygen with ethoxycarbonyl derivatives of tetrathiatriarylmethyl radicals

Article abstract of DOI:10.1021/jo0610560

Tetrathiatriarylmethyl (TAM) radicals are commonly used as oximetry probes for electron paramagnetic resonance imaging applications. In this study, the electronic properties and the thermodynamic preferences for O₂ addition to various TAM-type triarylmethyl (trityl) radicals were theoretically investigated. The radicals' stability in the presence of O₂ and biological milieu was also experimentally assessed using EPR spectroscopy. Results show that H substitution on the aromatic ring affects the trityl radical's stability (tricarboxylate salt 1-CO₂Na > triester 1-CO₂Et > diester 2-CO₂Et > monoester 3-CO ₂Et) and may lead to substitution reactions in cellular systems. We propose that this degradation process involves an arylperoxyl radical that can further decompose to alcohol or quinone products. This study demonstrates how computational chemistry can be used as a tool to rationalize radical stability in the redox environment of biological systems and aid in the future design of more biostable trityl radicals.

Full text of DOI:10.1021/jo0610560

Reactivity of Molecular Oxygen with Ethoxycarbonyl Derivatives of
Tetrathiatriarylmethyl Radicals
†
,‡
,†
‡
Shijing Xia, Frederick A. Villamena,* Christopher M. Hadad,* Periannan Kuppusamy,
‡
‡
,‡
Yunbo Li, Hong Zhu, and Jay L. Zweier*
Department of Chemistry, The Ohio State UniVersity, Columbus, Ohio 43210, and Center for Biomedical
EPR Spectroscopy and Imaging, The DaVis Heart and Lung Research Institute, and the
DiVision of CardioVascular Medicine, Department of Internal Medicine, College of Medicine,
The Ohio State UniVersity, Columbus, Ohio 43210
frederick.Villamena@osumc.edu; hadad.1@osu.edu; jay.zweier@osumc.edu
ReceiVed May 23, 2006
Tetrathiatriarylmethyl (TAM) radicals are commonly used as oximetry probes for electron paramagnetic
resonance imaging applications. In this study, the electronic properties and the thermodynamic preferences
for O
radicals’ stability in the presence of O
EPR spectroscopy. Results show that H substitution on the aromatic ring affects the trityl radical’s stability
tricarboxylate salt 1-CO Na > triester 1-CO Et > diester 2-CO Et > monoester 3-CO Et) and may
2
addition to various TAM-type triarylmethyl (trityl) radicals were theoretically investigated. The
2
and biological milieu was also experimentally assessed using
(
2
2
2
2
lead to substitution reactions in cellular systems. We propose that this degradation process involves an
arylperoxyl radical that can further decompose to alcohol or quinone products. This study demonstrates
how computational chemistry can be used as a tool to rationalize radical stability in the redox environment
of biological systems and aid in the future design of more biostable trityl radicals.
opment of electron paramagnetic resonance imaging (EPRI)5
-7
I. Introduction
of biological samples to provide improved image resolution and
Evaluating the concentration of O2 as well as O2-derived
quality, and this method has evolved to become an important
reactive species, including that of superoxide radical anion
4,8,9
tool for imaging free radicals in organs
of small animals,
and the entire body
-
(
O2‚ ) in in vivo and in vitro systems, is of utmost importance
10-12
tumors, and normal tissues.13-17
1-4
in the study of numerous pathophysiological processes. Over
the past decade, major efforts have been made toward the devel-
The development of paramagnetic materials, such as nitrox-
18
19,20
ides, triarylmethyl (trityl) radicals,
and, recently, spin-
21
labeled dendrimers, as probes for EPRI applications is critical
†
Department of Chemistry, The Ohio State University.
Center for Biomedical EPR Spectroscopy and Imaging, The Davis Heart
‡
and Lung Research Institute, and the Division of Cardiovascular Medicine,
Department of Internal Medicine, College of Medicine, The Ohio State
University.
(4) Zweier, J. L.; Flaherty, J. T.; Weisfeldt, M. L. Proc. Natl. Acad. Sci.
U.S.A. 1987, 84, 1404.
(5) Berliner, L. J. Applications of EPR imaging to materials, agriculture
and medicine. In Magnetic Resonance Microscopy: Methods and Applica-
tions in Material Science, Agriculture and Biomedicine; VCH: Weinheim,
Germany, 1992.
(1) Bilenko, M. V. Ischemia and Reperfusion of Various Organs: Injury,
Mechanisms, Methods of PreVention and Treatment; Nova Science Pub.,
Inc.: Huntington, New York, 2001.
(
2) Clerch, L. B., Massaro, D. J., Eds. Oxygen, Gene Expression and
Cellular Function; Marcel Dekker: New York, 1997; Vol. 105.
3) Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and
Medicine; Oxford University Press: Oxford, 1999.
(6) Berliner, L. J. In ViVo EPR (ESR): Theory and Applications; Kluwer
Academic Plenum: New York, 1993.
(
(7) Eaton, G. R.; Eaton, S. S.; Ohno, K. EPR Imaging and in ViVo EPR;
CRC Press: Boca Raton, FL, 1991.
10.1021/jo0610560 CCC: $33.50 © 2006 American Chemical Society
7
268
J. Org. Chem. 2006, 71, 7268-7279
Published on Web 08/22/2006

