A new synthesis of cystamine modified Eu3+ DOTAM-Gly-Phe-OH: A conjugation ready temperature sensitive MRI contrast agent

Article abstract of DOI:10.1039/b808282k

Several approaches towards asymmetrically derivatized peptide-decorated cyclens that yield lanthanide metal chelators, in which three of the nitrogen atoms of cyclen share a common substituent and the fourth nitrogen atom is differentially substituted, ha

Full text of DOI:10.1039/b808282k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
A new synthesis of cystamine modified Eu3+ DOTAM-Gly-Phe-OH:
a conjugation ready temperature sensitive MRI contrast agent†
Mojm´ır Suchy´,a,b Alex X. Li,b Robert Barthab and Robert H. E. Hudson*a
Received 16th May 2008, Accepted 9th June 2008
First published as an Advance Article on the web 11th July 2008
DOI: 10.1039/b808282k
Several approaches towards asymmetrically derivatized peptide-decorated cyclens that yield lanthanide
metal chelators, in which three of the nitrogen atoms of cyclen share a common substituent and the
fourth nitrogen atom is differentially substituted, have been evaluated. The most effective route
consisted of selective monoalkylation followed by peralkylation with a second different electrophile.
The unique substituent also possessed a masked sulfanyl group that was suitable for subsequent
chemoselective conjugation chemistry.
Introduction
Over the last two decades there have been dramatic developments
in the use of lanthanide(III) complexes as responsive MRI contrast
agents.1 A wide variety of MRI contrast agents have been
designed and used for the detection of various primary metabolites
(e.g. proteins, nucleic acids), enzymatic activities (e.g. caspase-
3, peroxidase, glucuronidase), and metal ions as well as other
important physiological parameters such as pH and temperature.1
Paramagnetic lanthanide(III) ions are known to induce a large
hyperfine shift of coordinated water protons and other exchange-
able protons present in the ligand framework.2 If the exchange
rate of these spin systems with bulk water is appropriately slow,
a separate MR signal may be observed for each state. Selective
saturation of these spins results in a method for the generation of
MRI contrast.2 A new class of MRI contrast agents employing
this technique is referred to as paramagnetic chemical exchange
saturation transfer (PARACEST). The structures of these agents
are often derived from DOTA (1) or DOTAM (2), Fig. 1.
We have recently designed, synthesized and evaluated the
PARACEST properties of a variety of DOTAM-based dipep-
tide decorated lanthanide(III) complexes.3 Among them, Eu3+
DOTAM-Gly-Phe-OH (3, Fig. 1) was found to be an MRI contrast
agent suitable for the molecular imaging of temperature.4 Tissue
temperature is an important physiological parameter, which often
varies between healthy and disease affected tissues.1 We are, in
particular, interested in using the agent 3 as a molecular probe
for the molecular imaging of brain tumors. A major obstacle
associated with the intended use of Eu3+ DOTAM-Gly-Phe-OH
(3) is its inability to cross various biological barriers (e.g. the blood
brain barrier). To overcome this problem, we decided to conjugate
Eu3+ DOTAM-Gly-Phe-OH (3) to a cell penetrating peptide5 via
Fig. 1 Structures of DOTA (1), DOTAM (2), Eu3+ DOTAM-G-
disulfide bond formation. It is expected that the disulfide bridge
ly-Phe-OH (3) and cystamine modified derivatives 4 and 5.
will be cleaved within the reductive intracellular environment thus
leaving the MRI contrast agent trapped inside. To achieve this
goal, we required a derivative of 3 bearing a primary sulfanyl (SH)
group. Cystamine-modified Eu3+ complexes 4a,b (Fig. 1) have been
aDepartment of Chemistry, The University of Western Ontario, London, ON,
identified as suitable synthetic targets and are derived from the
corresponding free ligands 5a,b (Fig. 1). Selective introduction of
the cystamine modified side chain present in the structure of 4
and 5 represents the major synthetic challenge associated with the
Canada, N6A 5B7. E-mail: robert.hudson@uwo.ca
bCentre for Functional and Metabolic Mapping, Robarts Research Institute,
The University of Western Ontario, London, ON, Canada, N6A 5K8
1
† Electronic supplementary information (ESI) available: Selected H and
13C NMR spectra. See DOI: 10.1039/b808282k
synthesis of desired ligands.
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The synthesis of asymmetrically substituted ligands 5a,b and
their Eu3+ complexes 4a,b has been achieved and moreover, the
PARACEST properties associated with complex 4b have been
determined and compared with those of Eu3+ DOTAM-Gly-Phe-
OH (3). In this paper we describe a detailed synthetic approach
leading to the synthesis of the sophisticated ligands 5a,b and
the Eu3+ complexes 4a,b and the evaluation of the PARACEST
properties of 4b.
(10).7 Our approach was to trialkylate 10 (Scheme 1) with N-
iodoacetyl-Gly-Phe-OEt (7), followed by coupling with Fmoc-
Phe-cystamine(Boc) (11, Scheme 1). As described later, the tri-
alkylation of 10 failed to produce the desired result and we turned
our attention to the final synthetic approach, which eventually
led us to the desired target 5. The key step in the retrosynthesis
of 5 then became the selective monoalkylation of cyclen (6) with
N-haloacetyl-Gly-Phe-OtBu (12, Scheme 1). This was followed
by peralkylation with N-iodoacetyl-Gly-Phe-OEt (7), selective
removal of the tBu ester group and coupling with mono-Boc-
cystamine (16), prepared according to a literature procedure.8
Results and discussion
Retrosynthetic analysis
To assemble the desired ligands 5 and their Eu3+ complexes 4,
synthetic approaches based on the selective alkylation of com-
mercially available cyclen or alkylation of some simple, easily
accessible derivatives of cyclen have been evaluated. The various
retrosynthetic disconnections that we explored are depicted in
Scheme 1.
