Large-scale synthesis of a persistent trityl radical for use in biomedical EPR applications and imaging

Article abstract of DOI:10.1016/j.bmcl.2007.10.030

Tetrathiatriarylmethyl radicals are ideal spin probes for biological electron paramagnetic resonance (EPR) spectroscopy and imaging. The wide application of trityl radicals as biosensors of oxygen or other biological radicals was hampered by the lack of affordable large-scale syntheses. We report the large-scale synthesis of the Finland trityl radical using an improved addition protocol of the aryl lithium monomer to methylchloroformate. A new reaction for the formal one-electron reduction of trityl alcohols to trityl radicals using neat trifluoroacetic acid is reported as well. Initial applications show that the compound is very sensitive to molecular oxygen. It has already provided high-resolution EPR images on large aqueous samples and should be suitable for a broad range of in vivo applications.

Full text of DOI:10.1016/j.bmcl.2007.10.030

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6801–6805
Large-scale synthesis of a persistent trityl radical for use
in biomedical EPR applications and imaging
Ilirian Dhimitruka,a,d Murugesan Velayutham,a,b Andrey A. Bobko,a
Valery V. Khramtsov,a Frederick A. Villamena,a,c
Christopher M. Hadadd and Jay L. Zweiera,b,
*
aCenter for Biomedical EPR Spectroscopy and Imaging, The Davis Heart and Lung Research Institute,
The Ohio State University, Columbus, OH 43210, USA
bDivision of Cardiovascular Medicine, Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, USA
cDepartment of Pharmacology, College of Medicine, College of Medicine, The Ohio State University, Columbus, OH 43210, USA
dDepartment of Chemistry, The Ohio State University, Columbus, OH 43210, USA
Received 12 September 2007; revised 9 October 2007; accepted 10 October 2007
Available online 17 October 2007
Abstract—Tetrathiatriarylmethyl radicalsareidealspinprobesforbiologicalelectronparamagneticresonance(EPR)spectroscopyand
imaging. The wide application of trityl radicals as biosensors of oxygen or other biological radicals was hampered by the lack of afford-
ablelarge-scalesyntheses. Wereportthelarge-scalesynthesisoftheFinlandtritylradicalusinganimprovedadditionprotocolofthearyl
lithium monomer to methylchloroformate. A new reaction for the formal one-electron reduction of trityl alcohols to trityl radicals using
neat trifluoroacetic acid is reported as well. Initial applications show that the compound is very sensitive to molecular oxygen. It has
already provided high-resolution EPR images on large aqueous samples and should be suitable for a broad range of in vivoapplications.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Reactive oxygen species (ROS) in low concentration
play a vital role in the regulation of physiological pro-
cesses and are central in the initiation of pathological
events in unregulated concentrations.1–3 It is well estab-
lished that the generation of ROS in cellular systems is a
result of chemical or enzymatic reduction of molecular
oxygen and is modulated by tissue oxygen levels.4–7
Therefore, it is critical to monitor the degree of intracel-
lular and tissue oxygenation in vitro and in vivo. Con-
ventional methods, such as optical and electrochemical
techniques, have been employed in the monitoring of
oxygen partial pressure (pO2) but are limited by their
invasiveness and the coverage of area being measured.
Electron paramagnetic resonance (EPR) spectroscopy
and imaging using water-soluble paramagnetic probes,
such as nitroxides and trityl radicals, as well as particu-
late-based probes, such as chars, india ink and lithium
phthalocyanine, have been employed for the measure-
ment of pO2 or dissolved O2 in the mouse tumor, brain,
and heart.8–10 EPR imaging has also been employed in
the investigation of drug delivery, toxicity of xenobiot-
ics, and tissue redox status.11–13 In contrast to conven-
tional methods, EPR spectroscopy and imaging are
non-invasive techniques. Furthermore, these techniques
can be applied to map oxygenation over a predefined
area in various tissues. In particular, trityl radicals have
been demonstrated to be very effective spin probes in
measuring the oxygen concentration in vivo, and
in vitro through line-width broadening analysis.14–17
Nycommed Innovations (now a subsidiary of GE
Healthcare) initially designed the sterically crowded tri-
tyl radicals, otherwise known by the acronym TAM (tet-
rathiatriarylmethyl), for use as contrast agents in
Overhauser Magnetic Resonance Imaging (Fig. 1).18–26
Gomberg’s work over 100 years ago proved that trityl
radicals are persistent at room temperature, hence the
interest in these compounds.27 An ideal spin probe
would exhibit a single, sharp EPR signal in order to
obtain maximum sensitivity under in vivo conditions.
To ensure practical use in biological systems, it is conve-
nient to have trityl radicals that are soluble in water.
By removing all sources of inhomogeneous hyperfine
Keywords: EPR spectroscopy and imaging; Trityl radical; Trityl alco-
hol; Tetrathiatriarylmethyl; TAM.
*
Corresponding author. Tel.: +1 614 247 7857; fax: +1 614 247
7845; e-mail: Jay.Zweier@osumc.edu
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.030

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I. Dhimitruka et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6801–6805
O
S
ONa
O
S
S
S
S
NaO
S
S
S
HO
HO
OH
OH
3
S
S
S
S
S
S
S
S
O
ONa
O
ONa
OX063
Finland Trityl
Gomberg's trityl radical
Figure 1. Representative trityl radicals used as spin probes.
couplings, the radicals exhibit extremely narrow signals,
allowing for high sensitivity (i.e. detection of nanomolar
concentration of radicals by EPR spectroscopy in bio-
logical media). The trityl radicals designed by Nycom-
med Innovations are extremely efficient spin probes;
however they are complex molecules, and their synthesis
in large scale has proven challenging.28 This limits their
utility, as biological, and especially in vivo animal stud-
ies and clinical applications require large quantities of
these spin probes. Herein, we report a procedure for
the large-scale synthesis of the Finland trityl radical
(radical 7 in this paper).
The direct synthesis of trityl alcohol 5 was extremely
slow because of the steric bulk of the aryl monomers.
The ketone 4 was isolated first, as an intermediate to-
ward the trityl alcohol 5.30 Subsequently it was reacted
with the 3 equiv of the aryl lithium monomer to afford
trityl alcohol 5 in very good yield (method A). This pro-
cedure involved lengthy chromatographic purifications;
however it gave us easy access to gram amounts of the
target compound 5.31 Next, tert-butyl ester 6, rather
than the ethyl ester described in the literature procedure,
was synthesized in good yield.32 The rationale was that
tert-butyl esters are not susceptible to multiple addition
and polymerization. Compound 6 was treated with neat
trifluoroacetic acid over 20 h at rt in order to cleave the
tert-butyl ester, and fortuitously during the same step
the radical 7 was generated in quantitative yield. Evap-
orating the trifluoroacetic acid afforded the pure radical
7 in quantitative yield. This formal one-electron reduc-
tion of the central carbon was quite surprising. Other
trityl alcohols that do not posses, the tert-butyl ester
functionality undergo formal one-electron reduction of
the central carbon using neat trifluoroacetic acid just
as easily and efficiently.33 We are looking into possible
explanations for this particular reaction. In order to
scale up to any desired amount of product, further mod-
ifications were introduced. The extremely slow addition
of methyl chloroformate afforded the trityl 5 from start-
Using the patent literature, and the procedure published
by Rawal and co-workers, milligram amounts of radical
7 were produced.29 The scale up of this literature proce-
dure did not give satisfactory results. The synthesis of
compound 2 proceeded as described in the literature
with some minor changes (Scheme 1). It was necessary
to reflux for at least one day, rather than 2 h in order
to synthesize 70 g of compound 2. Compound 3 was ob-
tained in large amount as reported in the literature, with
minor modifications.28,29 Ethanol was used to precipi-
tate pure compound 3 from the crude reaction mixture
instead of acetonitrile and tetrahydrofurane. However,
our attempts to produce the trityl alcohol 5 in large scale
were unsuccessful, resulting in very low yield.
O
2
Cl
Cl
Cl
Cl
c
a
S
S
S
S
S
S
S
S
S
S
S
S
b
1
4
3
2
OH
OH
3
3
3
S
S
S
S
S
S
S
f
e
S
S
S
d
S
S
OH
O
O
O
5
6
7
Scheme 1. Reagents and conditions: (a) Na, DMF, 2-methyl-2-propanethiol 61%; (b) HBF4 54% in Et2O, acetone, toluene 51%; (c) n-BuLi 2.5 M in
hexane 1 equiv in Et2O; than 0.3 equiv of methyl chloroformate; (d) n-BuLi 2.5 M in hexanes 3 equiv, 3 equiv of 3 in Et2O; than add the ketone 4;
70% in two steps; (e) n-BuLi 2.5 M in hexanes and TMEDA 10 equiv than excess Diboc 44%; (f) CF3COOH 99%.

I. Dhimitruka et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6801–6805
6803
ing material 3 in one step, as described in method B.31
The procedures have been optimized, and all products
are purified by precipitation, making possible the scale
up to any desired amount.
The radical 7 is a bulky molecule, surrounded by hydro-
phobic groups on most of its surfaces. However, the
three carboxylic groups render radical 7 to be soluble
in water. Due to aggregation, a lower concentration of
up to 2 mM was found suitable for practical applica-
tions. Three equivalents of hydrochloric acid were re-
quired to neutralize one equivalent of radical 7. This
number was calculated by potentiometric titration using
the amount of HCl used to neutralize the trisodium tri-
carboxylate form of the radical 7 (m(HCl)/7 ml/[radical
7] = 34.3 lmol/7 ml/1.6 mM ꢀ 3). The obtained number
supports the independent protonation of all three car-
boxyl groups of the radical 7 with similar pKa values
in agreement with the large distance between these
groups. Titration of the radical 7 was performed using
X-band EPR spectroscopy as well (Fig. 2).
Figure 3. The dependence of the EPR peak signal intensity of radical 7
on the pH of the sample (50 lM radical 7 in the presence of 1.5 mM
citrate buffer). The symbols (j) and (s) denote the data obtained
upon titration from alkaline pH into the direction of acidic pH and in
reverse direction, respectively.
The determination of the exact pKa value by potentio-
metric titration is complicated due to precipitate forma-
tion in acidic medium. Reversible EPR spectral changes
were observed with the appearance of a broad signal
with a line-width of 2.5 G in the acidic medium, appar-
ently due to reversible aggregation of the radical 7
(Fig. 2). To investigate the influence of pH of the solu-
tion on the EPR intensity as a result of aggregation
the experiment in Figure 3 was performed. The solid
linꢁes in Figure 3 represent the best fits for the function
[R ]pH = [R]0 ꢁ [R]0/(1 + 10(pHꢁpKagg)/q), where [R]0 is
total radical 7 concentration, [Rꢁ]pH is the concentration
of the anionic form of the radical 7 dissolved at given
pH value, pKagg is the pH value when half of the radical
7 molecules are aggregated, and q is a numerical coeffi-
cient. The fittings yield the pKagg and q values to be
equal to 3.7 and 0.37, respectively, for titration from ba-
sic to acidic medium. The corresponding values for titra-
tion from acidic to basic medium are 4.0 and 0.54,
respectively. The data support the conclusion that
[Rꢁ]pH = [R]0 if pH > 4.5, while in acidic conditions
[Rꢁ]pH decreases exponentially, becoming close to zero
at pH 3. The calculated partition coefficient of the rad-
ical 7 (defined as Kp=[Octanol]/[water]) at pH values
equal to 1, 7.4, and 13 was found to be P4. · 104,
0.27 0.15, and 0.3 0.1, respectively. The Kp values
provide further evidence for the observed low solubility
of the radical 7 in acidic solutions.
The oxygen partial pressure is one of the most important
parameters in the metabolic processes of the cell, thus it
plays a key role in physiological processes. Molecular
oxygen has two unpaired electrons, and the bimolecular
collision with a persistent radical (spin probe) of known
concentration causes the EPR line broadening of the
persistent radical. This line broadening is measured;
thus indirectly the concentration of oxygen in living tis-
sue can be ascertained. At biological pH (pH 7.4) we
estimate from the approximate pKa = 3.7 that the radi-
cal 7 exists in ionized form (7ꢁ). The EPR line-width
of the radical 7ꢁ was measured at different concentra-
tions of oxygen, diluted with argon. The EPR spectrum
in phosphate buffer (0.1 M, pH 7.4) consists of a single
sharp peak with a peak-to-peak line-width of 210 mG
in room air. The radical 7ꢁ exhibited excellent EPR
characteristics with an anaerobic line-width of 90 mG,
and 0.84 mG/mm Hg linear sensitivity to oxygen. The
line broadening effect is reversible upon exclusion of
oxygen by argon, and the line-width is restored to its ori-
ginal value of 90 mG (Fig. 4).
Figure 2. EPR spectra of 0.05 mM solution of the radical 7 in the
presence of 1.5 mM citrate buffer at different pH values. The
spectrometer settings were: sweep width, 30 G; time constant,
5.12 ms; conversion time, 40.96 ms; scan time, 83.89 s; 2048 points;
modulation amplitude, 0.2 G; and microwave power, 5.0 mW. The pH
of the samples was adjusted to the required value by the addition of
1 M aqueous HCl.
Radical 7ꢁ has already been used as a probe for EPR
imaging experiments in large aqueous samples.34 These
imaging experiments were performed in a home-built

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applications.
In conclusion, an efficient synthetic protocol for the
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and is very reproducible. Within its solubility range,
the ionized form of radical 7 (7ꢁ) exhibited excellent
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Acknowledgments
This work was supported by National Institutes of
Health Grants EB0890, EB4900, EB 03519, and
HL81248.
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.10.030.
29. Xia, S.; Villamena, F. A.; Hadad, C. M.; Kuppusamy, P.;
Li, Y.; Zhu, H.; Zweier, J. L. J. Org. Chem. 2006, 71, 7268.
30. Bis-(2,2,6,6-tetramethyl-benzo[1,2-d;4,5-d0]bis[1,3]dithiol-
4-yl)-methanone (4). To a solution of 3 (9 g, 31.4 mmol) in
350 ml of diethyl ether was added 12.6 ml of n-butyl
lithium (2.5 M in hexanes). The solution was stirred for at
least 2 h. Methyl chloroformate (0.97 ml, 12.5 mmol),
dissolved in 60 ml of diethyl ether, was added dropwise
over 120 min, and the solution was stirred over 24 h.
Saturated aqueous sodium bicarbonate was added, and
the organic phase was separated. The aqueous phase was
extracted again with 2· 100 ml of diethyl ether. The
combined organic phases were dried over sodium sulfate
and evaporated to afford the ketone 4 mixed with starting
material 3. The title compound 4 was isolated in 70% yield
(6.60 g) after flash chromatography on silica gel eluting
References and notes
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and Medicine; Oxford University Press: Oxford, 1999.
3. Zweier, J. L.; Talukder, H. Cardiovasc. Res. 2006, 70, 181.
4. Zweier, J. L.; Kuppusamy, P.; Williams, R.; Rayburn, B.
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Chem. 1989, 264, 18890.

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6805
1
first with hexanes to recover the starting material 3, than
with 11:1 hexanes-ethyl acetate. mp = 120–127 ꢁC; 1H
NMR (500 MHz, CDCl3) d = 7.19 (s, 2H), 1.78 (s, 12H);
13C (125 MHz, CDCl3) d = 193.6, 137.7, 137.6, 127.3,
119.4, 65.4, 31.2; MS (ESI, [M+Na]+): 620.9648
(observed), 620.9647 (calcd).
5 in 69% yield (8.5 g). mp = 145–154 ꢁC (dec); H NMR
(500 MHz, CDCl3) d = 7.19 (s, 3H), 6.22 (s, 1H), 1.82 (s,
9H), 1.80 (s, 9H), 1.77 (s, 9H), 1.70 (s, 9H); 13C (125 MHz,
CDCl3) d = 139.2, 138.3, 137.8, 137.3, 131.9, 118.2, 83.7,
64.1, 63.4, 34.8, 32.3, 29.2, 27.6.
32. Tris-(8-tert-butoxycarbonyl-2,2,6,6-tetramethyl-benzo-
[1,2-d;4,5-d0]bis[1,3]dithiol-4-yl)-methanol (6). To a solu-
tion of 5 (8.5 g, 9.6 mmol) in 40 ml of benzene and
TMEDA (14.5 ml, 96 mmol) was added slowly n-butyl
lithium (38.4 ml 2.5 M in hexanes, 96 mmol). The mixture
was stirred for 2 h at room temperature. The resulting
mixture was added slowly via syringe to solid di-tert-butyl-
dicarbonate (DiBoc, 136 g, 0.62 mol), which was previ-
ously charged in a 1 L dry flask, and placed at 0 ꢁC. The
reaction mixture was allowed to reach room temperature
and stirred for 2 days. A reflux condenser was placed on
the reaction flask, and methanol was added in small
portions on top, via the condenser, until no more reaction
was observed. The mixture was evaporated under high
vacuum, treated with 150 ml of methanol, and the yellow
precipitate was filtered and washed with small portions of
methanol. Compound 6 was obtained in 44% yield (5 g).
mp = 200–210 ꢁC (dec); 1H NMR (500 MHz, CDCl3)
d = 6.72 (s, 1H), 1.78 (s, 9H), 1.75 (s, 9H), 1.68 (s, 45H);
13C (125 MHz, CDCl3) d = 165.4, 141.2, 140.9, 140.3,
139.1, 133.8, 129.6, 122.9, 115.4, 84.2, 60.8, 34.0, 31.1,
29.3, 28.6, 28.4, 28.3; MS (ESI, [M+Na]+): 1207.1218
(observed), 1207.1198 (calcd).
31. Tris-(2,2,6,6-tetramethyl-benzo[1,2-d;4,5-d0]bis[1,3]dithiol-
4-yl)-methanol (5). Method A: To a solution of 3 (9 g,
31.4 mmol) in 350 ml of diethyl ether was added 12.6 ml of
n-BuLi (2.5 M in hexanes). The solution was stirred for at
least 2 h. The ketone 4 (5 g, 8.34 mmol) was added all at
once, and the resulting mixture was stirred for over 24 h.
Saturated aqueous sodium bicarbonate was added, and
the organic phase was separated. The aqueous phase was
extracted again with 2· 100 ml of diethyl ether. The
combined organic phases were dried over sodium sulfate
and evaporated. The mixture was purified by flash
chromatography on silica gel eluting first with hexanes
to remove the starting material 3, then with 20:1 to 10:1
hexanes-ethyl acetate. The title product 5 was isolated in
96% yield (7.1 g). Method B: To a solution of 3 (12 g,
41.9 mmol) in 450 ml of diethyl ether was added 16.8 ml of
n-BuLi (2.5 M in hexanes). The solution was stirred for at
least 2 h. Methyl chloroformate (1.29 ml, 16.7 mmol) was
dissolved in 60 ml of diethyl ether and added very slowly
via a syringe pump over 2 days. Saturated aqueous sodium
bicarbonate was added, and the organic phase was
separated. The aqueous phase was extracted again with
2· 100 ml of diethyl ether. The combined organic phases
were dried over sodium sulfate and evaporated. The thick
residue was treated with 200 ml of pentane and stirred
vigorously until a yellow precipitate was obtained. The
precipitate was filtered and dried to afford the title product
33. Bobko, A. A.; Dhimitruka, I.; Zweier, J. L.; Khramtsov,
V. V. J. Am. Chem. Soc. 2007, 129, 7240.
34. Deng, Y.; Petryakov, S.; He, G.; Kesselring, E.;
Kuppussamy, P.; Zweier, J. L. J. Magn. Res. 2007,
185, 283.