ReactiVity of Tetrathiatriarylmethyl Radicals
for the precise determination of O2 concentration and detection
logical matrix, rendering this probe ideal for in vitro and in vivo
EPR imaging applications. The perdeuterated analogue of
-
23
of superoxide radical anion (O2‚ ) in biological samples, includ-
ing cells and tissues.2
2-25
Trityl radicals have been the popular
1-CO2Na has also found applications in magnetic resonance
28
choice for EPRI applications as a result of their stability at
physiological pH and long relaxation times, which consequently
give rise to narrow EPR line widths. Moreover, trityl radicals
offer some advantages over nitroxides, that is, high analytical
resolution at µM concentrations, stability in cells and tissues,
and the flexibility to independently determine the concentrations
imaging and Overhauser-enhanced magnetic resonance imag-
2
9
ing.
Reddy et al. reported an elegant synthesis of 1-CO2Et and
the corresponding salt, 1-CO2Na, and these authors also evalu-
2
7
ated the sensitivities of these species to O2. However, the
underlying mechanism of decay for 1-CO2Na in the presence
of O2 was not reported. In a solid lattice, the reaction of the
-
of superoxide radical anion (O2‚ ) and O2 by EPR signal loss
and line broadening, respectively.25
triphenylmethyl (Ph3C ) radical with O2 results in the formation
•
of an equilibrium product, triphenylmethylperoxyl radical, Ph3-
•
30,31
COO (eq 1).
•
•
Ph C + O h Ph COO
(1)
3
2
3
In dichloromethane solution, a second-order reaction was
•
observed with Ph3C and O2, finally yielding benzophenone
3
2
(Ph2CdO) as a major product.
Trityl radicals have a long history in radical chemistry, ever
since the initial report by Gomberg in 1900 of the triphenyl-
•
26
methyl radical (Ph3C ). Tetrathiatriarylmethyl (TAM) radicals,
-CO2R, belong to a family of trityl radicals that exhibit a very
1
27
narrow line width (<100 mG), even in the presence of bio-
(
8) Zweier, J. L.; Kuppusamy, P. Proc. Natl. Acad. Sci. U.S.A. 1988,
5, 5703.
9) Yordanov, A. T.; Yamada, K.; Krishna, M. C.; Russo, A.; Yoo, J.;
English, S.; Mitchell, J. B.; Brechbiel, M. W. J. Med. Chem. 2002, 45,
283.
10) He, G.; Deng, Y.; Li, H.; Kuppusamy, P.; Zweier, J. L. Magn. Reson.
Med. 2002, 47, 571.
11) He, G.; Samouilov, A.; Kuppusamy, P.; Zweier, J. L. Mol. Cell.
Biochem. 2002, 234, 359.
12) Afeworki, M.; van Dam, G. M.; Devasahayam, N.; Murugesan, R.;
8
(
2
(
(
In this paper, we explored the electronic and thermodynamic
properties of TAM-type radicals with varying degrees of sub-
stitution (2-CO2Et and 3-CO2Et) and evaluated how these
properties can affect radical stability in the presence of O2 and
components of the biological milieu. We complement these
experimental efforts with computational chemistry so as to
understand and rationalize the radicals’ stability.
(
Cook, J.; Coffin, D.; Larsen, J. H.; Mitchell, J. B.; Subramanian, S.; Krishna,
M. C. Magn. Reson. Med. 2000, 43, 375.
(
13) Mitchell, J. B.; Russo, A.; Kuppusamy, P.; Krishna, M. C. Ann.
N.Y. Acad. Sci. 2000, 899, 28.
14) Mitchell, J. B.; Yamada, K.; Devasahayam, N.; Cook, J. A.;
Subramanian, S.; Krishna, M. C. J. Nutr. 2004, 134, 3210S.
15) Yamada, K. I.; Kuppusamy, P.; English, S.; Yoo, J.; Irie, A.;
Subramanian, S.; Mitchell, J. B.; Krishna, M. C. Acta Radiol. 2002, 43,
33.
16) Subramanian, S.; Yamada, K.; Irie, A.; Murugesan, R.; Cook, J.
(
(
4
II. Results and Discussion
(
A.; Devasahayam, N.; Van Dam, G. M.; Mitchell, J. B.; Krishna, M. C.
Magn. Reson. Med. 2002, 47, 1001.
A. EPR Spectroscopic Characterization. Tri- (1-CO2Et),
di- (2-CO2Et), and monoethoxycarbonyl (3-CO2Et) derivatives
of the TAM radical, as well as its trisodium carboxylate salt
(1-CO2Na) derivative, were synthesized and characterized by
minor modifications of the method reported by Reddy et al.27
Figure 1 shows the X-band EPR spectrum for each of the trityl
radical species with g values that range from 2.0041 to 2.0043,
(
17) Kuppusamy, P.; Afeworki, M.; Shankar, R. A.; Coffin, D.; Krishna,
M. C.; Hahn, S. M.; Mitchell, J. B.; Zweier, J. L. Cancer Res. 1998, 58,
562.
18) Matsumoto, K.; Krishna, M. C.; Mitchell, J. B. J. Pharmacol. Exp.
Ther. 2004, 310, 1076.
19) Bowmana, M. K.; Mailer, C.; Halpern, H. J. J. Magn. Reson. 2005,
72, 254.
20) Matsumoto, K.; English, S.; Yoo, J.; Yamada, K.; Devasahayam,
1
(
(
1
27
(
close to that reported for 1-CO2Et and 1-CO2Na of g ∼ 2.003.
N.; Cook, J. A.; Mitchell, J. B.; Subramanian, S.; Krishna, M. C. Magn.
Reson. Med. 2004, 52, 885.
The singlet peaks in Figure 1a,b are consistent with a trisubsti-
tuted trityl radical, while the doublet and triplet spectral features
for 2-CO2Et and 3-CO2Et, respectively, indicate the presence
of a hydrogen hyperfine splitting due to the unsubstituted aro-
matic ring. The observed hyperfine splitting constant for both
(
21) Yordanov, A. T.; Yamada, K.; Krishna, M. C.; Mitchell, J. B.;
Woller, E.; Cloninger, M.; Brechbiel, M. W. Angew. Chem. Int. Ed. 2001,
0, 2690.
22) Ilangovan, G.; Manivannan, A.; Li, H.; Yanagi, H.; Zweier, J. L.;
Kuppusamy, P. Free Radical Biol. Med. 2002, 32, 139.
23) Krishna, M. C.; English, S.; Yamada, K.; Yoo, J.; Murugesan, R.;
4
(
(
Devasahayam, N.; Cook, J. A.; Golman, K.; Ardenkjaer-Larsen, J. H.;
Subramanian, S.; Mitchell, J. B. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,
(28) Williams, B. B.; al Hallaq, H.; Chandramouli, G. V.; Barth, E. D.;
Rivers, J. N.; Lewis, M.; Galtsev, V. E.; Karczmar, G. S.; Halpern, H. J.
Magn. Reson. Med. 2002, 47, 634.
(29) Ardenkjaer-Larsen, J. H.; Laursen, I.; Leunbach, I.; Ehnholm, G.;
Wistrand, L.-G.; Petersson, J. S.; Golman, K. J. Magn. Reson. 1998, 133, 1.
(30) Ayers, C. L.; Janzen, E. G.; Johnston, F. J. J. Am. Chem. Soc. 1966,
88, 2610.
2
216.
24) Kutala, V. K.; Parinandi, N. L.; Zweier, J. L.; Kuppusamy, P. Arch.
Biochem. Biophys. 2004, 424, 81.
25) Rizzi, C.; Samouilov, A.; Kumar Kutala, V.; Parinandi, N. L.;
Zweier, J. L.; Kuppusamy, P. Free Radical Biol. Med. 2003, 35, 1608.
(
(
(
26) Gomberg, M. J. Am. Chem. Soc. 1900, 22, 757.
(31) Janzen, E. G.; Johnston, F. J.; Ayers, C. L. J. Am. Chem. Soc. 1967,
89, 1176.
(27) Reddy, T. J.; Iwama, T.; Halpern, H. J.; Rawal, V. H. J. Org. Chem.
2
002, 67, 4635.
(32) Wang, H.; Parker, V. D. Acta Chem. Scand. 1997, 51, 865.
J. Org. Chem, Vol. 71, No. 19, 2006 7269

