RGD-conjugated triarylmethyl radical as probe for electron paramagnetic imaging

Article abstract of DOI:10.1016/j.tetlet.2013.08.120

A stable RGD-conjugated triarylmethyl (TAM) radical 3 has been synthesized in a straightforward three-step sequence. CT-03 'the Finland trityl' which is a very popular contrast agent for electron paramagnetic resonance imaging has been covalently bound to the NH₂-ending of a pentapeptide commonly used to target integrins and confers tissue selectivity. Moreover, this new TAM radical is stable and sensitive to molecular oxygen.

Full text of DOI:10.1016/j.tetlet.2013.08.120

Tetrahedron Letters 54 (2013) 5924–5926
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
RGD-conjugated triarylmethyl radical as probe for electron
paramagnetic imaging
Benoît Driesschaert a,b, Philippe Levêque b, Bernard Gallez b, Jacqueline Marchand-Brynaert a,
⇑
a Institute of Condensed Matter and Nanosciences (IMCN), Molecules, Solids and Reactivity (MOST), Université catholique de Louvain, Place Louis Pasteur 1, L4.01.02, B-1348
Louvain-la-Neuve, Belgium
b Louvain Drug Research Institute (LDRI), Biomedical Magnetic Resonance Section (REMA), Université Catholique de Louvain, Avenue Mounier 73, B1.73.08, B-1200 Bruxelles, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A stable RGD-conjugated triarylmethyl (TAM) radical 3 has been synthesized in a straightforward three-
step sequence. CT-03 ‘the Finland trityl’ which is a very popular contrast agent for electron paramagnetic
resonance imaging has been covalently bound to the NH2-ending of a pentapeptide commonly used to
target integrins and confers tissue selectivity. Moreover, this new TAM radical is stable and sensitive
to molecular oxygen.
Received 15 July 2013
Revised 12 August 2013
Accepted 28 August 2013
Available online 6 September 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
EPR
Trityl radical
Oximetry
RGD
Low frequency electron paramagnetic resonance (EPR) imaging
is a technique that allows in vivo mapping of the distribution of a
paramagnetic species, and other physiological parameters such as
oxygen concentration, pH and redox status.1,2 The physical basis
of EPR is similar to the well known nuclear magnetic resonance
(NMR). However, the magnetic moment of the electron is 658
times greater than that of the proton, which makes EPR intrinsi-
cally more sensitive than NMR for the same probe concentration.
By contrast with magnetic resonance imaging (MRI) by NMR, there
is no naturally occurring paramagnetic species in sufficient con-
centration and long-enough half-life to be imaged in vivo by EPR
imaging (EPRI), with the exception of melanin.3,4 Thus an exoge-
nous paramagnetic spin probe is needed. Among the water soluble
radicals used in EPRI, the triarylmethyl (TAM) radicals bearing four
sulfur atoms on each phenyl ring such as CT-03 and Ox063 (Fig. 1)
are the most attractive.5
reported. Original Nycomed’s TAMs and subsequent chemically
modified TAMs have been used for EPR measurements of intracellu-
lar7 and extracellular8 O2 concentrations, pH,9,10 thiol,11 superoxide
anion,12 redox status,13 spin–spin interdistance,14 for EPRI15 and
employed as hyperpolarizing agents.6 However, to date, only tri-
arylmethyl radicals with unspecific distribution have been
reported.
Hereby, we describe a straightforward synthesis of the penta-
fluorophenol ester derivative 2 of CT-03. This activated form of
CT-03 can be used to bind the trityl core to a targeting moiety
designed to achieve tissue selectivity. Compound 2 has been used
to conjugate the TAM radical to the pentapeptide GRGDS
(Gly-Arg-Gly-Asp-Ser) including the RGD sequence, known to bind
some of the trans-membrane proteins called integrins which are
commonly used as targets for molecular imaging.16
These compounds initially developed to be used as hyperpolariz-
ing agents for Overhauser-enhanced magnetic resonance imaging
(OMRI)6 exhibit a good stability, a single narrow EPR line of less than
100 mG under anoxic conditions (0% oxygen), a low concentration-
dependent line-broadening and non toxic properties.5 Indeed, a nar-
row EPR line confers high sensitivity of detection, particularly useful
in vivo, since the intensity of the signal is inversely proportional to
the square of the line width. Yet, the synthesis of such complex mol-
ecules remains challenging and new structures are regularly
3
OH
OH
3
HO
HO
S
S
S
S
S
S
S
S
COOH
COOH
CT-03
OX063
⇑
Corresponding author.
E-mail address: jacqueline.marchand@uclouvain.be (J. Marchand-Brynaert).
Figure 1. Chemical structure of CT-03 and Ox063.
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.08.120

B. Driesschaert et al. / Tetrahedron Letters 54 (2013) 5924–5926
5925
matogram obtained under gradient elution (Fig. 2a), two peaks
with the same areas are obtained under isocratic elution
(Fig. 2b). These two peaks correspond to diasteroisomers associ-
ated with the helicoidal chirality of the trityl core.18,19
Indeed, the trityl core displays a propeller conformation where
all rings have the same direction of twist. Both right-handed (P)
and left-handed (M) propeller conformations exist and display
enantiomeric relationship. We have previously shown that for
the tetrathiatriarylmethyl core, the isomerization between the
two enantiomeric conformations is slow, which confers chiral
properties to these molecules.18,19 As GRGDS is an enantiomeri-
cally pure molecule, two diastereoisomers are obtained for the
conjugated compound 3.
3
(COCl)2
DIEA
3
S
S
S
S
S
S
S
C6F5OH
Pyridine
r.t., DCM
64 %
- 78°C to r.t.
DCM
S
COOH
COCl
82 %
CT-03
1
NH
3
3
S
S
S
S
S
NH2
HN
S
S
S
H
N
GRGDS
The EPR spectrum of 3 has been recorded at X-band in phos-
phate buffer saline (PBS) (Fig. 3). The spectrum shows a single
O
N
H
Na2CO3
DMSO
62%
O
F
O
O
F
F
HN
O
EPR line with a peak-to-peak linewidth
DBpp = 705 mG under air
room conditions (21% O2). The wider linewidth obtained for 3 than
for CT-03 can be explained by unresolved hyperfine splitting with
nitrogen atoms of the amide functions. This observation has been
already reported for dendritic TAM radicals.20 Bimolecular collision
between a TAM radical and molecular oxygen induces a broaden-
ing of the EPR line of the TAM spin probe. This phenomenon can
be used to assess oxygen concentration by EPR. According to the
Smoluchowski equation there is a linear relationship between
the linewidth and O2 concentration for a soluble spin probe. With
OH
O
O
F
HO
NH
O
F
2
N
H
O
OH
3
Scheme 1. Synthesis of 3.
0% of oxygen, the linewidth of 3 reaches
that compound 3 keeps its sensitivity to oxygen and can be used as
an EPR oxymetric probe (sensitivity: 0.47 mG/mmHg).
DBpp = 630 mG, indicating
The synthesis has been achieved as depicted in Scheme 1. CT-03
is synthesized according to a procedure described in the litera-
ture.17 Activation of the carboxylic acid functions of CT-03 by
oxalyl chloride and diisopropylethylamine (DIEA) leads to the
formation of 1 in 83% yield after filtration on a small silica gel path.
Then, esterification of 1 with pentafluorophenol and pyridine
furnishes 2 which is stable enough to be purified by column chro-
matography. This compound was recovered in a pure form in 64%
yield.
Compound 2 is an activated form of CT-03 and can be used to
conjugate the TAM radical to any carrier device. In order to prove
the concept, 2 was mixed in DMSO with a small excess of unpro-
tected pentapeptide GRGDS in the presence of sodium carbonate.
Triarylmethyl radical 3 coupled to three GRGDS peptides, most
probably through the reactive terminal NH2 function, was formed
with more than 90% conversion. Purification on semi-preparative
Recent studies about in vitro metabolism of CT-03 and Ox063
have shown that they could be reduced under anaerobic conditions
in the presence of liver microsomes to the corresponding triar-
ylmethane through formation of the anion.21 Furthermore, a TAM
cation is obtained through oxidation of radicals by peroxidases
and related hemoproteins.22 The cation can subsequently react
with nucleophiles such as water, peroxides, phosphines, thiols,
amines, etc. The redox potential of compound 3 has been measured
in PBS (pH 7.4) by cyclic voltammetry (CV) (Fig. 4) and compared
with CT-03.
The results show that compound 3 can be reduced more easily
than CT-03 but is harder to oxidize than CT-03 (Table 1). This can
be explained by the higher electron-attracting power of amide
functions of 3 by comparison with the carboxylate functions of
CT-03 in aqueous medium. Thus, radical 3 appears to be more sta-
ble than CT-03 under oxidation conditions.
HPLC carried out on reverse-phase gives
3 in a pure form
(Fig. 2a) in 62% yield. While only one peak is visible on the chro-
In conclusion, we have synthesized for the first time a tetrathi-
atriarylmethyl radical linked to three carrier molecules. Compound
3 has been synthesized by mixing pentafluorophenol ester 2 with
an unprotected pentapeptide sequence, followed by purification
Figure 2. HPLC chromatograms of 3 under gradient (a) and isocratic (b) elution.
Figure 3. EPR spectrum of 3 recorded in PBS (pH 7.4) at X-band.

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B. Driesschaert et al. / Tetrahedron Letters 54 (2013) 5924–5926
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.08.
120.
References and notes
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Table 1
Redox potentials (E1/2) relative to Ag/AgCl (KCl 3 M) determined by CV
Compound
Solvent
E1/2 oxa
E1/2 reda
CT-03
3
PBS
PBS
+0.434
+0.648
À0.642
À0.429
a
Calculated according to E1/2 = (Epa + Epc)/2.
by semi-preparative HPLC. The RGD-conjugated TAM 3 keeps the
sensitivity to oxygen. Its oxidation potential is higher than that
of CT-03 while its reduction potential is lower. Thus radical 3 could
be considered as a potential spin label possessing tissue selectivity
for EPR O2 measurements and for EPRI. Moreover, the activated es-
ter 2 is a valuable key-intermediate which can be used to bind
other carrier molecules to the TAM radical.
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Acknowledgements
We gratefully acknowledge funding from the Université
catholique de Louvain, the Fond de la Recherche Scientifique
(F.R.S-FNRS). J.M.B. is a senior research associate for the F.R.S-FNRS.
Professor Z. Mekhalif (F.U.N.D.P-Namur, Belgium) is gratefully
acknowledged for the recording of cyclic voltammograms.