Synthesis of MRI contrast agents derived from DOTAM-Gly-l-Phe-OH incorporating a disulfide bridge: Conjugation to a cell penetrating peptide and preparation of a dimeric agent

Article abstract of DOI:10.1016/j.bmcl.2010.07.070

A cell penetrating peptide conjugate and dimeric PARACEST MRI contrast agents, based on the DOTAM-Gly-l-Phe-OH scaffold have been prepared in moderate yields using diethyl azodicarboxylate (DEAD) or iodine-mediated disulfide bridge formation as a key step. Magnetic (PARACEST) properties of these agents have been evaluated.

Full text of DOI:10.1016/j.bmcl.2010.07.070

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5521–5526
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of MRI contrast agents derived from DOTAM-Gly-L-Phe-OH
incorporating a disulfide bridge: Conjugation to a cell penetrating peptide
and preparation of a dimeric agent
Mojmír Suchy a,b, Alex X. Li b, Robert Bartha b, Robert H. E. Hudson a,
*
´
a Department of Chemistry, The University of Western Ontario, London, ON, Canada N6A 5B7
b Centre for Functional and Metabolic Mapping, Robarts Research Institute, The University of Western Ontario, London, ON, Canada N6A 5K8
a r t i c l e i n f o
a b s t r a c t
Article history:
A cell penetrating peptide conjugate and dimeric PARACEST MRI contrast agents, based on the DOTAM-
Received 6 May 2010
Revised 15 July 2010
Accepted 16 July 2010
Available online 23 July 2010
Gly-L-Phe-OH scaffold have been prepared in moderate yields using diethyl azodicarboxylate (DEAD) or
iodine-mediated disulfide bridge formation as a key step. Magnetic (PARACEST) properties of these
agents have been evaluated.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
PARACEST
Europium
Contrast agent
TAT
Bimetallic
Magnetic resonance imaging (MRI) represents an important
diagnostic tool widely employed to obtain anatomical images of
soft tissues based upon the detection of protons in the body.1
Gd3+-based T1-shortening MRI contrast agents (CAs) are promi-
nently used in clinical diagnostics and decrease bulk water relaxa-
tion time constants through the rapid exchange of agent-bound
water with bulk water. However, there are several limitations
associated with their current use. Firstly, their presence is always
detected in an MR image; and secondly, designing Gd3+-com-
pounds sensitive to metabolic or environmental conditions that
maintain rapid water exchange and consequently high relaxivity
is difficult.
Recently, an alternative method of generating contrast in MRI
was introduced based on chemical exchange saturation transfer
(CEST).2 Subsequently, a large number of paramagnetic ion-con-
taining lanthanide(III) complexes, mainly involving Eu3+ and
Tm3+, have been prepared which are capable of inducing large
hyperfine shifts of coordinated water protons as well as other
exchangeable protons present in close proximity to the metal cen-
ter.3 The term PARACEST MRI CAs has been coined to describe
these molecules.4 When compared to classical Gd3+-derived MRI
CAs, the spectrum of information obtained using PARACEST MRI
CAs is potentially broader as the CEST effect is sensitive to the envi-
ronment. PARACEST CAs have been previously used to detect phys-
iological conditions such as tissue temperature5 and pH.6 Primary
metabolites such as glucose,7 polyarginine8 and lactate9 can also
be detected, along with biologically important phosphate anions10
and Zn2+ cations.11 PARACEST MRI CAs have been successfully ap-
plied toward the detection of enzymatic activities, for example:
cathepsin D;12 b-galactosidase;13 caspase-3;14 or urokinase
plasminogen activator.15
Despite the number of PARACEST MRI contrast agents recently
described, their detection in vivo16 remains a challenging task. This
can be attributed to rather low sensitivity as well as limited bio-
availability of these agents. To address these issues, we turned
our attention to a sulfanyl (thiol, SH) modified ligand 1 (Fig. 1),17
based on Eu3+ DOTAM-Gly-Phe-OH (2b, Fig. 1),18 a DOTAM-poly-
amide based PARACEST MRI CA exhibiting a high level of sensitiv-
ity within the physiological temperature range (36–40 °C). To
facilitate the vectorization of 2b and increase its bioavailability,
thiol 1 was conjugated to a cell penetrating TAT peptide19 by dieth-
ylazodicarboxylate (DEAD)-promoted disulfide bond formation20
followed by metalation with EuCl3Á6H2O. The resulting conjugate
(3, Fig. 1) is expected to undergo reductive disulfide cleavage by
endogenous thiols, such as glutathione, within the intracellular
compartment leaving the Eu3+ containing reporter group trapped
inside the cells for an extended period of time. Alternatively, treat-
ment with an exogenous thiol may facilitate excretion after a per-
iod of MR imaging.21
Disulfide linkages have been employed previously in the prepa-
ration of macromolecular complexes and peptide conjugates,
* Corresponding author. Fax: +1 519 661 3022.
E-mail address: rhhudson@uwo.ca (R.H.E. Hudson).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.070

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M. Suchy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5521–5526
5522
synthesized and have been shown to retain sensitivity on a per-
HO-Phe-Gly
O
Gly-Phe-OH
O
lanthanide basis.27 PANAM-based dendritic PARACEST CAs have
been prepared and used to detect flank tumor in vivo.16a,28 Morrow
and co-workers have evaluated Eu3+- and Nd3+-based bimetallic
complexes consisting of two DOTAM units joined by p-dibenzyl
linkage as potential carbonate or DNA responsive PARACEST MRI
CAs.29 We have recently prepared and evaluated both homo- (con-
N
N
N
N
O
O
HO-Phe-Gly
Gly-Phe-NH SH
1
HO-Phe-Gly
O
Gly-Phe-OH
taining two Eu3+) and heterobimetallic (containing Eu3+ and Tm3+
)
N
N
N
O
2a, Ln = Dy
Ln3+
N
2b, Ln = Eu
complexes featuring two different chelator subunits.30
O
O
In the present study, we report the synthesis and magnetic
properties associated with cell penetrating peptide conjugated
PARACEST MRI CA 3 and bimetallic complexes 4 (Fig. 1). The syn-
thetic methodology represents an alternative approach to conjuga-
tion of DOTA- and DOTAM-derived ligands with peptides that rely
mainly on amide bond formation between DOTA- or DOTAM-
derived carboxylic acids and N-terminus unprotected peptides
attached to a solid support.31
HO-Phe-Gly
Gly-Phe-OH
HO-Phe-Gly
O
Gly-Phe-OH
N
N
Eu3+
N
O
O
O
N
HO-Phe-Gly
Gly-Phe-NH
3
S
S
Sulfanyl-modified ligand 1 has been prepared following the
experimental protocol recently established in our laboratory.17
We initially attempted to conjugate ligand 1 to the cell penetrating
TAT peptide (‘N’-YG-RKK-RRQ-RRR-‘C’) by solid phase peptide syn-
thesis. The fully protected peptide (for the protecting groups see
ref.32) attached to Wang’s resin was derivatized with commer-
cially available Fmoc-Cys(Mmt)-OH. The acid labile Mmt protect-
ing group was selectively removed (1% TFA in CH2Cl2), followed
by treatment with ligand 1 preactivated with diethylazodicarboxy-
late (DEAD).20 Cleavage of the peptide from the resin (5% TES in
wet TFA) followed by HPLC purification afforded the desired disul-
fide-bridged conjugate in unacceptable (<5%, based on ligand 1)
yield, despite many attempts to modify the reaction conditions.
To overcome this problem we turned to solution phase conjugation
chemistry as indicated in Scheme 1.33 Thus, Fmoc-Cys(Trt)-OH (6)
was coupled (HBTU, DIPEA, and DMF) to the resin bound, fully pro-
tected peptide 7. Peptide 8 was obtained after cleavage from the
resin and HPLC purification (Scheme 1). Treatment of peptide 8
with ligand 1 under the conditions of DEAD-promoted asymmetri-
cal disulfide bond formation afforded conjugate 9 in 27% yield
(based on 1) after HPLC purification. With ligand 9 in hand we per-
formed the metalation with EuCl3Á6H2O (Scheme 1). This reaction
was found to be surprisingly slow, requiring large excess of the
Eu3+ salt (20 equiv), elevated reaction temperature (60 °C) and
prolonged reaction time (2–3 days). A time course study of the
metalation of 9 with EuCl3Á6H2O can be found in Supplementary
NH2
RRR-QRR-KKR-GY
HO-Phe-Gly
O
Gly-Phe-OH
N
N
N
Ln3+
N
O
O
O
O
4a, Ln = Dy
4b, Ln = Eu
HO-Phe-Gly
HO-Phe-Gly
Gly-Phe-NH
Gly-Phe-OH
O
N
N
N
O
S
Ln3+
N
S
O
O
HO-Phe-Gly
Gly-Phe-NH
HO-Phe-Gly
O
Gly-Phe-OH
O
N
N
Eu3+
O
O
N
N
S
S
HO-Phe-Gly
Gly-Phe-NH
5
NH2
Figure 1. Structures of ligands and complexes 1–5.
although disulfide bond formation was not always the key step. For
instance, an approach using cystamine as a linker wherein the
b-amino group was used for amide bond formation has been
described for the conjugation of Gd3+-derived MRI CAs with
data. HPLC purification afforded desired Eu3+ DOTAM-Gly-
OH-TAT peptide conjugate 3 in 65% yield (Scheme 1). It is worth
L-Phe-
poly(L L
-glutamic acid)21 and poly( -arginine).22 A Gd3+-based DOTA
has been conjugated to reduced bovine serum albumin (BSA) or
silica nanoparticles by disufides formed through an intermediate
methanethiosulfonate derivative.23 Also carbon–sulfur bond forma-
tion was exploited when a sulfanyl modified DOTA ligand was
attached to phosphopeptides by Michael addition to dehydroalani-
nyl or b-methyldehydroalaninyl residues.24
The presence of a free SH group in 1 also prompted us to explore
the preparation of a homodimeric ligand via symmetrical disulfide
bond formation. The resulting bifunctional disulfide-bridged ligand
was envisioned to undergo a double metalation with Dy3+ or Eu3+
salts to form a bimetallic PARACEST MRI CAs 4a,b (Fig. 1) expected
to possess higher sensitivity, compared to monometallic CAs 2,
based on literature precedent. Sherry and co-workers used free-
radical polymerization and prepared a series of DOTAM-based lin-
ear polymers which upon metalation with Eu3+ salts afforded
PARACEST MRI CAs with enhanced sensitivities.25 A large number
of PARACEST CAs have also been shown to be conjugated to adeno-
virus, although diminished viral bioactivity was observed when the
number of ligands attached to the virus exceeded ꢀ1000.26 Several
dendritic pH responsive multimeric PARACEST MRI CAs have been
+
Fmoc-Cys(Trt)-OH
H2N-YG-RKK-RRQ-RRR
7
i, HBTU, DIPEA, DMF
ii, TFA, TES
6
8
Fmoc-CYG-RKK-RRQ-RRR
1
, DEAD, Et3N, DMF
27%, based on 1
HO-Phe-Gly
O
Gly-Phe-OH
N
N
N
N
O
O
EuCl3 6H2O
3
MeCN/H2O, 65%
O
HO-Phe-Gly
Gly-Phe-NH
9
S
S
NH2
RRR-QRR-KKR-GY
O
Scheme 1. Synthesis of Eu3+ DOTAM-Gly-
L
-Phe-OH peptide conjugate 3.

´
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M. Suchy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5521–5526
pointing out, that excess EuCl3Á6H2O was efficiently removed by
HPLC as confirmed by Xylenol Orange test.34 The resulting peptide
conjugate was characterized by ESI-HRMS.33
therefore be an Eu3+-derived complex of 1. We have previously
shown,17 that synthesis of such complex is troublesome due to
the oxidation during the treatment with EuCl3Á6H2O. In Ref.17
we used a model compound, complex 5 (Fig. 1) possessing a pro-
tected SH group and were able to determine its PARACEST proper-
ties. The CEST effect due to bound water (ca. 45 ppm) was found to
be slightly lower (ca. 35% at 10 mM) compared to parent CA 2b.17
Considering our plans to use the peptide conjugate 3 as biocompat-
ible temperature probe for MRI, we have also established the rela-
tionship between the temperature and the chemical shift of bound
water in complex 5. As observed for the unmodified parent com-
pound 2b, a linear relationship within the physiological tempera-
ture range was obtained as depicted in Supplementary data.
Due to the presence of two lanthanide(III) metals in CAs 4, they
were expected to posses higher sensitivity compared to parent
monomeric structures 2. A PARACEST effect (<5% at ca. À750 ppm)
Synthesis of CAs 4a,b was accomplished as depicted in
Scheme 2.35 DEAD-promoted oxidation of 1 was found to produce
multiple products and was therefore abandoned. However, thiol 1
smoothly underwent disulfide formation upon treatment with Br2
or I2 in the presence of Et3N and the reaction was further optimized
using I2 as an oxidizing agent. The presence of the disulfide 10 as a
major product was confirmed by UPLC-ESI-HRMS.35 Fortunately,
no HPLC purification of crude disulfide 10 was required at this
stage. The crude disulfide 10 was subjected to metalation with Eu-
Cl3Á6H2O (Scheme 2). Similar to the metalation of conjugate 9, a
large excess of EuCl3Á6H2O (20 equiv), prolonged reaction time
and elevated temperature (4–5 days at 70 °C) was required to
achieve the metalation of 10. The desired product, PARACEST
MRI CA 4b was isolated by semi-preparative HPLC (Fig. 2) in 30%
yield (based on 1) and was characterized by ESI-HRMS.35 The
Dy3+ based CA 4a was prepared (in 36% yield based on 1) similarly
(Scheme 2) by treatment with DyCl3Á6H2O. The excess Ln3+ salts
were efficiently removed during the HPLC purification. The ab-
sence of free Ln3+ in CAs 4 was verified using a Xylenol Orange
test.34
After the desired contrast agents were prepared, we evaluated
their PARACEST properties. Peptide conjugate 3 is expected to un-
dergo an intracellular reduction of the disulfide bond mediated by
the thiols normally present within the body (e.g., glutathione). The
actual CA trapped within an intracellular environment would
Gly-Phe-OH
HO-Phe-Gly
O
N
N
N
N
O
O
O
Br2 or I2, Et3N
MeOH/MeCN HO-Phe-Gly
HO-Phe-Gly
Gly-Phe-NH
Gly-Phe-OH
10
1
N
N
N
N
O
O
O
O
S
S
HO-Phe-Gly
Gly-Phe-NH
EuCl3 6H2O
DyCl3 6H2O
MeCN/MeOH
MeCN/MeOH
30%, based on 1
36%, based on 1
4a
4b
Scheme 2. Synthesis of dimeric PARACEST MRI CAs 4.
Figure 3. CEST spectra of the bimetallic PARACEST MRI CA 4b at two temperatures
(10 mM) at 28 °C (top) and 37 °C (middle) and the dependence of the bound water
chemical shift on temperature (bottom).
Figure 2. CEST spectrum of 4a (23.4 mM in water containing 20% MeCN, room
temperature, saturation power B1 30 T, presaturation time TS 3.9 s).
l

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M. Suchy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5521–5526
5524
due to bound water3a was observed in the corresponding spectrum
(at 23.4 mM) of Dy3+ derived dimer 4a (Fig. 2). This observation is
consistent with the results obtained for Dy3+ DOTAM.3a Due to the
fact, that peaks associated with bound water in complexes 2a (see
Supplementary data) and 4a (Fig. 2) are rather broad, it was difficult
to establish a relationship between the number of Dy3+ centers and
the PARACEST signal. It appears that more than two Dy3+ centers will
be required to create a PARACEST MRI CA with enough sensitivity for
in vivo applications. The experimental conditions used to acquire
the CEST spectrum of 4a would indeed preclude any in vivo study.
To obtain basic information about the sensitivity of 4b, its CEST
spectrum (at 10 mM) was acquired at 28 °C and 37 °C (Fig. 3). A
strong (>50%) CEST effect due to bound water present in the coor-
dination sphere of 43a was observed. Since the CEST effect of CA 2b
acquired under identical experimental conditions18b is smaller (ca.
40%), the increase in the saturation transfer is attributed to the
presence of the additional paramagnetic Eu3+. CA 4b is not com-
pletely soluble in water at 10 mM, therefore an addition of MeCN
(20%) was necessary to solubilize it completely. As discussed below
no MeCN was required at concentrations of 1 mM or lower. To rule
out the possibility that the presence of MeCN affected the magnetic
properties of CA 4b, CEST spectra of the original monometallic CA
2b18 were also acquired in water and in water containing 20%
MeCN at 10 mM concentration. These results confirmed that the
presence of MeCN had a negligible effect on the overall detection
sensitivity of CA 1. This observation is consistent with literature re-
ports wherein it is accepted practice to use MeCN to solubilize
PARACEST CAs for in vitro characterization.36
and chemical shift was established. The CEST spectra of 4b (at
10 mM concentration, temperature range 28–40 °C) in water con-
taining 20% MeCN were acquired using previously established
experimental conditions18b (pH 7.0, B0 9.4 T, B1 14
lT, saturation
time 10 s). There was a linear correlation between the temperature
and the chemical shift of the bound water pool in the CEST spec-
trum, Figure 3.
Previously developed temperature responsive PARACEST MRI
CAs suffer from low sensitivities. The required high concentration
of CA will likely be incompatible with in vivo studies. In order to
further elucidate the detection sensitivity of CA 4b, we measured
its CEST spectra at low concentrations (1 and 0.5 mM, at 37 °C)
and compared them to those of CA 2b under identical conditions,
Figure 4. It is important to point out, that the addition of MeCN
was not required to achieve these low concentrations. Both agents
were found to exhibit the CEST effect due to bound water present
in their coordination spheres. Interestingly, the CEST effect ob-
served with dimeric CA 4b was found to be about 1.6 times greater
(13% vs 8% at 1 mM; 6% vs 3.6% at 0.5 mM) than the CEST effect ob-
served with CA 2b. The increased sensitivity correlates with the
number of Eu3+ cations present in the molecule, whilst maintaining
the desirable temperature dependent properties. It is reasonable to
expect a less than twofold increase in the CEST effect when com-
paring 2b and 4b. Several factors may generally result in a lower
than expected increase in CEST effect including altered T1 relaxa-
tion, altered exchange rates, the nonlinear nature of the concentra-
tion dependence of the CEST effect at high concentrations, and the
expected less than unity proportionality of the CEST effect with
concentration at low concentrations.37 Although changes in T1
relaxation induced by the agent are likely not a factor for the Eu(III)
With the intention to use the bimetallic CA 4b for MRI based
temperature mapping, the correlation between the temperature
Figure 4. Comparison of the monomeric (2b) and dimeric europium-containing compounds (4b). CEST spectra (at 37 °C) of CAs 2b (solid line) and 4b (dashed line) at 10 mM
(top left); 1 mM (bottom left) and 0.5 mM (top right). Expanded CEST spectrum at 0.5 mM (bottom right).

´
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M. Suchy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5521–5526
20. For the recent review on disulfide bond formation see: Witt, D. Synthesis 2008,
2491.
21. Lu, Z. R.; Wang, X.; Parker, D. L.; Goodrich, K. C.; Buswell, H. R. Bioconjugate
Chem. 2003, 14, 715.
based compounds, particularly at the low concentrations used in
the current study, the other mechanisms may each contribute to
some extent.
22. Major, J. L.; Meade, T. J. Acc. Chem. Res 2009, 42, 893. and references cited
therein.
23. Thonon, D.; Jacques, V.; Desreux, J. F. Contrast Med. Mol. Imaging 2007, 2, 24.
24. Mattila, K.; Siltainsuu, J.; Balaspiri, L.; Ora, M.; Lönnberg, H. Org. Biomol. Chem.
2005, 3, 3039.
25. Wu, Y.; Zhou, Y.; Ouari, O.; Woods, M.; Zhao, P.; Soesbe, T. C.; Kiefer, G. E.;
Sherry, A. D. J. Am. Chem. Soc. 2008, 130, 13854.
26. Vasalaitiy, O.; Gerard, R. G.; Zhao, P.; Sun, X.; Sherry, A. D. Bioconjugate Chem.
2008, 19, 598.
27. Pikkemaat, J. A.; Wegh, R. T.; Lamerichs, R.; van de Molengraaf, R. A.; Langereis,
S.; Burdinski, D.; Raymond, A. Y. F.; Jansse, H. M.; de Waal, B. F. M.; Willard, N.
P.; Meijer, E. W.; Grüll, H. Contrast Med. Mol. Imaging 2007, 2, 229.
28. Ali, M. M.; Yoo, B.; Pagel, M. D. Mol. Pharm. 2009, 6, 1409.
29. Nwe, K.; Andolina, C. M.; Huang, C. H.; Morrow, J. R. Bioconjugate Chem. 2009,
20, 1375.
30. Suchy´, M.; Li, A. X.; Bartha, R.; Hudson, R. H. E. Tetrahedron Lett. 2010, 51, 1087.
31. For the recent review on conjugation of DOTA-derived ligands with peptides
see: De León-Rodríguez, L. M.; Kovacs, Z. Bioconjugate Chem. 2008, 19, 391.
32. General experimental procedures: All reagents were commercially available,
unless otherwise stated. Protected peptide 7 (protecting groups R-Pbf, Q-Trt, K-
Boc, Y-tBu, ‘N’-terminal Fmoc) attached to Wang’s resin was custom
synthesized. All solvents were HPLC grade and used as such, except for
In summary we have shown, that sulfanyl modified ligand 1 can
be used as a starting material for the synthesis of peptide conju-
gated as well as dimeric PARACEST MRI CAs based on a DOTAM-
Gly-L-Phe-OH scaffold. A linear correlation between the tempera-
ture and the chemical shift of bound water was observed with
model complex 5 and dimeric Eu3+-based complex 4b. Considering
the significant increase in sensitivity of CA 4b compared to the
monomeric structures 2b and 5, this compound may be useful
for the purpose of temperature mapping. The increase of sensitivity
in the case of Dy3+ derived dimer 4a is modest, indicating that
more than two metal centers are required to boost the sensitivity
in to a practically useful range. Studies on cellular uptake of CA 3
are currently in progress and will be reported in due course.
Acknowledgment
The authors thank the Ontario Institute for Cancer Research
(OICR) for the financial support of this work.
water (18.2 M
X cm millipore water). Solvents were removed under reduced
pressure in a rotary evaporator. Aqueous solutions were lyophilized. HPLC
analysis and purification was carried out using a radial compression module
C18 300 Å column (particle size 15
lm; 8 Â 100 mm cartridge) monitored by a
Supplementary data
diode array detector (wavelength range 190–600 nm). Mobile phase: Method
A: 90% H2O/10% MeCN—100% MeCN over 20 min, linear gradient, flow rate
3 mL/min. UPLC analysis was carried out using a C18 column (particle size
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.07.070.
1.7
lm; 2.1 i.d. Â 50 mm) and monitored by a diode array (wavelength range
190–600 nm) and HR-ESI-TOF-MS detectors. Mobile phase: Method B: 100%
H2O—25% H2O/75% MeCN over 3 min, linear gradient, flow rate 0.25 mL/min;
mass spectra (MS) were obtained on TOF mass spectrometer, using electron
spray (ESI) for ionization. CEST spectra associated with CAs 4 and 5 have been
acquired on 400 MHz (9.4 T) NMR spectrometer in water or in 80% water/20%
MeCN as described previously.18b Temperature was controlled by blowing
warm air over each phantom using a small animal monitoring and gating
system and monitored using a fibre optic probe placed in a separate adjacent
phantom.
References and notes
1. For the recent reviews on MRI CAs see: (a) Yoo, B.; Pagel, M. D. Front. Biosci.
2008, 13, 1733; (b) Geraldes, C. F. G. C.; Laurent, S. Contrast Med. Mol. Imaging
ˇ
2009, 4, 1; (c) Kubícek, V.; Tóth, É.. In Advances in Inorganic Chemistry; Elsevier
Academic Press: San Diego, 2009; Vol. 61; (d) Terreno, E.; Delli Castelli, D.;
Viale, A.; Aime, S. Chem. Rev. 2010, 110, 3019.
33. Synthesis of Eu3+ DOTAM-Gly-
L-Phe-OH peptide conjugate 3: In a manual peptide
synthesis vessel, resin (150 mg) containing the protected peptide (7,
7.62 Â 10À5 mol) was treated with 20% piperidine in DMF (2 mL, repeated
twice) for 5 min. The resin was consecutively washed with dry DMF and CH2Cl2
2. For the seminal paper introducing this idea see: Ward, K. M.; Aletras, A. H.;
Balaban, R. S. J. Magn. Reson. 2000, 143, 79.
3. For the recent reviews on PARACEST MRI CAs see: (a) Woods, M.; Woessner, D.
E.; Sherry, A. D. Chem. Soc. Rev. 2006, 35, 500; (b) Sherry, A. D.; Woods, M. Annu.
Rev. Biomed. Eng. 2008, 10, 391; (c) Terreno, E.; Delli Castelli, D.; Aime, S.
Contrast Med. Mol. Imaging 2010, 5, 78; (d) Wu, Y.; Evbuomwan, M.; Melendez,
M.; Opina, A.; Sherry, A. D. Future Med. Chem. 2010, 2, 351; (e) Viswanathan, S.;
Kovács, Z.; Green, K. N.; Ratnakar, S. J.; Sherry, A. D. Chem. Rev. 2010, 110, 2960.
4. Zhang, R. S.; Merrit, M.; Woessner, D. E.; Lenkinski, R.; Sherry, A. D. Acc. Chem.
Res. 2003, 36, 783.
(ca. 2 Â 10 mL each, repeated twice). In
a
separate vial DIPEA (110
l
L,
a
6.30 Â 10À4 mol) and HBTU (107 mg, 2.84 Â 10À4 mol) were added to
cooled (0 °C) solution of Fmoc-Cys(Trt)-OH (6, 185 mg, 3.15 Â 10À4 mol) in
DMF (700 lL). The resulting mixture was added (after 10 min at 0 °C) to the
washed resin in a peptide vessel followed by agitation for 1.5 hour at rt. The
resin was washed as described above and was treated with cleavage cocktail
(5 mL, for 1.5 h at rt) prepared by dissolving water (300 lL) and TES (200 lL) in
TFA (10 mL). The TFA solution was concentrated to ca. 10% of its original
volume, was cooled to 0 °C, cold (À20 °C) Et2O (20 mL) was added and the
mixture was set aside for 1 h at À20 °C. The resulting mixture was transferred
to centrifuge tubes, was centrifuged (1000 rpm for 3 min), the supernatant was
decanted and the pellet was dissolved in water (3 mL). The aqueous solution
thus obtained was lyophilized to afford 54 mg of the crude Fmoc-CYG-RKK-
RRQ-RRR. This material was purified by semi-preparative HPLC (Method A),32
fractions were concentrated to leave the peptide 8 (25.8 mg, 19%) as colorless
solid; HPLC: tR 8.0 min. A stock solution of diethylazodicarboxylate (DEAD,
5. (a) Terreno, E.; Delli Castelli, D.; Cravotto, G.; Milone, L.; Aime, S. Invest. Radiol.
2004, 39, 235; (b) Zhang, S.; Malloy, C. R.; Sherry, A. D. J. Am. Chem. Soc. 2005,
127, 17572.
6. (a) Aime, S.; Delli Castelli, D.; Terreno, E. Angew. Chem., Int. Ed. 2002, 41, 4334;
(b) Aime, S.; Barge, A.; Delli Castelli, D.; Fedeli, F.; Mortilarro, A.; Nielsen, F. U.;
Terreno, E. Magn. Reson. Med. 2002, 47, 639.
7. (a) Zhang, S.; Trokowski, R.; Sherry, A. D. J. Am. Chem. Soc. 2003, 125, 15288; (b)
Trokowski, R.; Zhang, S.; Sherry, A. D. Bioconjugate Chem. 2004, 15, 1431.
8. Aime, S.; Delli Castelli, D.; Terreno, E. Angew. Chem., Int. Ed. 2003, 42, 4527.
9. Aime, S.; Delli Castelli, D.; Fedeli, F.; Terreno, E. J. Am. Chem. Soc. 2002, 124,
9364.
10. Huang, C. H.; Morrow, J. R. J. Am. Chem. Soc. 2009, 131, 4206.
11. Trokowski, R.; Ren, J.; Kalman, F. K.; Sherry, A. D. Angew. Chem., Int. Ed. 2005, 44,
6920.
0.1 M) was prepared by dissolving DEAD (16 ll) in dry DMF (1 ml). DEAD
solution in DMF (0.1 M, 14.4
tube containing dry DMF (100
l
l, 1.44 Â 10À5 mol) was added to a centrifuge
l
l), followed by the addition of Et3N (40 ll,
2.88 Â 10À4 mol) and cooling to 0 °C. In a separate Eppendorf tube sulfanyl
modified DOTAM-Gly-Phe-OH 1 (18.4 mg, 1.44 Â 10À5 mol) was dissolved in
dry DMF (350 ll) and the solution obtained was added dropwise (over 2 min,
12. Suchy´, M.; Ta, R.; Li, A. X.; Wojciechowski, F.; Pasternak, S. H.; Bartha, R.;
with stirring) to a previously prepared DEAD and Et3N solution (kept at 0 °C).
The ice bath was removed and stirring continued for 10 min at rt after which
time the mixture obtained was then transferred to another centrifuge tube
containing peptide 8 (25.8 mg, 1.44 Â 10À5 mol). The resulting suspension was
stirred for 48 h at rt, then it was diluted with water (ca. 4 ml) and finally
lyophilized to afford 32 mg of the residue. This residue was purified by semi-
preparative HPLC (Method A),32 fractions were concentrated to leave DOTAM-
Gly-Phe-TAT disulfide conjugate 9 (11.2 mg, 27%) as colorless solid; HPLC: tR
7.9 min. HRMS (ESI) m/z: found 2940.5361 [M+H]+ (2940.5215 calcd for
Hudson, R. H. E. Org. Biomol. Chem. 2010, 8, 2560.
13. Chauvin, T.; Durand, P.; Bernier, M.; Meudal, H.; Doan, B. T.; Noury, F.; Badet,
B.; Beloeil, J. C.; Tóth, É. Angew. Chem., Int. Ed. 2008, 47, 4370.
14. (a) Yoo, B.; Pagel, M. D. J. Am. Chem. Soc. 2006, 128, 14032; (b) Yoo, B.; Raam, M.
S.; Rosenblum, R. M.; Pagel, M. D. Contrast Med. Mol. Imaging 2007, 2, 189.
15. Yoo, B.; Sheth, V. R.; Pagel, M. D. Tetrahedron Lett. 2009, 50, 4459.
16. (a) Ali, M. M.; Liu, G.; Shah, T.; Flask, C. A.; Pagel, M. D. Acc. Chem. Res. 2009, 42,
915. and references cited therein.; For the recent example from our laboratory
see: (b) Jones, C. K.; Li, A. X.; Suchy´, M.; Hudson, R. H. E.; Menon, R. S.; Bartha, R.
Magn. Reson. Med. 2010, 63, 1184.
C129H203N46O30S2). EuCl3Á6H2O (41 mg, 1.12 Â 10À4 mol) was added to
a
solution of DOTAM-Gly-Phe-TAT disulfide conjugate
9
(16.5 mg,
17. Suchy´, M.; Li, A. X.; Bartha, R.; Hudson, R. H. E. Org. Biomol. Chem. 2008, 6, 3588.
18. (a) Wojciechowski, F.; Suchy´, M.; Li, A. X.; Azab, H. A.; Bartha, R.; Hudson, R. H.
5.60 Â 10À6 mol) in water (1 ml). The mixture was incubated for 72 h at
60 °C, then cooled to rt and was subjected for purification by semi-preparative
HPLC (Method A),32 fractions were concentrated to leave Eu3+ DOTAM-Gly-
Phe-TAT disulfide conjugate 3 (11.2 mg, 65%) as colorless solid; HPLC: tR
7.8 min. HRMS (ESI) m/z: found 3090.4192 [MÀ2H]+ (3090.4068 calcd for
´
E. Bioconjugate Chem. 2007, 18, 1625; (b) Li, A. X.; Wojciechowski, F.; Suchy, M.;
Jones, C. K.; Hudson, R. H. E.; Menon, R. S.; Bartha, R. Magn. Reson. Med. 2008,
59, 374.
19. For the recent review on the use of cell penetrating peptides in drug delivery
see: Stewart, K. M.; Horton, K. L.; Kelly, S. O. Org. Biomol. Chem. 2008, 6, 2242.
C129H200N46O30S2Eu).

´
M. Suchy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5521–5526
5526
34. Barge, A.; Cravotto, G.; Gianolio, E.; Fedeli, F. Contrast Med. Mol. Imaging 2006, 1,
addition of MeCN (600 ll) and stirring for 120 h at 70 °C.
184–188.
Dy3+ DOTAM-Gly-Phe-OH derived symmetrical disulfide dimer 4a was purified
by semi-preparative HPLC (Method A),32 fractions were concentrated to leave
colorless solid (13.6 mg, 38%); HPLC: tR 16.6 min. HRMS (ESI) m/z: found
2877.9750 [MÀ2H]+ (2877.9696 calcd for C124H159N26O30S2Dy2).
Eu3+ DOTAM-Gly-Phe-OH derived symmetrical disulfide dimer 4b was purified
by semi-preparative HPLC (Method A),32 fractions were concentrated to leave
colorless solid (10.8 mg, 30%); HPLC: tR 10.1 min. HRMS (ESI) m/z: found
2856.9219 [MÀ4H]+ (2856.9190 calcd for C124H154N26O30S2Eu2).
36. (a) Woods, M.; Woessner, D. E.; Zhao, P.; Pasha, A.; Yang, M. Y.; Huang, C. H.;
Vasalitiy, O.; Morrow, J. R.; Sherry, D. A. J. Am. Chem. Soc. 2006, 128, 10155; (b)
Huang, C. H.; Morrow, J. R. Inorg. Chem. 2009, 48, 7237.
35. Synthesis of dimeric PARACEST MRICAs4: Et3N (7
l
l, 5.00 Â 10À5 mol) was added to
a solutionof sulfanyl modified DOTAM-Gly-Phe-OH1 (32 mg, 2.50 Â 10À5 mol) in
MeOH (1 ml) and the mixture was cooled to 0 °C. A solution (0.1 M in MeCN) of I2
(250
l
l, 2.50 Â 10À5 mol) was added dropwise (over 2 min) with stirring, the
stirring continued for 10 min at 0 °C and then for 48 h at rt. Solvent was
evaporated and crude DOTAM-Gly-Phe-OH derived symmetrical disulfide
dimer 10 was used for the metalation without further purification. Its
presence was confirmed by UPLC analysis as described in Ref.32; HRMS (ESI)
m/z: found 2558.1382 [M+H]+ (2558.1313 calcd for C124H161N26O30S2). Crude
dimer 10 was slowly (ca. 4 h required) dissolved in water (1.5 ml at 70 °C with
stirring). EuCl3Á6H2O (183 mg, 5.00 Â 10À4 mol) or DyCl3Á6H2O (188 mg,
5.00 Â 10À4 mol) were added to separate reaction mixtures, followed by the
37. Woessner, D. E.; Zhang, S.; Merritt, M. E.; Sherry, A. D. Magn. Reson. Med. 2005,
53, 790.