Xia et al.
was bubbled through DMSO (or water for 1-CO2Na) solutions.
This difference in the ∆∆Bpp between the ester and the carbox-
ylated trityl radicals could be due to the higher solubility of O2
34
in DMSO compared to that in water. Purging the O2-saturated
trityl radical solution with argon gave line widths similar to
that observed originally in the absence of O2, thereby demon-
strating that the O2 with trityl radical interaction is reversible.
B. Computational Analysis of Structures, Properties, and
Decomposition Pathways. The measurement of O2 in biological
systems requires long and continuous monitoring. Hence, an
investigation of the stability of trityl radicals in the presence of
O2 is critical because O2 reaction with trityl radicals can lead
to the degradation of the radical probe. To gain insight into the
stability of these radicals in the presence of O2, the electronic
as well as the thermodynamic properties of the trityl radical
were theoretically investigated. Figure 2 shows the charges and
spin (R-â) populations for various TAM-type compounds, as
•
well as for Ph3C , using the natural population analysis (NPA)
partitioning scheme at the B3LYP/6-311+G**//B3LYP/6-31G*
level. These data show that, for TAM-type compounds, there
is a 0.62 electron localization at the central carbon that is higher
compared to the spin density (population) on the central carbon
•
predicted for Ph3C of 0.56 e. However, the spin density of the
para-C atoms in TAM-type compounds is in the range of 0.07-
•
0
.09 e, but higher (0.12 e) in Ph3C .
A closer look at the molecular structures of 1-CO2Na and
•
Ph3C showed a significant difference in their respective aro-
matic ring conformations, that is, the trityl radical 1-CO2Na
has a substantial out-of-plane ring twisting compared to that of
•
35
the Ph3C radical. This difference in conformations may have
a considerable effect on the electron density distribution and,
thereby, could affect spin relaxation that can result in EPR
spectral line broadening.
The preference for O2 addition to the trityl radical was inves-
tigated thermodynamically as to whether the O2 addition occurs
at the central or para-carbon atoms (Scheme 1). Table 1 shows
the thermodynamic values for O2 addition to various trityl
radicals to form the peroxyl adduct (steps A and B), followed
by subsequent H-atom abstraction (steps C and D) from various
H-atom donors to form the hydroperoxyl adduct at the B3LYP/
FIGURE 1. CW X-band EPR spectra of various trityl radicals in
DMSO under anaerobic conditions. (a) 1-CO Et; (b) 1-CO Na (in
O); (c) 2-CO Et; and (d) 3-CO Et. See Experimental Section for
the EPR parameters.
2
2
H
2
2
2
6
-311+G**//B3LYP/6-31G* level of theory. Results show that,
in general, O2 addition to TAM-type radicals is endothermic
steps A and B in Table 1), and the formation of the peroxyl
2
-CO2Et and 3-CO2Et trityl radicals of aH,para ) 2.30 G in
DMSO is close to our predicted isotropic hyperfine splitting of
.11 or 2.22 G at the B3LYP/6-311+G**//B3LYP/6-31G* level
of theory in the gas phase and also similar to that reported ex-
(
2
adduct at the aromatic ring C is preferred by 2.5 to 6.5 kcal/
mol compared to peroxyl adduct formation at the central carbon
atom for all of the partially substituted TAM radicals. However,
peroxyl adduct formation at the central carbon atom is preferred
by 1.4 to 2.5 kcal/mol for the fully substituted trityl radicals
such as 1-CO2Et, 1-CO2H, and 1-CO2Na for the gas-phase
calculations. The formation of the peroxyl adduct at the central
carbon in Ph3C is exothermic by -4.0 kcal/mol and is preferred
by 17.7 kcal/mol relative to O2 addition to the aromatic ring’s
carbon atom. An examination of the charge and spin populations
•
33
perimentally for Ph3C of aH,para ) 2.745 G in toluene solution.
Anaerobic peak-to-peak widths in DMSO for 1-CO2Et,
-CO2Et, and 3-CO2Et range from ∆Bpp ) 380 to 470 mG
2
and are broader compared to the ∆Bpp ) 111 mG observed for
1
0
1
-CO2Na in aqueous solution using a modulation amplitude of
.01 G. Reddy et al.27 previously reported line widths for
-CO2Et and 1-CO2Na of ∼270 mG (in pyridine) and 84 mG
•
(in water), respectively, at modulation amplitudes of 0.025-
0.086 G. It should be noted, however, that the slight broadening
•
for Ph3C and 4-CO2Et shows only a small difference in their
observed in this study may be due to the presence of trace
amounts of O2 because the capillary tubes were only sealed with
inert clay after bubbling with Ar. The effect of O2 on the spectral
profile of various trityl radicals was investigated as well. Results
show that there is a significant line width broadening by about
respective charge (∼0.05 e) and spin population (∼ 0.06 e) at
•
the central C atom. The spin population on the para-C of Ph3C ,
however, is higher by only 0.03 e compared to that of 4-CO2Et,
with no difference in their respective atomic charges. Based on
∆
∆Bpp ) 0.8-0.9 G for all of the ester-derivatized trityl radicals
(
34) Che, Y.; Tokuda, K.; Ohsaka, T. Bull. Chemi. Soc. Jpn. 1998, 71,
51.
35) Bowman, M. K.; Mailer, C.; Halpern, H. J. J. Magn. Reson. 2005,
172, 254.
and about ∆∆Bpp ) 0.2 G broadening for 1-CO2Na when O2
6
(
(33) Trapp, C.; Kulkarni, S. V. J. Phys. Chem. 1984, 88, 2703.
7270 J. Org. Chem., Vol. 71, No. 19, 2006

ReactiVity of Tetrathiatriarylmethyl Radicals
FIGURE 2. Spin populations (R-â) and atomic charges (in parentheses) of various trityl radicals at the B3LYP/6-311+G**//B3LYP/6-31G*
level in the gas phase using the NPA partitioning scheme.
SCHEME 1. Molecular Oxygen Reaction with Trityl Radicals by Addition to the Center Carbon Atom of the Trityl Radical
(A) or by Addition to the Aromatic Ring of the Trityl Radical (B)
•
the electronic properties predicted for Ph3C and 4-CO2Et, the
steric effects are also important. The effect of solvation on the
O2 addition to 1-CO2H and 1-CO2Na was investigated by
•
preference of O2 addition to the central C of Ph3C appears to
be due mainly to a steric effect. Although it has been previ-
ously3 shown that O2 adds preferentially to the carbon centers
in delocalized radicals bearing the highest spin density, this
behavior does not apply completely for trityl radicals for which
6,37
(
36) Pratt, D. A.; Mills, J. H.; Porter, N. A. J. Am. Chem. Soc. 2003,
1
25, 5801.
(37) Merle, J. K.; Hadad, C. M. J. Phys. Chem. A 2004, 108, 8419.
J. Org. Chem, Vol. 71, No. 19, 2006 7271

Xia et al.
TABLE 1. Predicted Reaction Energies ∆E0 (kcal/mol) for the Formation of Various Trityl Radical-OOH Adducts at the B3LYP/
6
-311+G**//B3LYP/6-31G* Level in the Gas Phase
b
reaction energies ∆E0 (in kcal/mol)
step C
step D
step A + C
step B + D
entrya
Ph3C•
step A
step B
H2O
DMSO
MeSH
H2O
DMSO
MeSH
H2O
DMSO
MeSH
H2O
DMSO
MeSH
-4.0
24.2
15.4
22.8
14.3
24.0
22.5
23.9
19.7
13.7
26.7
25.3
24.2
22.8
25.9
17.7
17.4
17.2
35.5
36.6
37.2
36.8
37.0
38.4
38.5
35.9
37.3
20.7
21.9
20.9
22.0
20.7
23.7
23.8
21.2
22.5
2.1
3.2
4.1
3.4
3.9
5.1
5.1
2.5
3.9
36.7
34.0
34.2
32.2
34.8
34.3
35.0
35.2
35.2
21.9
19.3
17.9
17.5
18.5
19.5
20.3
20.5
20.4
3.3
0.7
1.1
-1.2
1.7
0.9
1.6
1.8
1.8
31.5
60.8
52.6
59.6
51.3
62.4
61.0
59.8
57.0
16.7
46.1
36.3
44.8
35.0
47.7
46.3
45.1
42.2
-1.9
27.4
19.5
26.2
18.2
29.1
27.6
26.4
23.6
50.4
60.7
59.5
56.4
57.6
60.2
52.7
52.6
52.4
35.6
46.0
43.2
41.7
41.3
45.4
38.0
37.9
37.6
17.0
27.4
26.4
23.0
24.5
26.8
19.3
19.2
19.0
1
-CO2H
c
1
1
1
1
2
3
4
-CO2H
-CO2Na
-CO2Na
-CO2Et
-CO2Et
-CO2Et
-CO2Et
a
c
See Figure 2 for the respective structure. b See Scheme 1 for the reactions for steps A through D. c In aqueous system, calculated at the B3LYP/6-
3
11+G**//B3LYP/6-31G* level using the PCM model for water.
employing the polarizable continuum model (PCM) for water
as compared to that of the gas-phase energetics. The reactions
for O2 addition to the aromatic ring show no significant differ-
ence, while O2 addition reactions to the central carbon atom
are preferred by 8∼10 kcal/mol in water compared to that of
the gas-phase calculations. We have noted similar solvation
effects for peroxyl radicals earlier.38
Table 1 also shows that the energetics for H-atom abstraction
by the peroxyl radical adducts from H2O, DMSO, or MeSH to
form the aryl hydroperoxyl adduct (steps C and D). Although
H-atom abstraction is an endothermic process, abstraction is
more preferred with DMSO as a H-atom donor compared to
that with H2O and most preferred with MeSH. The overall
energetics (steps A + C and B + D) for the formation of hydro-
peroxyl adducts in the various solvents are highly endothermic.
For all of the trityl radicals, the formation of the aromatic
hydroperoxyl adduct (step B + D) is more preferred than the
formation of the central carbon hydroperoxyl adduct (A + C)
in the gas phase. The partially substituted trityl radicals are more
favored to form aryl hydroperoxyl adducts than the fully
substituted trityl radicals. This indicates that the stability of these
radicals appears to be correlated with the number of H’s on the
aryl ring. However, based on the computational results, the
trityl salt, the TS may have a different correlation with its prod-
uct. These thermodynamic results are not exclusive, and other
pathways for decomposition may be possible.
C. Experimental Stability of Trityl Radicals in Solution:
EPR, MS, and Product Studies. The stability in solution of
various trityl radicals was investigated. Results show that each
argon-purged solution of the esterifed trityls (1-CO2Et, 2-CO2Et,
and 3-CO2Et) gave a half-life of ∼4 days, while the carboxylate
salt, 1-CO2Na, did not decay for at least 17 days. This slow
decomposition of the esterified trityls could be accounted for
by the slow diffusion of O2 into the solution. However, in the
presence of air, the half-life of 3-CO2Et was significantly shorter
(∼1 day), while the half-lives of 2-CO2Et, 1-CO2Et, and 1-
CO2Na were the same as in the absence of O2. Product analysis
of each of the trityl radicals’ solutions using electrospray ioniza-
tion (ESI) and matrix-assisted laser desorption ionization-time-
of-flight (MALDI-TOF) mass spectrometry, before and after
exposure to O2, was carried out (Figure 3). These analyses reveal
that the same spectral pattern was observed for trityl solutions
with ESI before and after exposure to O2 (Table 2). It should
be noted, however, that ESI is an atmospheric pressure method,
is an oxidizing environment, and may lead to the formation of
peroxyl adducts. To differentiate whether the observed mass
spectra were due to O2 addition in the gas or liquid phase, trityl
1-CO2Na trityl salt is more preferred to form aryl hydroperoxyl
adducts than the fully ester-substituted trityl radicals 1-CO2Et.
This is not consistent with the stability study results noted below.
Our calculations, of course, are thermodynamic results and
cannot be used directly to represent kinetic results. Because of
the computational expense, searching for the transition state (TS)
of the reactions was not practical. Also, TSs for O2 addition to
carbon-center radicals are often difficult to locate, and the
corresponding wave functions usually suffer from significant
18
radical solutions were bubbled with O-18 labeled O2 ( O2) and
18
the MS were acquired. Unfortunately, the peak for the O-
labeled product was too small to be observed, which could be
due to the reversibility of O-16 O2 addition to these trityl
radicals.
MALDI-TOF spectra for the carboxylate salt radical 1-CO2Na
show only a very small peak corresponding to the OH adduct
(Figure 3c), while OOH and OH adducts were produced from
38b
spin contamination, thereby rendering the results to be suspect.
1
-CO2Et, 2-CO2Et, and 3-CO2Et (Figure 3a,b and Table 2).
Based on Hammond’s postulate, the TS should be late on the
potential energy surface and should resemble the product,
peroxyl radical adduct, or hydroperoxyl adduct. We evaluated
the reactions by the energy difference between product and
starting material, not the activation barrier that would actually
control the reaction process. A possible explanation for the
inconsistency between computational and experimental results
is that, for the ester-substituted trityl radicals, their TSs have a
similar correlation with the products and the thermodynamic
energies are consistent with their kinetic values, while for the
This result is consistent with the EPR observation that, in the
presence of O2, 1-CO2Na exhibited the greatest stability com-
pared to the three ester derivatives. The stability of these
radicals, 1-CO2Na > 1-CO2Et > 2-CO2Et > 3-CO2Et, appears
to be correlated with the number of hydrogens on the aryl ring,
as we predicted by the energetic calculations in the gas phase.
Scheme 2 suggests an explanation consistent with this correla-
tion and shows the MS fragmentation pattern and the possible
origin of the OH adduct, as well as the quinone analogue. The
scheme shows that the OH adduct could originate from the O-O
homolytic cleavage of the peroxyl moiety. The formation of an
(
38) (a) Liu, J.; Hadad, C. M.; Platz, M. S. Org. Lett. 2005, 7, 549. (b)
Fadden, M. J.; Barckholtz, C.; Hadad, C. M. J. Phys. Chem. A 2000, 104,
004.
(M + 17) ion is evident in the analysis of all of the ester trityl
3
radicals 1-CO2Et, 2-CO2Et, and 3-CO2Et, while a quinone-
7272 J. Org. Chem., Vol. 71, No. 19, 2006