The first approach we attempted was thought to be the most
straightforward at the outset and was based on the selective
trialkylation of cyclen (6) with N-iodoacetyl-Gly-Phe-OEt (7)3
followed by the alkylation of the remaining secondary amine with
N-iodoacetylGly-Phe-cystamine(Boc) (8), Scheme 1. However, we
were not able to achieve satisfactory results, which necessitated
considering alternative routes. The next possibility investigated
was the assembly of ligand 5 via N-formylcyclen (9), which can
be obtained from cyclen (6) in two steps.6 Subsequent sequential
alkylation of 9 with N-iodoacetyl-Gly-Phe-OEt (7); removal of
the formyl group; and alkylation with N-iodoacetyl-Gly-Phe-
cystamine(Boc) (8, Scheme 1) would yield the desired ligand.
Unfortunately, this route had to be abandoned after the N-formyl
group was found to resist removal.
Synthesis of alkylation agents 7, 8 and 12
N-Iodoacetyl-Gly-Phe-OEt (7) was prepared from N-chloro-
acetylglycine (13) and H-Phe-OEt·HCl according to a recently
established literature procedure.3a A similar approach was used
to prepare electrophiles 12 via the reaction of 13 with H-Phe-
OtBu·HCl (14, Scheme 2). N-Chloroacetyl-Gly-Phe-OtBu (12a)
was obtained in excellent yield (98%), followed by the Finkelstein
reaction (NaI, acetone) to furnish N-iodoacetyl-Gly-Phe-OtBu
(12b) in 56% yield (Scheme 2).
N-Chloroacetylglycine (13) was subjected to NHS–DCC-
mediated coupling with H-Phe-OH (15) followed by coupling with
mono-Boc-cystamine8 (16, Scheme 3). The crude N-chloroacetyl-
Gly-Phe-cystamine(Boc) was obtained, containing ca. 20% of
N,N -dicyclohexylurea (as determined by 1H NMR). This material
was treated with NaI in acetone to afford N-iodoacetyl-Gly-Phe-
cystamine(Boc) (8, Scheme 3) in an acceptable yield of 33% (over
three steps, based on 13).
Synthesis of Fmoc-Phe-cystamine(Boc) (11)
Another easily accessible cyclen-derived precursor investi-
gated for the synthesis of ligand 5 was N-cyclenacetic acid
The EDC·HCl–NHS mediated coupling of Fmoc-Phe-OH
(17) with mono-Boc-cystamine8 (16) was used to prepare
Scheme 1 Comprehensive retrosynthetic analysis of cystamine modified ligands 5.
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Scheme 2 Synthesis of N-haloacetyl-Gly-Phe-OtBu (12).
Scheme 5 Alkylation of N-formylcyclen (9) with N-iodoacetyl-Gly-
Phe-OEt (7) and subsequent saponification of 18 to yield 19.
of the formyl group, followed by alkylation with N-iodoacetyl-
Gly-Phe-cystamine(Boc) (8, Scheme 1).
The formyl group is a well known nitrogen atom protective
group and several methods for its removal have appeared in the
literature. For this work, we attempted to remove the formyl group
from intermediate 18 employing oxidative,9 reductive,10 acidic,11
or basic12 conditions. All of these conditions have proved to be
unsuitable for the reasons given below.
Scheme 3 Synthesis of N-iodoacetyl-Gly-Phe-cystamine(Boc) (8).
Fmoc-Phe-cystamine(Boc) (11) dipeptide in moderate yield (51%),
Scheme 4.
Attempts to remove the formyl group under oxidative con-
ditions (m-CPBA in THF, or H2O2 in EtOH–H2O9) led only
to intractable mixtures while catalytic hydrogenation10 led to
complete recovery of starting material. Hydrolysis of the formyl
group was tried under acidic conditions (HCl in EtOH–H2O,
SOCl2 in EtOH or p-TSA in EtOH),11 but we were plagued by
partial hydrolysis of the ester groups present in 18. When 18 was
treated with HCl in EtOH–H2O, a modest amount of impure
product was obtained that showed removal of the formyl group;
however, this result could neither be reliably reproduced nor scaled
up despite many attempts. It was also found that the reaction of
this impure material with N-iodoacetyl-Gly-Phe-cystamine(Boc)
(8, Scheme 1) was rather sluggish and for both of these reasons,
this approach was abandoned. Finally, basic conditions were
investigated as a way to remove the protecting group. Treatment
of 18 with K2CO3 in aqueous EtOH even at elevated temperatures
(up to reflux) resulted in recovery of the starting material while
complete decomposition was observed when NaOEt was used as
a base. Classical saponification12 conditions (NaOH in EtOH–
H2O) led to the formation of macrocycle 19 (Scheme 5), in which
the hydrolysis of the ester functionalities took place, while the
formyl group remained. Attempts to promote the removal of
Scheme 4 Synthesis of Fmoc-Phe-cystamine(Boc) (11).
Alkylation of N-formylcyclen (9) and N-cyclenacetic acid (10)
N-Formylcyclen (9) can be obtained from cyclen (6) first by
reacting 6 with dimethylformamide dimethylacetal, followed by
hydrolysis of unstable tricyclic amine.6 With N-formylcyclen in
hand, we turned our attention to the peralkylation of 9 with
N-iodoacetyl-Gly-Phe-OEt (7). The reaction proceeded smoothly
and peralkylated cyclen 18 was isolated in 92% yield (Scheme 5).
The intermediate triester 18 was expected to undergo the removal
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the formyl group under identical conditions but using elevated
temperatures (up to 50 ◦C) led to massive decomposition (HPLC
inspection). The above mentioned experiments clearly indicated
that N-formylcyclen (9) is not a suitable precursor in the synthesis
of desired ligands 5a,b and their Eu3+ complexes (Fig. 1).
Finding that we were unable to remove the formyl group from 18
we turned our attention to N-cyclenacetic acid (10) as a starting
point. Acid 10 can be obtained by alkylation of cyclen (6) with
bromoacetic acid.7 We envisioned the peralkylation of 10 with
N-iodoacetyl-Gly-Phe-OEt (7), followed by coupling with Fmoc-
Phe-cystamine(Boc) (11, Scheme 1) to furnish the remaining
side chain. Somewhat unexpectedly, we observed alkylation of
the carboxylic functionality with N-iodoacetyl-Gly-Phe-OEt (7)
leading to the formation of a complex and inseparable mixture of
the desired N-alkylation product and O-alkylated side product,13
precluding the use of 10 for the synthesis of ligand 5.
Selective trialkylation and monoalkylation of naked cyclen
Selective trialkylation of unprotected cyclen (6) with N-iodoacetyl-
Gly-Phe-OEt (7) followed by the alkylation of the remaining
nitrogen atom with N-iodoacetyl-Gly-Phe-cystamine(Boc) (8,
Scheme 1) conceptually was the most straightforward route
towards assembling the ligand 5. In the recent literature,14 there are
several reports describing selective trialkylation of cyclen, followed
by the separation of the desired product by chromatography.
It is worth mentioning that the alkylation agents used to carry
out the above mentioned transformations usually possess simpler
structures as compared to N-iodoacetyl-Gly-Phe-OEt (7). We have
found that treatment of cyclen (6) with N-iodoacetyl-Gly-Phe-
OEt (7) using Et3N as a base leads to the formation of a complex
mixture. Analysis of the reaction mixture by HPLC (Microsorb-
CN column) indicated the presence of the desired trialkylated
cyclen (75%) accompanied with the products of peralkylation
(20%, this material was compared with an authentic sample3a
available in our laboratory) and dialkylation (5%). Under these
conditions, the products of the reaction were well resolved and
we therefore attempted scale up the reaction and to carry out
gravity chromatography, using nitrile bonded phase silica gel.
Unfortunately, the chromatographic separation could not be
reproduced on a larger scale and in fact we observed the desired
product to be essentially irreversibly adsorbed onto the stationary
phase.
The synthesis of the ligand 5 was eventually accomplished, when
selective monoalkylation15 of cyclen was used as a key step in the
synthesis. Cyclen (6) was monoalkylated with N-haloacetyl-Gly-
Phe-OtBu in chloroform (12, Scheme 6). The reaction proceeded
in moderate yields (12a, 48%; 12b, 23%), affording the monoalky-
lated cyclen 20 (Scheme 6). The product of monoalkylation
was isolated by normal phase column chromatography without
difficulty. Peralkylation of 20 with N-iodoacetyl-Gly-Phe-OEt (7)
afforded the orthogonally protected ester 21 in excellent yield
(99%, Scheme 6). As described below, compound 21 was later
on converted to the desired ligands 5a,b.
Scheme 6 Preparation of orthogonally protected mono-t-butyl ester 21.
to HBTU-mediated coupling with mono-Boc-cystamine8 (16).
Protected ligand 22, bearing all the necessary substitution was
isolated in 78% yield (based on 21), Scheme 7.
Dithiotreitol (DTT) mediated reduction16 of 22 followed by
saponification of the ester functionalities afforded ligand 5a in
64% overall yield. Studies on the conjugation of compound 5a
as a modified DOTAM-Gly-Phe-OH subunit to cell penetrating
peptides and gold nanoparticles are currently in progress and the
results of these studies will be described in due course.
We were also interested in preparing the Eu3+ complexes of
ligands 5 (compounds 4) in order to compare the PARACEST
properties (see below) of these modified ligands with those of
parent compound Eu3+ DOTAM-Gly-Phe-OH (3). It was found
that the treatment of ligand 5a with EuCl3·6H2O did not afford the
desired complex 4a. The formation of the disulfide bridge between
the two molecules of ligand 5a was found to be an unwanted
side reaction and a complex mixture, containing ligand 5a, the
disulfide dimer as well as their Eu3+ complexes was obtained.
Although we were keenly interested in pursuing this compound,
frustratingly, the reaction did not proceed to completion even us-
ing a large excess (20 equivalents) of EuCl3·6H2O, thus precluding
the isolation of the disulfide dimer and its Eu3+ complex. The
problem of oxidative disulfide formation was solved by preparing
the ligand 5b via Boc-deprotection and subsequent saponification
of 22 (Scheme 7). Metalation of 5b proceeded smoothly affording
the complex 4b (purified by size exclusion chromatography as
described previously3) in 58% yield (Scheme 7). The absence of free
Eu3+ was confirmed by negative xylenol orange test.17 Complex
4b has been characterized by 1H NMR and MS (ESI-TOF)
spectroscopies. DTT-mediated reduction of 4b (Scheme 7) was
attempted, leading to the formation of Eu3+ complex 4a (confirmed
by MS (ESI-TOF) spectroscopy) accompanied with significant
amount of Eu3+ DOTAM-Gly-Phe-OH (3). This unwanted side
product apparently results from the hydrolysis of the amide
Having prepared the orthogonally protected derivative 21 in
reasonable quantities and purity, we focused our attention on
completing the synthesis of ligands 5. The tert-butyl ester group of
21 was removed by treatment with TFA in dichloromethane and
the crude monocarboxylic acid derivative obtained was subjected
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Scheme 7 Synthesis of target molecules: ligands 5 and their Eu3+ complexes 4.
◦
bond between H-Phe-OH and cystamine. Our experience with
molecules described above indicates that this amide bond is quite
prone to hydrolysis. Complex 4b indeed serves as a suitable
model compound for a subsequent PARACEST properties study,
whereas magnetic studies with impure complex 4a have not been
carried out. Although complexes 4a and 4b are shown as eight
coordinate (Scheme 7), in aqueous solution the europium centre
also coordinates a molecule of water. It is this bound water
which, under appropriate conditions of slow exchange, gives rise
a signal distinguishable from that of bulk water. These signals
are observed in the Z-spectrum in which bulk water appears at
d = 0 ppm. For complexes such as compound 4b, the bound
water signal appears at approximately 40–45 ppm, while the
signal for other exchangable protons such as the amide N–H
protons is much closer to and often obscured by the bound water
signal.3a
The observed PARACEST effect (35%, ca. 45 ppm, at 37 C)
with cystamine modified complex 4b was found to compare
reasonably well with that associated with Eu3+ DOTAM-Gly-Phe-
OH (3, 44%, ca. 45 ppm). This finding implies that the cystamine
modified complex 4b, and likely close structural analogues such
as 4a and peptide conjugates, preserve the desirably strong
PARACEST properties that are the basis of generating MRI
contrast.
Conclusions
Various synthetic approaches have been investigated towards
the preparation of asymmetrically peptide-decorated cyclens that
possess suitable functionality for chemoselective conjugation to
other molecular entities. The challenge was to leave as much of the
ligand sphere as possible undisturbed to preserve the PARACEST
properties observed for the parent Eu3+ DOTAM-Gly-Phe-OH,
as previous model studies showed that this was necessary.3b The
inclusion of a sulfanyl group (−SH) was viewed as a useful
functional group that could be addressed chemoselectively. The
sulfanyl group adds to the versatility of the molecule as it is reactive
towards Michael acceptors, iodoacetyl compounds or can undergo
oxidative disulfide formation. It is this latter manifestation of
reactivity that we intend to exploit for the formation of peptide–
chelator complexes.
PARACEST properties of complex 4b
The magnetic resonance PARACEST properties of complex 4b
were investigated at physiological temperature (37 ◦C) using a
previously established experimental protocol.3a,4 The data ob-
tained were compared (Fig. 2) with the PARACEST properties of
Eu3+ DOTAM-Gly-Phe-OH (3), a new PARACEST MRI contrast
agent with the potential ability to perform temperature mapping.4
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Fig. 2 Z-spectrum of a 10 mM, pH 7.0 solution of Eu3+ DOTAM-Gly-Phe-OH (3, left) and cystamine modified complex 4b (right) acquired on a 9.4 T
NMR spectrometer using a 10 s, 14 lT saturation pulse at 37 ◦C.
Synthetic routes based on the peralkylation of easily accessible
cyclen derivatives [N-formylcyclen (9), N-cyclenacetic acid (10)]
permitted us to explore some new chemistry, yet did not lead
to a workable synthesis of ligands 5 and their Eu3+ complexes.
Selective trialkylation of cyclen (6) with N-iodoacetyl-Gly-Phe-
OEt (7) was also found to be problematic. Ultimately, the new
synthetic route to ligands 5a,b and their Eu3+ complexes rested on
a selective monoalkylation of cyclen (6) with N-haloacetyl-Gly-
Phe-OtBu (12) as a key step. This was followed by peralkylation
with 7, removal of the OtBu group and HBTU-mediated coupling
with mono-Boc-cystamine (16). After some protecting group
manipulations, intermediate 22 was converted into ligands 5a,b.
These were successfully loaded with metal by treatment with
EuCl3·6H2O resulting in the formation of complexes 4a,b. Among
them, complex 4b was obtained in pure form and its magnetic
properties have been investigated. The PARACEST properties as-
sociated with complex 4b compare favourably with Eu3+ DOTAM-
Gly-Phe-OH (3). It can be envisioned that conjugation of cell
penetrating peptide with ligand 5a, followed by introduction of
Eu3+ will provide a new advanced PARACEST MRI contrast
agent with the potential to cross various biological barriers (e.g. the
blood brain barrier) while retaining desirable magnetic properties.
The development of a cell penetrating peptide conjugate of
complex 5a and its Eu3+ complex is underway and will be described
later.
or by 5% solution of ninhydrin in AcOH/EtOH (compound 4b).
Thin layer chromatography (TLC) was carried out on Al backed
silica gel plates, compounds were visualized by UV light or I2
vapors. Melting points were obtained on Fisher–Johns apparatus
and are uncorrected. Specific rotations [a]D were determined at
ambient temperature using a 5 mL, 10 cm path length cell; the
units are 10−1 deg cm2 g−1 and the concentrations are reported
in g 100 mL−1. HPLC analysis was carried out using a high
performance liquid chromatograph equipped with an automatic
injector, diode array detector (wavelength range 190–600 nm),
degasser and a Microsorb-CN column (particle size 5 lm; 4.6
id × 200 mm). Mobile phase: 75% H2/25% MeCN–25% H2O/75%
MeCN over 35 min, linear gradient, flow rate 1 mL min−1. NMR
1
spectra were recorded on a 400 MHz spectrometer; for H (400
MHz), d values were referenced as follows CDCl3 (7.26 ppm);
DMSO-D6 (2.49 ppm); D2O (4.75 ppm) for 13C (100 MHz) CDCl3
(77.0 ppm); DMSO-D6 (39.5 ppm). Mass spectra (MS) were
obtained using electron impact (EI) or electrospray ionization
(ESI) techniques.
NHS–DCC mediated coupling of N-chloroacetylglycine (13)
with H-Phe-OtBu·HCl (14). NHS (575 mg, 5 mmol) and DCC
(1.341 g, 6.5 mmol) were added (at 0 ◦C) to a solution of N-
chloroacetylglycine (13, 758 mg, 5 mmol) in dry CH2Cl2 (40 mL).
The mixture was stirred for 30 min at 0 ◦C, H-Phe-OtBu·HCl
(14, 1.289 g, 5 mmol) and Et3N (1.4 mL, 10 mmol) added and
the stirring continued for 18 h at rt. The mixture was cooled to
−20 ◦C, precipitate was filtered off, the fi◦lter was washed with
a small amount (ca. 15 mL) of cold (−20 C) dichloromethane,
the combined filtrate was washed with 1 M HCl (30 mL) and the
aqueous phase was extracted with CH2Cl2 (20 mL). The organic
phase was dried, concentrated and the residue was subjected to
FCC on 50 g SiO2 (CH2Cl2–MeOH, 9 : 1). The residue obtained
after concentrating the eluate contained a small amount of N,N ꢀ-
dicyclohexylurea (DCU), which was removed by re-dissolution in
a minimal amount of acetone (ca. 10 mL) and set aside for 18 h
Experimental
General experimental procedures
All amino acids (naturally occurring L isomers) and reagents
were commercially available, unless otherwise stated. All solvents
were HPLC grade and used as such, except for CH2Cl2 and
THF (dried over Al2O3, in a solvent purification system) and
water (18.2 MX cm millipore water). Organic extracts were
dried with Na2SO4 and solvents were removed under reduced
pressure in a rotary evaporator. Flash column chromatography
◦
at −20 C. The precipitate that formed (DCU) was removed by
˚
(FCC) was carried out using silica gel, mesh size 230–400 A.
filtration through a cotton plug placed in a Pasteur pipette. The
solvent was removed to give N-chloroacetyl-Gly-Phe-OtBu (12a,
1.734 g, 98%). Slightly yellow oil; [a]2D5 +44 (c 0.79, CH2Cl2). HPLC:
tR 13.7 min; 1H NMR (CDCl3) d 7.43 (t, D2O exch., J = 4.5 Hz,
1H); 7.23 (m, 3H); 7.12 (m, 2H); 6.85 (d, D2O exch., J = 8 Hz,
Size exclusion chromatography was carried out on BIO-GEL
P2, 45–90 lm mesh resin (20 g, column size 15 × 2 cm per
0.1 mmol of compound). Ten fractions (10 ml each) were collected.
Fractions were identified by UV/I2 vapours (compounds 4a, 19)
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[a]2D5 +19 (c 0.78, CH2Cl2). HPLC: tR 13.3 min; 1H NMR (DMSO-
D6) d 8.41 (m, D2O exch., 1H); 8.18 (m, D2O exch., 2H); 7.21
(m, 5H); 6.98 (m, D2O exch., 1H); 4.42 (m, 1H); 3.73 (dd, J =
16.5, 6 Hz, 1H); 3.68 (s, 2H); 3.57 (dd, J = 16.5, 6 Hz, 1H); 3.26
(m, 4H); 2.97 (dd, J = 13, 5 Hz, 1H); 2.72 (m, 5H); 1.35 (s, 9H);
13C NMR (DMSO-D6) d 170.8, 168.1, 167.9, 155.5, 137.7, 129.1,
128.1, 126.3, 77.8, 54.1, 42.4, 39.4, 38.0, 37.8, 37.5, 36.9, 28.2.
HRMS (ESI) m/z: found 625.1000 [M + H]+ (625.1015 calcd for
C22H34IN4O5S2).
1H); 4.73 (m, 1H); 4.00 (d, J = 2 Hz, 2H); 3.92 (ddd, J = 6.5, 5.5,
1.5 Hz, 2H); 3.07 (dd, J = 14, 4.5 Hz, 1H); 3.02 (dd, J = 14, 4.5 Hz,
1H); 1.38 (s, 9H); 13C NMR (CDCl3) d 170.3, 167.6, 166.5, 135.8,
129.3, 128.3, 126.9, 82.5, 53.6, 42.9, 42.2, 37.8, 27.8. HRMS (EI)
m/z: found 354.1341 (354.1346 calcd for C17H23ClN2O4). LRMS
(EI) m/z (rel abundance): 354 [M + H]+ (10), 298 (32), 224 (25),
148 (100), 120 (100), 99 (21).
N-Iodoacetyl-Gly-Phe-OtBu (12b). NaI (1.35 g, 9 mmol) was
added to a stirred solution of N-chloroacetyl Gly-Phe-OtBu (12a,
1.065 g, 3 mmol) in acetone (20 mL). The mixture was stirred
for 18 hours at rt, was concentrated to ca. one-third of its original
volume, diluted with EtOAc (50 mL) and washed with 10% Na2SO3
solution (50 mL). The aqueous phase was extracted with EtOAc
(30 mL), the organic phase was dried, concentrated, and the
residue was subjected to FCC on 30 g SiO2 (hexanes–acetone, 1 :
1) to afford 752 mg (56%) of N-iodoacetyl Gly-Phe-OtBu (12b).
Slightly yellow oil; [a]D25 +29 (c 0.69, CH2Cl2). HPLC: tR 13.8 min;
1H NMR (CDCl3) d 7.26 (m, 3H); 7.18 (m, D2O exch., 1H); 7.15
(m, 2H); 6.71 (d, D2O exch., J = 8 Hz, 1H); 4.74 (m, 1H); 3.93
(d, J = 5 Hz, 2H); 3.71 (s, 2H); 3.10 (dd, J = 14, 6.5, Hz, 1H);
3.05 (dd, J = 12, 6 Hz, 1H); 1.40 (s, 9H); 13C NMR (CDCl3) d
170.2, 167.9, 167.8, 135.9, 129.4, 128.4, 127.0, 82.6, 53.8, 43.6,
38.0, 27.9. HRMS (EI) m/z: found 446.0695 (446.0703 calcd for
C17H23IN2O4). LRMS (EI) m/z (rel abundance): 446 [M + H]+
(10), 390 (56), 224 (20), 198 (10), 120 (100), 99 (15).
Fmoc-Phe-cystamine(Boc) (11). NHS (115 mg, 1 mmol) and
EDC·HCl (383 mg, 2 mmol) were added to a solution of Fmoc-
Phe-OH (17, 387 mg, 1 mmol) in dry THF (5 mL). The mixture was
stirred for 18 h at rt, was concentrated, the residue was dissolved
in water (40 mL) and was extracted with EtOAc (40 + 2 × 20 mL).
The organic extract was dried and the solvent was evaporated to
leave the corresponding NHS-ester. This material was dissolved in
dry THF (5 mL) followed by a dropwise addition (over a period of
10 min, at 0 ◦C) of a solution of mono-Boc-cystamine (16, 252 mg,
1 mmol) and Et3N (210 lL, 1.5 mmol) in dry THF (5 mL). The
stirring continued for 3 h at 0 ◦C, the mixture was diluted with brine
(60 mL), and was extracted with EtOAc (40 + 20 mL). The organic
extract was dried and then concentrated to leave crude compound
11, which was purified by FCC on 50 g SiO2 (CH2Cl2–MeOH,
19 : 1) followed by crystallization from dichloromethane–hexanes
solution.
Fmoc-Phe◦-cystamine(Boc) (11, 318 mg, 51%), colorless crystals;
mp 154–155 C; [a]2D5 +13 (c 0.80, DMSO). HPLC: tR 11.0 min; 1H
NMR (DMSO-D6) d 8.21 (m, D2O exch., 1H); 7.87 (m, 2H); 7.63
(m, 2H); 7.42 (m, 2H); 7.23 (m, 7H); 6.98 (m, D2O exch., 1H); 4.15
(m, 3H); 3.33 (m, 3H); 3.19 (m, 2H); 2.96 (dd, J = 13.5, 4.5 Hz,
1H); 2.75 (m, 5H); 1.35 (s, 9H); 13C NMR (DMSO-D6) d 171.5,
155.8, 155.5, 143.8, 143.7, 142.6, 140.7, 139.4, 138.1, 137.4, 129.3,
129.2, 128.9, 128.1, 128.0, 127.6, 127.3, 127.0, 126.2, 125.4, 125.3,
121.4, 120.0, 109.7, 77.8, 65.6, 56.2, 46.6, 39.4, 38.0, 37.7, 37.6,
37.1, 28.2. HRMS (EI) m/z: found 621.2296 (621.2331 calcd for
C33H39N3O5S2). LRMS (EI) m/z (rel abundance): 621 [M + H]+
(10), 308 (10), 223 (16), 178 (79), 120 (100), 91 (10).
N-Iodoacetyl-Gly-Phe-cystamine(Boc) (8). DCC (318 mg,
2.2 mmol) was added (at 0 ◦C) to a stirred suspension of N-
chloroacetylglycine (13, 212 mg, 1.4 mmol) and NHS (161 mg,
1.4 mmol) in dry CH2Cl2 (7 mL). The cooling bath was removed
and the mixture was stirred for 18 h at rt. The solvent was
evaporated, the residue was suspended in dry THF (6 mL), H-
Phe-OH (15, 231 mg, 1.4 mmol) and Et3N (390 lL, 2.8 mmol)
were added and the mixture was stirred for 2 h at rt. The reaction
mixture was then diluted with 1 M HCl (50 mL) and extracted
with EtOAc (40 + 3 × 20 mL). The combined organic extracts
were dried, concentrated and the residue was dissolved in dry
◦
THF (14 mL). The solution obtained was cooled to 0 C, NHS
(161 mg, 1.4 mmol) and DCC (347 mg, 1.7◦mmol) were added
and the mixture was stirred for 30 min at 0 C, followed by the
addition of mono-Boc-cystamine8 (16, 353 mg, 1.4 mmol) and
Et3N (390 lL, 2.8 mmol). The cooling bath was removed and the
mixture was stirred for 18 h at rt. The mixture was concentrated
to ca. one-third of its original volume, was diluted with brine
(50 mL) and was extracted with EtOAc (40 + 2 × 20 mL).
The combined organic extracts were dried, concentrated and the
residue obtained contained ca. 20% of N,Nꢀ-dicyclohexylurea (by
1H NMR). The residue was dissolved in acetone (8 mL) and NaI
(341 mg, 2.3 mmol) was added and then the mixture was stirred
for 18 h at rt. Subsequently, the reaction mixture was concentrated
to ca. one-third of its original volume and was diluted with EtOAc
(30 mL), washed with 10% Na2SO3 solution (20 mL) and the
aqueous phase was extracted with EtOAc (20 mL). The organic
phase was dried and concentrated to give crude N-iodoacetyl
compound 8, which was purified by FCC on 50 g SiO2 (CH2Cl2–
MeOH, 19 : 1) and then recrystallized from dichloromethane–
hexanes solution.
Alkylation of N-formylcyclen (9) with N-iodoacetyl-Gly-Phe-
OEt (7). DIPEA (260 lL, 1.5 mmol) and N-iodoacetyl-Gly-
Phe-OEt3 (7, 628 mg, 1.5 mmol) were added to a solution of
N-formylcyclen6 (9, 100 mg, 0.5 mmol) in MeCN (10 mL). The
mixture was stirred for 18 h at 50 ◦C, cooled to rt and then diluted
with EtOAc (40 mL). The organic solution was washed with water
(40 mL) and the aqueous phase was extracted with EtOAc (20 mL).
The organic extract was dried and was concentrated and the
resulting oil was triturated (twice) with a small amount (ca. 15 mL)
of hexane to leave N-formyl-N-triacetyl-Gly-Phe-OEt cyclen (18,
494 mg, 92%). Colorless solid; [a]2D5 +55 (c 0.55, CH2Cl2). HPLC:
tR 21.8 min; 1H NMR (DMSO-D6) d 8.26 (m, D2O exch., 2H); 7.87
(m, D2O exch., 2H); 7.09 (m, 15H); 4.35 (m, 3H); 3.89 (m, 6H);
3.62 (m, 6H); 3.26–2.38 (br m, 28H); 0.96 (m, 9H); 13C NMR
(DMSO-D6) d 171.9, 171.4, 170.9, 170.7, 170.5, 170.4, 169.6,
169.1, 168.9, 168.3, 163.3, 137.0, 136.9, 129.1, 128.3, 126.6, 60.6,
53.7, 52.5, 41.8, 41.7, 37.0, 36.9, 13.9. HRMS (ESI) m/z: found
1071.5514 [M + H]+ (1071.5515 calcd for C54H75N10O13).
N-Iodoacetyl-Gly-Phe-cystamine(Boc) [8, 288 mg, 33% based
Saponification of N-formyl-N-triacetyl-Gly-Phe-OEt cyclen
(18). NaOH (1 M, 980 lL) was added to a solution of
on N-chloroacetylglycine (13)], colorless crystals; mp 130–131 ◦C;
3594 | Org. Biomol. Chem., 2008, 6, 3588–3596
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N-formyl-N-triacetyl-Gly-Phe-OEt cyclen (18, 107 mg, 0.1 mmol)
in EtOH (3 mL). The mixture was stirred for 18 h at rt, EtOH
was evaporated, the aqueous residue was cooled to 0 ◦C and
the pH was adjusted to 6 (1 M HCl). The obtained solution
was subjected to size exclusion chromatography as described in
the general experimental procedures; fractions containing the
product were combined and were lyophilized to leave N-formyl-
N-triacetyl-Gly-Phe-OH cyclen (19, 76 mg, 78%). Colorless solid;
Removal of tBu group from 21, followed by HBTU-mediated
coupling with mono-Boc-cystamine (16). TFA (1 mL) was added
to a solution of N-monoacetyl-Gly-Phe-OtBu-N-triacetyl-Gly-
Phe-OEt cyclen (21, 408 mg, 0.3 mmol) in CH2Cl2 (2 mL). The
mixture was stirred for 18 h at rt, was concentrated, the residue
was dissolved in EtOAc (40 mL) and the solution obtained was
washed with saturated NaHCO3 solution (20 mL). The aqueous
phase was extracted with EtOAc (20 mL), and the organic phase
was separated, dried and concentrated to leave N-monoacetyl-
Gly-Phe-OH-N-triacetyl-Gly-Phe-OEt cyclen (342 mg, 87%) of
sufficient purity (HPLC inspection) for the next step. This material
was dissolved in dry CH2Cl2 (6 mL), the solution obtained was
cooled to 0 ◦C followed by the addition of DIPEA (91 lL,
0.52 mmol) and HBTU (99 mg, 0.26 mmol). The mixture was
stirred for 15 min at 0 ◦C, a solution of mono-Boc-cystamine8 (16,
66 mg, 0.26 mmol) in dry CH2Cl2 (2 mL) was added dropwise (over
the period of 1 min), the cooling bath was removed and the mixture
was stirred for 4 h at rt. The solvent was evaporated, the residue
was dissolved in EtOAc (50 mL) and the solution obtained was
consecutively washed with saturated NaHCO3 solution (30 mL),
1 M HCl (30 mL) and saturated NaHCO3 solution (20 mL).
The organic phase was dried and was concentrated to leave a
colorless solid, which was purified by trituration (twice) with a
small amount (ca. 15 mL) of hexane to leave N-monoacetyl-Gly-
Phe-cystamine(Boc)-N-triacetyl-Gly-Phe-OEt cyclen (22, 358 mg,
90%). Colorless solid; [a]2D5 +30 (c 0.67, CH2Cl2). HPLC: tR
23.2 min; 1H NMR (DMSO-D6) d 8.44 (m, D2O exch., 2H); 8.21
(m, D2O exch., 3H); 7.22 (m, 20H); 4.45 (m, 4H); 3.99 (m, 6H);
1
[a]2D5 −7 (c 0.72, MeOH). HPLC: tR 9.4 min; H NMR (D2O) d
7.20 (m, 16H); 4.36 (m, 3H); 3.75 (m, 6H); 3.45–2.84 (br m, 28H);
13C NMR (DMSO-D6) d 178.1, 173.4, 170.5, 170.3, 166.5, 138.2,
138.0, 129.7, 128.9, 127.1, 56.5, 54.0, 53.3, 51.8, 50.5, 42.5, 42.0,
39.6, 37.8. HRMS (ESI) m/z: found 987.4575 [M + H]+ (987.4576
calcd for C48H63N10O13).
Selective monoalkylation of cyclen (6) with N-haloacetyl-
Gly-Phe-OtBu (12) to give N-monoacetyl-Gly-Phe-OtBu cyclen
(20). Separate solutions of N-chloroacetyl-Gly-Phe-OtBu (12a,
562 mg, 1.57 mmol) or N-iodoacetyl-Gly-Phe-OtBu (12b, 705 mg,
1.57 mmol) in CHCl3 (2 mL) were added dropwise (over a 10 min
period) to a stirred solution (at 0 ◦C) of cyclen (6, 541 mg,
3.14 mmol) in CHCl3 (4 mL). The slow addition of electrophile and
careful control of the temperature is needed to have reproducible
results. The mixtures were stirred for 2 h at 0 ◦C and 2 h at rt;
a clear solution was obtained with N-chloroacetyl-Gly-Phe-OtBu
(12a); it was concentrated and the residue was subjected to FCC
on 15 g SiO2 (CH2Cl2–MeOH–NH4OH, 80 : 20 : 1). Evaporation
of the eluate afforded N-monoacetyl-Gly-Phe-OtBu cyclen (20,
368 mg, 48%). A white precipitate formed when N-iodoacetyl-
Gly-Phe-OtBu (12b) was used, it was filtered off, the filtrate was
concentrated and the residue was subjected to FCC as described
above, leaving N-monoacetyl-Gly-Phe-OtBu cyclen (20, 176 mg,
23%). Colorless oil; [a]D25 +19 (c 0.52, MeOH). HPLC: tR 7.5 min;
1H NMR (DMSO-D6) d 8.48 (m, D2O exch., 1H); 7.23 (m, 5H);
4.34 (m, 1H); 3.76 (s, 2H); 3.48 (br m, D2O exch., 1H); 3.22 (s, 2H),
2.92 (m, 2H); 2.67 (m, 16H); 1.29 (s, 9H); 13C NMR (DMSO-D6)
d 171.1, 170.5, 169.2, 137.2, 129.2, 128.2, 126.5, 80.7, 58.0, 54.3,
51.4, 48.6, 46.7, 45.4, 44.8, 41.3, 37.0, 30.7, 27.5. HRMS (ESI)
m/z: found 491.3349 [M + H]+ (491.3346 calcd for C25H43N6O4).
3.71 (m, 8H); 3.20–2.31 (br m, 40H); 1.36 (s, 9H); 1.04 (m, 9H); 13
C
NMR (DMSO-D6) d 171.8, 171.7, 171.3, 170.8, 170.3, 168.9, 168.8,
168.5, 168.3, 166.3, 155.5, 137.5, 137.0, 136.8, 129.2, 129.1, 128.2,
128.1, 126.6, 77.8, 60.5, 59.8, 57.0, 53.7, 47.6, 41.4, 38.2, 37.0, 33.4,
32.3, 28.2, 25.4, 25.3, 24.7, 24.5, 14.1, 13.9. HRMS (ESI) m/z:
found 1539.7303 [M + H]+ (1539.7380 calcd for C75H107N14O17S2).
DTT-mediated reduction and saponification of 22. DTT
(358 mg, 2.32 mmol) was added to a solution of N-monoacetyl-
Gly-Phe-cystamine(Boc)-N-triacetyl-Gly-Phe-OEt cyclen (22,
358 mg, 0.23 mmol) in EtOH (8 mL). The mixture was stirred
for 18 h at rt, was concentrated, the residue was dissolved in
EtOAc (40 mL), and the solution obtained was washed with
saturated NaHCO3 solution (20 mL). The aqueous phase was
extracted with EtOAc (20 mL), the organic phase was dried and
then concentrated to leave a solid residue (N-monoacetyl-Gly-
Phe-NH(CH2)2SH-N-triacetyl-Gly-Phe-OEt cyclen) that was used
in the next step without further purification. The solid residue
obtained was dissolved in THF (4.5 mL), NaOH solution (1M,
4.5 mL) was added and the mixture was stirred vigorously for
2 h at rt. THF was evaporated, the aqueous solution was cooled
to 0 ◦C, was acidified (1 M HCl, pH 5) and was set aside for
2 h at 0 ◦C. Water was decanted, the oily product deposited
on the flask walls was washed with water, was coevaporated
with toluene (ca. 20 mL) and was triturated (twice) in hexanes
(ca. 15 mL) to leave N-monoacetyl-Gly-Phe-NH(CH2)2SH-N-
triacetyl-Gly-Phe-OH cyclen (5a, 189 mg, 64% based on 22).
Colorless solid; [a]2D5 −5 (c 0.94, MeOH). HPLC: tR 11.6 min;
1H NMR (DMSO-D6) d 8.27 (m, D2O exch., 6H); 7.20 (m, 20H);
4.40 (m, 4H); 3.71 (m, 8H); 3.51–2.15 (br m, 36H); 13C NMR
(DMSO-D6) d 173.1, 169.2, 168.3, 137.7, 137.6, 137.5, 129.3,
Alkylation of N-monoacetyl-Gly-Phe-OtBu cyclen (20) with N-
iodoacetyl-Gly-Phe-OEt (7). DIPEA (385 lL, 2.2 mmol) and
N-iodoacetyl-Gly-Phe-OEt3 (7, 919 mg, 2.2 mmol) were added to
a solution of N-monoacetyl-Gly-Phe-OtBu cyclen (20, 359 mg,
0.73 mmol) in MeCN (8 mL). The mixture was stirred for 18 h
◦
at 70 C, was cooled to rt and was diluted with EtOAc (30 mL).
The organic solution was washed with water (30 mL), the aqueous
phase was extracted with EtOAc (20 mL) and the organic phase
was dried and concentrated. The resulting oil was triturated
(twice) in a small amount (ca. 15 mL) of hexanes to leave N-
monoacetyl-Gly-Phe-OtBu-N-triacetyl-Gly-Phe-OEt cyclen (21,
986 mg, 99%). Colorless solid; [a]2D5 +28 (c 0.91, CH2Cl2). HPLC:
1
tR 23.0 min; H NMR (DMSO-D6) d 8.28 (m, D2O exch., 2H);
8.05 (m, D2O exch., 2H); 7.22 (m, 20H); 4.41 (m, 4H); 4.00 (m,
6H); 3.71 (m, 8H); 3.01–2.87 (br m, 16H); 2.60–2.53 (br m, 16H);
1.27 (s, 9H); 1.05 (m, 9H); 13C NMR (DMSO-D6) d 171.8, 171.3,
170.4, 168.8, 168.3, 166.2, 137.0, 136.8, 129.2, 129.1, 128.2, 128.1,
126.6, 126.5, 80.8, 60.5, 59.7, 54.0, 53.6, 41.5, 37.2, 37.0, 27.5, 13.9.
HRMS (ESI) m/z: found 1361.7184 [M + H]+ (1361.7186 calcd
for C70H97N12O16).
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129.2, 128.3, 128.2, 126.4, 126.3, 56.0, 55.9, 53.9, 51.1, 50.9, 50.4,
47.6, 45.4, 42.2, 41.7, 41.6, 41.5, 37.8, 36.9, 33.4, 32.3, 28.2, 27.0.
HRMS (ESI) m/z: found 1280.5720 [M + H]+ (1280.5774 calcd
for C62H82N13O15S).
Acknowledgements
We thank the Ontario Institute for Cancer Research (OICR), for
providing the operating funds and equipment needed to perform
this work.
Boc-deprotection and saponification of 22. TFA (1.5 mL) was
added to a solution of N-monoacetyl-Gly-Phe-cystamine(Boc)-
N-triacetyl-Gly-Phe-OEt cyclen (22, 328 mg, 0.21 mmol) in
CH2Cl2 (3 mL), the mixture was stirred for 30 min at rt and
was concentrated to leave N-monoacetyl-Gly-Phe-cystamine-N-
triacetyl-Gly-Phe-OEt cyclen that was used for the next step
without further purification. This material was dissolved in THF
(1.7 mL) and NaOH solution (2.5 M, 1.7 mL) was added. The
mixture was stirred vigorously for 2 h at rt. THF was evaporated,
the aqueous solution was cooled to 0 ◦C, was acidified (1 M HCl,
pH 5) and was set aside for 2 h at 0 ◦C. Water was decanted, an oily
product deposited on the flask walls was washed with water, was
coevaporated with toluene (ca. 20 mL) and was triturated (twice) in
hexanes (ca. 15 mL) to leave N-monoacetyl-Gly-Phe-cystamine-
N-triacetyl-Gly-Phe-OH cyclen (5b, 240 mg, 83% based on 22).
Colorless solid; [a]2D5 +9 (c 0.59, MeOH). HPLC: tR 13.0 min; 1H
NMR (DMSO-D6) d 8.32 (m, D2O exch., 6H); 7.19 (m, 20H); 4.38
(m, 4H); 3.78–2.77 (br m, 48H); 13C NMR (DMSO-D6) d 172.9,
172.8, 171.8, 171.1, 168.8, 168.6, 137.6, 137.4, 129.2, 128.2, 128.1,
126.5, 126.4, 57.0, 53.8, 50.8, 41.6, 38.1, 36.9, 34.0, 33.4, 32.9, 32.4.
HRMS (ESI) m/z: found 1355.5850 [M + H]+ (1355.5917 calcd
for C64H87N14O15S2).
References and notes
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references cited therein.
2 S. Zhang, M. Merritt, D. E. Woessner, R. E. Lenkinski and A. D.
Sherry, Acc. Chem. Res., 2003, 36, 783–790.
3 (a) F. Wojciechowski, M. Suchy´, A. X. Li, H. A. Azab, R. Bartha and
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5 For the recent reviews on the use of cell penetrating peptides in drug
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Prasad, S. K. