Synthesis, structure, and EPR characterization of deuterated derivatives of Finland trityl radical

Article abstract of DOI:10.1016/j.bmcl.2010.05.006

Substituted trityl radicals are important spin probes for functional electron paramagnetic resonance spectroscopy and imaging including oxygen and pH mapping in vivo. Here we report the synthetic procedure for large scale synthesis of deuterated Finland t

Full text of DOI:10.1016/j.bmcl.2010.05.006

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3946–3949
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, structure, and EPR characterization of deuterated derivatives
of Finland trityl radical
*
Ilirian Dhimitruka, Olga Grigorieva, Jay L. Zweier, Valery V. Khramtsov
Davis Heart and Lung Research Institute and the Divisions of Pulmonary Critical Care Allergy & Sleep Medicine and Cardiovascular Medicine,
Department of Internal Medicine of the College of Medicine, The Ohio State University, Columbus, OH 43210, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Substituted trityl radicals are important spin probes for functional electron paramagnetic resonance
spectroscopy and imaging including oxygen and pH mapping in vivo. Here we report the synthetic pro-
cedure for large scale synthesis of deuterated Finland trityl radical with superior EPR spectral properties
and higher sensitivity towards oxygen concentrations in solution. Additionally Finland trityl radicals
substituted with linkers suitable for attaching peptide, or other synthetic precursors have been synthe-
sized. The effect of deutero-substitution on EPR spectra of homologous derivatives has been evaluated.
The compounds are potential candidates for targeted spin probes in EPR imaging.
Received 22 March 2010
Accepted 4 May 2010
Available online 7 May 2010
Keywords:
Electron paramagnetic resonance
EPR imaging
Ó 2010 Elsevier Ltd. All rights reserved.
EPR oximetry
Trityl radicals
Deuterated trityl radicals
Triarylmethyl radicals, TAMs, and nitroxyl radicals, NRs, repre-
sent two main classes of soluble paramagnetic materials used for
EPR spectroscopy and imaging applications. TAMs have advantages
over NRs in extraordinary stability toward tissue redox processes,
longer relaxation time, and narrower line width making them par-
ticularly attractive for imaging applications.1 However, undevel-
oped chemistry of the TAM radicals limits the number of
available structures and their functional applications.
Triphenylmethyl radical was the first organic free radical syn-
thesized by Gomberg more than hundred years ago.2 Nevertheless,
only recently the compounds with sterically protected trivalent
carbon regained attention as the basic structural fragment for the
synthesis of stable organic radicals. By the late 1990s, Nycomed
Innovation AB refined Gomberg’s original trityl radical in order to
avoid hydrogen hyperfine coupling and enhance its stability and
water solubility.1,3 A new family of trityl spin probes, tetrathiatri-
arylmethyls, bearing four sulfur atoms on the phenyl ring was
developed. The most representative members are TAM derivatives
containing carboxyl group, namely cTAM, deuterated cTAM, and
more hydrophilic Oxo63 derivative (see Scheme 1).
case, the long relaxation time of TAMs makes them easily saturat-
able by radio frequency irradiation, and provides an advantage over
NR for PEDRI applications by allowing enhancement of sensitivity
and resolution with less heating of the sample.1,8,9 Moreover, TAM
radicals, due to their long relaxation time, are the superior probes
for pulsed EPR/EPRI. Applications of TAM radicals include EPR oxim-
etry,1,8,9 recently reported sensitivity to the superoxide radical an-
ion10,11 and pH12,13 and their use as hyperpolarizing agents in
dynamic nuclear polarization (DNP)-enhanced NMR14 and MRI.15
The extraordinary stability in vivo, very narrow single EPR line of
about 100 mG or less, and oxygen-induced line broadening make
TAMs the most efficient soluble oxygen probes. Oxygen-induced
broadening of the TAMs in water is about (300–500) mG/mM of oxy-
gen1,13 similar to that for the NRs. On the other hand, the concentra-
tion broadening of the TAMs is about 10–30 mG/mM1 which is one
order of magnitude less than that for the NRs.16 These properties
make TAM radicals superior oximetric probes for in vivo EPR, EPRI,
and PEDRI applications.1,8
The synthetic chemistry of TAM probes, while it is still in its in-
fancy, is becoming a fast developing area of research. First reports
on the synthesis of TAMs useful for EPRI and PEDRI have appeared
in the patent literature.3 The published procedures have been diffi-
cult to duplicate, and the large scale synthesis of these compounds
has proven to be very challenging. Recently creative efforts have
been done for the synthesis of these complex molecules.4,5,17 We
have published a large scale synthesis of the Finland trityl, cTAM,
based on a few modifications of the original literature.4 Fluorinated
TAMs18 possessing a high affinity to fluorous media were designed
for assessment of tumor oxygenation using biocompatible
The EPR spectra of these TAM derivatives1,4,5 display a very nar-
row single line which is generally not broadened by interaction with
proteins and other biological molecules, making them particularly
attractive for imaging applications using EPR imaging, EPRI, and pro-
ton electron double resonance imaging, PEDRI (also known as
Overhouser magnetic resonance imaging, OMRI).6,7 In the latter
* Corresponding author. Tel.: +1 614 688 3664; fax: +1 614 293 4799.
E-mail address: valery.khramtsov@osumc.edu (V.V. Khramtsov).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.006

I. Dhimitruka et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3946–3949
3947
C
C
C
3
HO
HO
OH
OH
3
S
S
S
S
S
S
S
S
3
=
OH
Finland Trityl-cTAM
Scheme 1. Representative structures of trityl radicals.
O
OH
OX063
O
Gomberg Trityl
OH
3
S
S
S
S
O
D3C
D3C
CD3
CD3
S
S
ii
D3C
D3C
i
S
S
S
S
CD3
CD3
+
D3C
CD3
S
S
1
2
OH
3
3
S
S
S
S
D3C
D3C
iv
CD3
CD3
iii
S
S
S
S
D3C
D3C
CD3
CD3
O
OH
O
O
3
deuterated Finland trityl, cTAM*
*
Scheme 2. Synthesis of deuterated Finland trityl radical, cTAM . Reagents: (i) HBF4, toluene, 70%; (ii) n-BuLi, Et2O, CH3COCl, 70%; (iii) n-BuLi, TMEDA, DiBoc, 44%; (iv)
CF3COOH, 95%.
3
3
S
S
S
S
X3C
X3C
S
S
S
S
X3C
X3C
CX3
CX3
CX3
CX3
+
HO
N
Cl
O
O
OH
O
N
Cl
X=H, cTAM
X=D, cTAM*
X=H, aTAM
X=D, aTAM*
Scheme 3. Reagents: HBTU, DMAP, TEA, DMF, 65%.
perfluorocarbon emulsions. Recently we developed TAM structures
with dual function pH and oxygen sensitivity,12,13 and probes with
enhanced sensitivity to oxygen due to doublet spectral pattern.19
Dendritic20 and ester21 derivatives of Finland trityl were also re-
ported as potentially valuable probes with enhanced stability and
ability for intracellular delivery.
The isotopic substitution of the 36 methyl protons in the three
aryl groups of TAMs for deuterons result in significant decrease of
their EPR linewidth which is important for the applications of
TAMs as functional probes.1 However, until now the synthesis of
several deuterated TAMs were published only in patented litera-
ture.3 Herein, we report the improved procedure for the synthesis
of deuterated Finland trityl (cTAM*) and several new deuterated
cTAM derivatives with the linkers attached to the carboxylic
groups allowing for further functional modification. The effect of
the isotopic substitution on the EPR spectra is described.
To synthesize cTAM* a slightly modified procedure previously
developed in our group for large scale synthesis of cTAM radical
was applied4 (see Scheme 2).
An introduction of new groups and/or linkers in the TAM struc-
ture allows for adjustment of their functional properties, such as
solubility, stability and sensitivity to oxygen and pH.13,17,18,20 The
aTAM derivative with positively charged ammonium group and
*
its deuterated analog, aTAM , were synthesized as shown in
Scheme 3. The carboxylic moiety of cTAM was activated by
standard peptide coupling conditions using O-(benzotriazol-1-yl)-
N,N,N0,N0-tetramethyluronium hexafluorophosphate, HBTU, N,N-
dimethylaminopyridine, DMAP, and triethylamine, TEA, followed
by the addition of choline chloride.
An additional series of TAMe derivatives based on glycolic acid es-
ter, followed by addition of choline chloride was synthesized as
shown in Scheme 4. The compound cTAMe and its deuterated analog
cTAMꢀe with carboxylic end group, and aTAMe and aTAMꢀe with
ammonium end group were obtained. The structures and EPR spec-
tral parameters are summarized in Table 1 and detailed procedures
and EPR spectra are available in the supporting information.
*
The EPR peak-to-peak linewidth, Dpp, of cTAM was found to be
65 mG in anoxic solution in agreement with the previously re-
ported data.1 This 1.5-fold line narrowing compared with 95 mG
linewidth of cTAM corresponds to more than twofold increase in
spectral intensity, and provides significant advantage for EPR/EPRI
applications.
The exchange of proton with deuterons observed during the
first step was less than a few percent. The degree of deuteration
of the product 2 in all preparations exceeded 95% according to
1H NMR data and was even larger (>97%) at larger scale synthesis.

3948
I. Dhimitruka et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3946–3949
3
3
3
S
S
S
S
X3C
X3C
S
S
S
S
CX3
CX3
i, ii
X3C
X3C
S
S
S
S
X3C
X3C
CX3
CX3
iii
CX3
CX3
OH
O
O
O
O
O
N
OH
Cl
O
X=H, cTAM
X=D, cTAM*
O
X=H, aTAMe
X=D, aTAMe*
O
X=H, cTAMe
X=D, cTAMe*
Scheme 4. Reagents: (i) HBTU, DMAP, TEA, DMF glycolic acid, tert-butylester; (ii) trifluoroacetic acid 98% over two steps; (iii) HBTU, DMAP, TEA, DMF, choline chloride, 60%.
Table 1
ate. Interestingly, the aH hyperfine splitting is almost twice larger
EPR spectral parameters of the cTAM, aTAM, aTAMe, cTAMe, and their deuterated
for aTAM (110 mG) with positively charged ammonium group com-
analogs obtained from EPR measurements in anoxic solutions
pared with cTAMe (60 mG) with carboxyl group in agreement with
Dtpp
, 5 mG
TAM
Dpp
,
5 mG
aH (CH2), 2 mG
expected higher electron attracting inductive effect of the ammo-
nium function. An increase of the linker length between aryl and
ammonium groups in the compound aTAMe resulted in decrease
of aH hyperfine splitting from 110 to 70 mG. Note that the hyperfine
structure from methylene protons was not resolved for the non-deu-
terated cTAMe and aTAMe derivatives with aH values being signifi-
cantly lower than the linewidth of the individual spectral
components (see Table 1). For these compounds aH values were first
determined from the corresponding deuterated radicals and then
used for calculation of aH and Dpp values of non-deuterated com-
pounds from their spectral simulation (see Fig. 1 for the aTAMe).
The origin of multiplet spectral structure was further confirmed by
(resolved line)
(unresolved multiplet)
cTAM
95
65
—
—
—
—
cTAM*
aTAM
92
60
110
110
—
—
aTAM*
cTAMe
cTAMe
100
70
60
60
220
ꢁ190a
*
aTAMe
aTAMe
100
60
70
70
230
—
*
a
The spectral line was only partially resolved.
the overnight incubation of aTAMꢀ in CF3COOD/D2O solution, which
e
resulted in partial substitution of the methylene protons for deute-
rons and corresponding disappearance of the multiplet structure
(see Fig. 1c for the EPR spectrum of the aTAMꢀeꢀ).
The EPR spectra of the cTAM and its synthesized derivatives
show similar oxygen-induced line broadening about 70 mG/20%
[O2]. Among ꢀthe deuterated cTAM derivatives the compounds
ꢀꢀ
*
cTAM , cTAMe, and aTAMe demonstrate single EPR line in anoxic
solution with the narrowest line for cTAM*. The cTAM* radical,
therefore, has an advantage in oxygen sensitivity and simplicity
of the EPR spectrum. The multiplet character of the EPR spectra
of aTAM and aTAMꢀe derivatives make them less attractive for
*
EPR oximetric applications. Nevertheless, they have their own
advantages compared with Finland trityl derivatives containing
carboxylic groups. First, as it was previously shown4 cTAM has ten-
dency to aggregation associated with protonation of its carboxyl
groups and facilitated in environments with low pH and low polar-
ity, for example, in the presence of biomembranes. On the other
hand, aTAM derivatives show pH-independent solubility in aque-
ous solution in mM range of concentration. Apparently, the pres-
ence of positively charged ammonium group in the structure of
aTAM derivatives prevents their aggregation and, therefore, makes
them potentially less toxic. Second, in general the EPR oximetric
probes with multiplet spectral pattern possess higher sensitivity
to oxygen at low oxygen tension compared with single-line EPR
probes due to the opportunity to follow changes in peak intensity
ratio of partially resolved components rather than EPR line-
width.19,22 Recently we described the cTAM derivative with en-
hanced sensitivity to oxygen down to 1 mmHg due to its
partially overlapped doublet EPR spectrum.19 Therefore, aTAM*
and aTAMꢀe derivatives may have an advantage when accurate
measurements of low oxygen concentrations are required, for
example, to locate area of hypoxia (615 mmHg23) or even to detect
a threshold of true anoxia (61.5 mmHg).
Figure 1. X-band EPR spectra of 1 mM aTAMe (a), aTAMꢀe (b), and aTAMeꢀꢀ (c)
measured in anoxic DMSO solutions radicals at room temperature. The spectra are
shown with the same receiver gain. Spectral parameters were as follows:
microwave power, 0.63 mW; modulation amplitude, 0.01 G; sweep width, 2 G;
number of points, 1024. The simulated EPR spectra (a) and (b) are shown by dotted
lines.
Similar narrowing effect of deutero-substitution on the EPR line-
width was observed for aTAM, cTAMe, and aTAMe (see Table 1 for the
linewidth of the resolved line). However, additional hyperfine split-
ting (septet with peak intensity ratio 1:6:15:20:15:6:1) appears
from six protons of methylene group, aH, adjacent to aryl carboxyl-
In summary, we describe the synthesis of several new TAM rad-
icals. The deuterated Finland trityl, cTAM*, was synthesized
according to a modified protocol. The synthesis is very efficient
in multigram scale. cTAMe structures, which can be used for

I. Dhimitruka et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3946–3949
3949
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further derivatization such as in designing peptide-TAM bioconju-
gates have been also synthesized. The EPR characterization of new-
ly synthesized compounds should help in predicting the EPR
hyperfine pattern of other derivatized TAMs.
Acknowledgments
The authors thank Drs. Andrey Bobko, Olga Efimova, and De-
nis Komarov for the help in spectra acquisition and analysis. This
work was partially supported by the NIH Grants R21 HL091423,
R01 HL38324, EB0890, and EB4900.
15. Gallagher, F. A.; Kettunen, M. I.; Day, S. E.; Hu, D. E.; Ardenkjaer-Larsen, J. H.;
Zandt, R.; Jensen, P. R.; Karlsson, M.; Golman, K.; Lerche, M. H.; Brindle, K. M.
Nature 2008, 453, 940.
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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.2010.05.006.
18. Driesschaert, B.; Charlier, N.; Gallez, B.; Marchand-Brynaert, J. Bioorg. Med.
Chem. Lett. 2008, 18, 4291.
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