ReactiVity of Tetrathiatriarylmethyl Radicals
FIGURE 3. Product ESI mass spectra of (a) 3-CO
2
Et and (b) 1-CO
2
Et and MALDI-TOF mass fingerprint of (c) 1-CO
2
2
Na upon exposure to O .
TABLE 2. ESI and MALDI-TOF Mass Fingerprints of Various Trityl Radicals in the Presence of O2a
Mobs
(Mcalcd)
M + Naobs
(M + Nacalcd)
M + Na + O-Hobs
(M + Na + O-Hcalcd)
M + Na + Oobs
(M + Na + Ocalcd)
M + Na + OHobs
(M + Na + OHcalcd)
M + Na + OOHobs
(M + Na + OOHcalcd)
entry
1
2
3
-CO2Et
1083.036
1083.036)
1011.030
1011.011)
938.993
1106.026
(1106.023)
1034.000
(1034.001)
961.981
not observed
(1121.010)
not observed
(1048.988)
976.946
1121.998
(1122.018)
1049.988
(1049.996)
977.970
1123.054
(1123.026)
1050.999
(1051.004)
978.937
1139.066
(1139.021)
1067.047
(1067.000)
994.976
(
(
-CO2Et
-CO2Et
(938.990)
(961.980)
(976.967)
(977.975)
(978.983)
(994.978)
a
1-CO2Na: 999.110 (‚C(ArCO2H)3), 1021.115 ((ArCO2Na)‚C(ArCO2H)2), 1038.117 ((ArCO2Na)‚C(ArCO2H)2 + OH).
like product (M + 15) was observed only for 3-CO2Et. While
-CO2Et has one aromatic ring with a H, a species with a mass
of (M + 15) was not observed and is probably due to the smaller
number of unsubstituted aromatic rings in 2-CO2Et compared
to that in 3-CO2Et.
the M residue, trace (M + 16) and (M + 17) ions can also be
seen from the M residue for both decomposed samples. Further-
more, the relative intensities of (M + 16) and (M + 17) ions to
(M + 15) are higher in the M residue of 2-CO2Et than in that
of 3-CO2Et. This also supports our previous assumption that
the more unsubstituted aromatic rings, as in 3-CO2Et versus
2-CO2Et, will form more quinone-like (M + 15) products. The
MS results also indicate that the proposed radical pathway
mechanism (Scheme 2a) may be a route for the decomposition
of the trityl radical. There may also be a redox pathway in which
trityl radicals lose their EPR signal by being oxidized or reduced
to the cation or anion, respectively (Scheme 2b). (However, we
should note that all attempts to obtain negative ion ESI-MS data
were unsuccessful, and the MS results are most consistent with
the formation of cationic species.) The formed trityl cation (or
anion) could then form other products by reactions with
nucleophiles (or electrophiles) and/or O2. UV-vis spectra of
2
We then performed a product analysis for the decomposed
2-CO2Et and 3-CO2Et samples and for which both samples
possessed a lack of EPR signals. The products were separated
by preparative HPLC, and the major products were analyzed
by UV-vis, NMR, and high-resolution MS. Based on the MS
results for both purified trityl reaction mixtures, quinone-like
products (M + 15) and trityl cation or anion (M) species are
the major products. Even though these two products can be
separated completely by HPLC because of their different polar-
ity, a trace quinone-like compound could always be detected
from the M residue for both trityl compounds (Figure 4). Based
on the MS pattern for the quinone-like compound (M + 15) in
J. Org. Chem, Vol. 71, No. 19, 2006 7273

Xia et al.
SCHEME 2. Proposed Mechanism for Decomposition of an Unsubstituted Trityl Radical. (a) Radical and (b) Charged
Molecule Pathways
the quinone-like products have λmax peaks that are shifted to
the red to 515 nm compared with their corresponding radicals
is possibly due to the enhanced conjugation between the
aromatic rings for the quinone-like products. The trityl cation
or anion products, however, show blue shifts of their UV-vis
(493 nm for 2-CO2Et and 481 nm for 3-CO2Et). This red shift
7274 J. Org. Chem., Vol. 71, No. 19, 2006

ReactiVity of Tetrathiatriarylmethyl Radicals
FIGURE 4. Product ESI mass spectra of M residues (trityl cation or anion) of (a) 2-CO
2
Et and (b) 3-CO
2
Et decomposition products.
J. Org. Chem, Vol. 71, No. 19, 2006 7275

Xia et al.
TABLE 3. Predicted EAs, IPs, BDEs, and Energies of Protonation
of Various Trityl Radicals at the B3LYP/6-311+G**//B3LYP/6-31G*
Level
EA
kcal/mol
(eV)
IP
kcal/mol
(eV)
Ccentral-H
BDE (0 K)
kcal/mol
∆Eacid
(0 K)
kcal/mol
Ar3C•
radical
Ph3C•
32.7 (1.42) 137.8 (5.98)
38.8 (1.68) 118.1 (5.12)
79.4 (3.44) 109.8 (4.76)
60.7 (2.63) 139.0 (6.03)
57.6 (2.50) 137.2 (5.95)
54.4 (2.36) 136.2 (5.91)
50.3 (2.18) 134.8 (5.85)
80.4
77.1
80.2
77.2
77.8
77.9
78.1
362.8
353.4
313.4
331.7
335.3
338.6
342.9
1
1
1
2
3
4
-CO2Na
-CO2Na (aq)a
-CO2Et
-CO2Et
-CO2Et
-CO2Et
FIGURE 5. EPR signal intensity of 1-CO
in rat H9C2 cardiomyocytes after 1, 2, 3, 4, and 5 h of incubation and
washing using phenol-red-free DMEM medium. (The 2-CO Et and
-CO Et trityl radicals did not show an EPR signal after 1 h of
incubation and washing.)
2 2
Na (O) and 1-CO Et (b)
a
At the B3LYP/6-311+G**//B3LYP/6-31G* level using PCM.
2
3
2
TABLE 4. Redox Potentials E1/2 of 1-CO2Na and 1-CO2Et
Determined by Cyclic Voltammetry Relative to Ag/4 M AgCl
a
E1/2 (ox1)
E1/2 (ox2)
(V)
(∆Ep [mV])
E1/2 (red1)
(V)
(∆Ep [mV])
spectra to 486 nm for 2-CO2Et and 473 nm for 3-CO2Et,
(
V)
respectively. The NMR analysis was less-conclusive, as only
c
compound
solvent
(∆Ep [mV])
1
H NMR could be obtained (see Supporting Information).
TEMPO
TEMPO
aqueous
CH3CN
0.52 (90)
0.66 (85)
0.45 (60)
0.59 (60)
1.42d
D. Stability of Trityl Radicals in Intracellular Studies. So
far, our studies show that the 3-CO2Et trityl radical is the least-
stable of all of the trityl radicals investigated in the presence of
O2 alone. To test if these theoretical and experimental studies
can be used to assess biostability of the trityls, their intracellular
stability in rat H9C2 cardiomyocytes was investigated. The
ability of these compounds to penetrate into cells, as well as
their overall biostability, was assessed for the four trityl radicals
by incubating the compounds in the presence of rat H9C2
cardiomyocytes at various time intervals and subsequent washing
of the cells to remove extracellular trityl radicals. After washing,
the cells became colored (i.e., green or orange, which are the
original colors of the trityl radicals), indicating that there is
absorption of these compounds inside the cell. EPR data reveal
that, among all of the trityl radicals used in the study, only the
trisubstituted radicals, that is, 1-CO2Na and 1-CO2Et, exhibited
intracellular stability (Figure 5). Despite the hydrophobic nature
of the cell membrane, high permeability to cells was observed
for the more polar 1-CO2Na, and this may be due to the nature
of the carrier solvent used, that is, 1 µL of DMSO per mL of
medium. The 2-CO2Et and 3-CO2Et trityl radicals, which have
at least one H on the aryl rings, showed only negligible EPR
peak intensity after 1 h of incubation with cells and suggests
that these trityl radicals are susceptible to biodegradation in
cellular systems.
1
1
-CO2Na
-CO2Na
aqueous
0.83 (50)
0.97 (50)
0.94e
-0.65 (60)
-0.51 (60)
-1.52 (400)
CH3CNb
1-CO2Et
CH3CN
a
Scan rate ) 100 mV/s. b Using TEMPO to calibrate aqueous solution to
acetonitrile. c ∆E ) |E - Epc|. Anodic peak only. e Cathodic peak only.
d
p
pa
lowest compared to the TAM-type radicals, indicating that the
fully unsubstituted trityl radicals are the most difficult to be
reduced. We, therefore, suggest that the loss of EPR signal in
the decomposition of the trityl radicals leads to two processes:
(1) formation of the trityl cation, which subsequently reacts with
nucleophiles, and (2) O2-addition to the radical center, which
subsequently forms the corresponding quinone product.
CV was performed to obtain the redox potentials for the
carboxylate salt 1-CO2Na and triester 1-CO2Et (Table 4). The
experimental results showed that the carboxylate salt is easier
to oxidize and to reduce than the triester. This is consistent with
the computational results using the PCM calculations for the
carboxylate salt and with the gas-phase calculations for the tri-
ester. Because the trityl radical salt (1-CO2Na) is more stable
than the triester (1-CO2Et) in organic and biological solutions
and 1-CO2Na is more easily oxidized and reduced, the
decomposition of the trityl radical is more likely to be a radical
process (Scheme 2a). It is possible that the redox reaction
proceeds via an equilibrium; recall that the oxidation/reduction
of 1-CO2Na is reversible, but is not reversible for 1-CO2Et
(Table 4). Also, from the CV data (Table 4), the carboxylate
salt 1-CO2Na is easier to oxidize than the 1-CO2Et triester.
Therefore, we conjecture that the rate-determining step for
decomposition involves the corresponding trityl radical for the
1-CO2Na species, proceeding via peroxyl radical formation, but
the rate-determining step for the ester radicals, such as 1-CO2Et,
may involve the radical or the irreversibly formed cation. Such
a scenario would be consistent with the stability of the
carboxylate salt 1-CO2Na being greater than the corresponding
esters (1-CO2Et), as well as the greater propensity to oxidize
and to reduce the irreversibly formed cation. Such a scenario
E. Computational and Cyclic Voltammetry (CV) Studies
of the Oxidation/Reduction of the Trityl Radicals. To gain
insights into the nature of the degradation process, the suscep-
tibility of the trityl radicals to oxidation or reduction was
investigated by calculating their respective electron affinities
(EAs) and ionization potentials (IPs), as shown in Table 3. The
partially substituted TAM radicals, such as 2-CO2Et, 3-CO2Et,
and 4-CO2Et, gave lower EA and IP values compared to those
of the fully substituted TAM radicals (with the exception of
the carboxylate salt, 1-CO2Na), indicating that the partially
substituted TAM radicals are harder to reduce and easier to
oxidize compared to the fully substituted ones. The IP for the
•
simplest trityl radical Ph3C is intermediate, while the EA is
7276 J. Org. Chem., Vol. 71, No. 19, 2006

ReactiVity of Tetrathiatriarylmethyl Radicals
would be consistent with the stability of the carboxylate salt
studies show that fully substituted trityl radicals are biostable
compared to the partially substituted ones. Predicted EAs and
IPs indicate that for TAM-type compounds, the less that the
trityl radical is substituted, the easier it is oxidized. This study
demonstrates how computational chemistry can be used as a
tool to assess radical stability in complex systems and aid in
the future design of more biostable trityl adducts.
1-CO2Na. However, which pathway, Scheme 2a (radical) or
2
b (ionic), is the main cause of the decomposition of the trityl
radicals is not fully clarified.
F. Stability of Trityl Radicals: Bond Dissociation Ener-
gies. If the trityl radicals were decomposing by radical processes,
it is important to understand the stability of the carbon-centered
radical in this homologous series. A simple hypothesis would
•
be that the more stable trityl radical (Ar3C ) would be derived
IV. Experimental Section
from the parent aryl methane (Ar3C-H) derivative that had the
lowest C-H bond dissociation energy (BDE). To assess the
relative stability of various TAM radicals, the Ccentral-H BDE
was calculated for the compounds under study (Table 3). The
calculations reveal that there is less than 1.5 kcal/mol difference
in the BDEs for generating these diverse radicals and indicates
that the unpaired electron on all of the TAM radicals is stabi-
lized, as supported by the spin density (population) results shown
in Figure 2. Ph3C-H, however, gave the highest BDE value of
2 2
Although the preparations of 1-CO Na and 1-CO Et have been
described in the literature,27 slight modifications in the reaction
conditions were utilized.
Tris(8-ethoxycarbonyl-2,2,6,6-tetramethylbenzo[1,2-d;4,5-d′]-
bis[1,3]dithiol-4-yl)methanol (8), Bis(8-ethoxycarbonyl)tris-
2,2,6,6-tetramethylbenzo[1,2-d;4,5-d′]bis[1,3]dithiol-4-yl)meth-
anol (9), 8-Ethoxycarbonyl-tris(2,2,6,6-tetramethylbenzo[1,2-
d;4,5-d′]bis[1,3]dithiol-4-yl)methanol (10). To a stirred solution
of TMEDA (250 µL, 1.66 mmol) in benzene (1 mL) was added
2.5 M n-BuLi in pentane (0.67 mL, 1.67 mmol) dropwise at 0 °C.
After being stirred for 30 min, a solution of 7 (193 mg, 0.2 mmol)
in benzene (1 mL) was added to the above solution dropwise at
(
8
7
0.4 kcal/mol compared to the TAM-type radicals of 77.2-
8.1 kcal/mol, thereby indicating that substitution of the aromatic
ring has a stabilizing effect on the radical formation and dis-
favors protonation (∆Eacid) of their respective carbanion ana-
logues (Table 3). The partially substituted TAM radicals, such
as 2-CO2Et, 3-CO2Et, and 4-CO2Et, gave higher BDE and
0
°C. After being stirred at 35-45 °C for 45 min, the resulting
solution was added to a solution of diethyl carbonate (1.04 mL,
.62 mmol) in benzene (1 mL) that was maintained at 0 °C. After
the mixture was stirred at 45 °C for 2 h, saturated aqueous KH
4
PO was added. The organic layer was separated, dried over MgSO ,
8
2
-
∆
Eacid values compared to those of the fully substituted TAM
4
radicals (with the exception of the carboxylate salt, 1-CO2Na),
indicating that the H on the aromatic ring does not stabilize the
trityl radical center and may contribute to the instability of the
trityl radical.
and concentrated in vacuo. The red residue was crystallized from
MeCN (4 mL) to give 90 mg of a crude orange solid. Flash chro-
matography on silica gel, using diethyl ether/hexanes (1:20) and
then gradually increasing to ethyl acetate/hexanes (1:5) as eluent,
provided the following: (1) compound 8 (45 mg, 25%) as orange
crystals that contained a small amount of inseparable impurities;
III. Conclusions
1
mp > 280 °C (gradually turned black, dec); H NMR (400 MHz,
3
CDCl ) δ 1.45 (t, 9H), 1.65 (s, 18H), 1.73 (s, 9H), 1.76 (s, 9H),
Over the years, the use of trityl radicals as probes for EPRI
applications has become increasingly important. An understand-
ing of the degradation pathways of trityl radicals is critical in
the design of probes with improved stability. Electronic proper-
ties as well as the thermodynamics of O2 addition to various
TAM-type radicals were theoretically investigated. Results show
that the presence of hydrogen as a substituent on the aromatic
ring affects the stability of the trityl radical by making the
intermediate more susceptible to O2 addition at the aryl carbon
bearing the H. Addition of O2 to the aromatic ring is more
favored in TAM-type trityl radicals, while addition to the central
4
2
1
1
.43 (m, 6H), 6.76 (s, 1H); 13C NMR (100 MHz, CDCl
8.6, 29.2, 31.9, 33.8, 60.85, 60.93, 62.3, 84.3, 121.3, 134.0, 139.3,
40.3, 141.4, 141.8, 166.2; IR (CHCl ) 3335, 2970, 1699, 1239,
3
) δ 14.3,
3
-
1
+
217, 1018, 728 cm ; MS (ESI, [M + Na] , m/z) 1123.030 05
(
observed), 1123.025 363 (calcd); (2) compound 9 (25 mg, 15%)
as orange crystals that contained a small amount of inseparable
impurities; mp > 265 °C (gradually turned black, dec); H NMR
(400 MHz, CDCl ) δ 1.45 (td, 6H), 1.63-1.82 (m, 36H), 4.42 (m,
1
3
4H), 6.68 (s, 1H), 7.18 (s, 1H); 13C NMR (100 MHz, CDCl
) δ
4.2, 14.3, 27.5, 27.7, 29.3, 29.6, 30.1, 30.8, 31.6, 33.0, 34.0, 34.6,
4.9, 60.71, 60.74, 61.2 (d), 61.8, 62.3, 63.2, 63.8, 84.1, 118.5,
3
1
3
•
121.24, 121.26, 130.6, 134.6, 134.7, 137.0, 137.7, 138.0, 139.1,
carbon atom is more preferred in Ph3C because of a steric effect.
1
1
1
39.2, 139.6, 140.2, 140.7, 141.4, 141.7, 141.86, 141.89, 166.22,
Mass spectrometric analyses reveal the formation of a quinone-
type product in partially substituted trityl radicals in the presence
of O2. The formation of the quinone intermediate after reaction
with O2, as observed for 3-CO2Et, could be a potential precursor
to the formation of gluthathione or Michael adducts with other
nucleophiles that are present in cells, as demonstrated by previ-
+
66.24; MS (ESI, [M + Na] , m/z) 1051.007 82 (observed),
051.004 233 (calcd); and (3) compound 10 (15 mg, 9%) as orange
crystals that contained a small amount of inseparable impurities;
1
mp > 255 °C (gradually turned black, dec); H NMR (400 MHz,
CDCl ) δ 1.45 (t, 3H), 1.62-1.82 (m, 36H), 4.43 (m, 2H), 6.55 (s,
3
1H), 7.17 (t, 2H); 13C NMR (100 MHz, CDCl ) δ 14.3, 27.3, 27.9,
3
3
9-43
ous studies
that show the addition of certain thiols, such
28.3, 29.1, 29.7, 31.6, 33.0, 33.3, 33.6, 34.3, 34.6, 35.0, 60.7, 62.34,
6
1
1
2.39, 63.46, 63.49, 63.54, 63.63, 83.9, 118.3, 118.4, 121.3, 130.7,
as glutathione, to quinones. Redox chemistry is also evident
and suggests the formation of the trityl cation. Cell permeability
31.7, 135.6, 136.9, 137.2, 137.5, 137.6, 137.9, 139.04, 139.06,
+
39.20, 139.28, 140.8, 141.8, 142.0, 166.3; MS (ESI, [M + Na] ,
m/z): 978.984 81 (observed), 978.983 103 (calcd).
(
39) Seung, S.; Lee, J.; Lee, M.; Park, J.; Chung, J. Chem.-Biol. Interact.
998, 113, 133.
40) Karczewski, J. M.; Peters, J. G. P.; Noordhoek, J. Biochem.
Pharmacol. 1999, 57, 27.
41) Briggs, M. K.; Desavis, E.; Mazzer, P. A.; Sunoj, R. B.; Hatcher,
S. A.; Hadad, C. M.; Hatcher, P. G. Chem. Res. Toxicol. 2003, 16, 1484.
42) Sachdeva, B.; Thomas, B.; Wang, X.; Ma, J.; Jones, K. H.; Hatcher,
P. G.; Cornwell, D. G. Chem. Res. Toxicol. 2005, 18, 1018.
43) Wang, X.; Thomas, B.; Sachdeva, R.; Arterburn, L.; Frye, L.;
Hatcher, P. G.; Cornwell, D. G.; Ma, J. Proc. Natl. Acad. Sci. U.S.A. 2006,
03, 3604.
Tris(8-ethoxycarbonyl-2,2,6,6-tetramethylbenzo[1,2-d;4,5-d′]-
1
bis[1,3]dithiol-4-yl)methyl Ester (1-CO
tris(2,2,6,6-tetramethylbenzo[1,2-d;4,5-d′]bis[1,3]dithiol-4-yl)-
methyl Ester (2-CO Et), 8-Ethoxycarbonyl-tris(2,2,6,6-tetra-
methylbenzo[1,2-d;4,5-d′]bis[1,3]dithiol-4-yl)methyl Ester (3-
CO Et), Tris-(8-carboxyl-2,2,6,6-tetramethylbenzo-[1,2-d;4,5-
d′]bis[1,3]dithiol-4-yl)methyl Sodium Salt (1-CO Na). To a stirred
solution of 8 (11 mg, 0.01 mmol) in CH Cl (2 mL) was added
BF ‚Et O (10 µL, 0.08 mmol) dropwise at room temperature. After
2
Et), Bis(8-ethoxycarbonyl)-
(
(
2
(
2
2
(
2
2
1
3
2
J. Org. Chem, Vol. 71, No. 19, 2006 7277

Xia et al.
being stirred for 1 h, the resulting dark green-blue solution was
treated with a solution of SnCl (3.2 mg, 0.017 mmol) in tetra-
hydrofuran (THF) (0.4 mL). After 10 min, saturated aqueous KH
PO was added. The organic layer was separated, dried over
Na SO , and concentrated in vacuo to give 10 mg of the crude
trityl radical 1-CO Et as an orange-brown solid: IR 2960, 2924,
General instrument settings are as follows: microwave power,
3
2
3 mW; modulation amplitude, 0.03 G; receiver gain, 3.5 × 10 ;
2
-
modulation frequency, 6 kHz; scan time, 40 s; and time constant,
82 ms. Total volume of all solutions used for EPR measurements
was 50 µL and was loaded into 50 µL micropipets that were sealed
with clay.
4
2
4
2
-
1
2
4
1
855, 1704, 1490, 1452, 1367, 1234, 1110, 1044 cm ; UV 249,
11, 495 nm; MS (ESI, [M + Na] , m/z) 1106.020 81 (observed),
106.022 623 (calcd).
Mass Spectrometric Analysis. ESI/MS. ESI mass spectrometric
+
analyses were performed for 1-CO
The trityl solution (∼1 mM) in THF was initially purged with Ar
or O gas for 4 min before mixing with THF/CH OH solvent that
2 2 2
Et, 2-CO Et, and 3-CO Et.
To a stirred solution of 9 (10 mg, 0.01 mmol) in CH
was added BF ‚Et O (10 µL, 0.08 mmol) dropwise at room
temperature. After being stirred for 1 h, the resulting dark green-
blue solution was treated with a solution of SnCl (3.2 mg, 0.017
mmol) in THF (0.4 mL). After 10 min, saturated aqueous KH PO
was added. The organic layer was separated, dried over Na SO
and concentrated in vacuo to give 10 mg of the crude trityl radical
2 2
Cl (2 mL)
2
3
3
2
was previously saturated with NaCl. This combination provided
the necessary cationization for charging in the electrospray process
for ion generation. Mass analysis was then performed in positive
ion detection mode.
2
2
4
2
4
,
MALDI-TOF/MS. MALDI-TOF was used for 1-CO
the mass spectrometer operated in linear, positive ion mode and
with a N laser for laser desorption. Samples were prepared in 0.1%
2
Na, with
2
1
4
1
2
-CO Et as an orange-brown solid: IR 2958, 2923, 2855, 1702,
-
1
2
488, 1453, 1366, 1237, 1148, 1109, 1043 cm ; UV 247, 405,
TFA at an approximate concentration of 50 pmol/µL. 2,5-Dihy-
droxybenzoic acid was prepared as a saturated solution in 50%
ACN/0.1% TFA (in water). Allotments of 1 mL of matrix and 1 mL
+
93 nm; MS (ESI, [M + Na] , m/z) 1034.000 46 (observed),
034.001 493 (calcd).
To a stirred solution of 10 (9.5 mg, 0.01 mmol) in CH
was added BF ‚Et O (10 µL, 0.08 mmol) dropwise at room
temperature. After being stirred for 1 h, the resulting dark green-
blue solution was treated with a solution of SnCl (3.2 mg, 0.017
mmol) in THF (0.4 mL). After 10 min, saturated aqueous KH PO
was added. The organic layer was separated, dried over Na SO
and concentrated in vacuo to give 9 mg of the crude trityl radical
2 2
Cl (2 mL)
of ∼1 mM aqueous solution of 1-CO
2 2
Na (purged with Ar or O gas
for 4 min) were thoroughly mixed together; 0.5 mL of this was
spotted on the target plate and allowed to dry prior to MS analysis.
3
2
2
Cell Studies. Rat cardiac H9C2 cells were cultured in DMEM
2
4
supplemented with 10% fetal bovine serum, 100 U/ml of penicillin,
2
4
,
2
and 100 µg/mL of streptomycin in 150 cm tissue culture flasks at
2
37 °C in a humidified atmosphere of 5% CO . The cells were fed
3
1
4
9
2
-CO Et as an orange-brown solid: IR 2957, 2923, 2853, 1703,
-
1
every 2-3 days and subcultured once they reached 80-90%
confluence. For determination of penetration of the trityl radicals
into cells, H9C2 cells were incubated with the trityl compounds
introduced by 10 µL (1 mM) in 10:1 DMSO to water solution for
various time points. Cells were then collected and washed twice
with PBS prior to EPR measurement.
605, 1452, 1365, 1239, 1149, 1109, 1020 cm ; UV 239, 449,
+
81 nm; MS (ESI, [M + Na] , m/z): 961.981 35 (observed),
61.980 363 (calcd).
2
The crude solid 1-CO Et was dissolved in dioxane (0.2 mL),
and 1 M aqueous KOH (0.1 mL) was added. The resultant dark
orange-brown solution was heated at 50 °C for 2 h. After being
cooled to room temperature, the reaction mixture was diluted with
water and washed twice with ether. The aqueous layer was acidified
with 1 M HCl, and the resulting orange-brown precipitate was
Cyclic Voltammetry Measurements. Cyclic voltammetry was
performed on a potentiostat and computer-controlled electro-
analytical system. Electrochemical measurements were carried out
in a 5 mL cell equipped with a gold working electrode (1.6 mm
diameter), a platinum-wire auxiliary electrode, and a Ag/AgCl
reference electrode. The gold electrode was cleaned before each
run by rubbing (circular motion for approximately 10 s) on a polish-
ing pad with crystal solution and then washed with water-acetone.
Solutions were degassed by bubbling with the argon gas. Back-
ground current (solvent with supporting electrolyte) corrections were
done for all measurements. Half-wave potentials were calculated
pc
extracted with ether. The ether layer was separated, dried over Na
SO , and concentrated in vacuo. The residue was dissolved in 0.1 M
aqueous NaOH (0.3 mL) and concentrated in vacuo to give 9 mg
of trityl radical 1-CO
Na as a dark green-yellow solid: IR ∼3400
broad), 2923, 1585, 1425, 1260, 1150, 879 cm ; UV 277, 469 nm.
2
-
4
2
-1
(
V. Computational Methods
Density functional theory44,45 was applied in this study to deter-
according to the relation E1/2 ) (Epa + E )/2.
4
6-49
mine the optimized geometry of each species.
All calculations
were performed using Gaussian 0350 at the Ohio Supercomputer
Center. Optimized geometries were obtained at the B3LYP/6-31G*
level. Electron spin densities (populations) were obtained from a
NPA approach using single-point energies evaluated at the B3LYP/
-311+G** level with 6d functions. The effect of solvation on
the gas-phase calculations was also investigated using the PCM.
(50) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K.
N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.;
Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.;
Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.;
Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li,
X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.;
Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.;
Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.;
Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich,
S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A.
D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A.
G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.;
Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham,
M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.;
Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian
03, revision B.04; Gaussian, Inc.: Pittsburgh, PA, 2003.
51
6
52-56
Because the vibrational frequency calculation for each trityl radical
could not be finished in more than 14 days of CPU time, we
employed the bottom-of-the-well energies to compare the respective
radicals.
EPR Measurements. EPR measurements were carried out on
an X band spectrometer with HS resonator at room temperature.
(
44) Labanowski, J. W.; Andzelm, J. Density Functional Methods in
Chemistry; Springer: New York, 1991.
45) Parr, R. G.; Yang, W. Density Functional Theory in Atoms and
Molecules; Oxford University Press: New York, 1989.
(
(51) Reed, A. E.; Weinhold, F. A.; Curtiss, L. A. Chem. ReV. 1998, 98,
899.
(
(
(
46) Becke, A. D. Phys. ReV. A: At., Mol., Opt. Phys. 1988, 38, 3098.
(52) Tomasi, J.; Persico, M. Chem. ReV. 1994, 94, 2027.
47) Becke, A. D. J. Chem. Phys. 1993, 98, 5648.
(53) Cossi, M.; Barone, V.; Cammi, R.; Tomasi, J. Chem. Phys. Lett.
1996, 255, 327.
48) Lee, C.; Yang, W.; Parr, R. G. Phys. ReV. B: Condens. Matter 1988,
3
7, 785.
(54) Barone, V.; Cossi, M.; Tomasi, J. J. Chem. Phys. 1997, 107, 3210.
(55) Barone, V.; Cossi, M.; Tomasi, J. J. Comput. Chem. 1998, 19, 404.
(56) Cossi, M.; Barone, V. J. Chem. Phys. 1998, 109, 6246.
(49) Hehre, W. J.; Radom, L.; Schleyer, P. V.; Pople, J. A. Ab Initio
Molecular Orbital Theory; John Wiley & Sons: New York, 1986.
7278 J. Org. Chem., Vol. 71, No. 19, 2006

ReactiVity of Tetrathiatriarylmethyl Radicals
Acetonitrile and phosphate-buffered saline were used without
further purification. Tetrabutylammonium perchlorate (TBAP) and
Acknowledgment. This work was supported by NIH grants
HL38324, HL63744, and HL65608. C.M.H. acknowledges
support from the NSF-funded Environmental Molecular Science
Institute (CHE-0089147). The Ohio Supercomputer Center
2,2,6,6-tetrmethylpiperidine-1-oxyl (TEMPO) were used as pur-
chased.
(
OSC) is acknowledged for generous computational support of
All measurements were performed in dry acetonitrile with 0.1
M TBAP as the supporting electrolyte for triester trityl radical
-CO Et or in phosphate aqueous solution for carboxylate salt trityl
radical 1-CO Na. The concentration of the investigated compounds
was about 1 mM. Voltage was cycled between -2.0 and 0.0 V
beginning at 0.0 V, between 0.0 and 2.0 V beginning at 0.0 V, and
this research.
1
2
Supporting Information Available: Geometries, energies,
enthalpies, and free energies for all compounds and their corre-
sponding adducts and H NMR, EPR, and MS spectra. This material
is available free of charge via the Internet at http://pubs.acs.org.
2
1
-2.0 and 2.0 V beginning at 0.0 V.
JO0610560
J. Org. Chem, Vol. 71, No. 19, 2006 7279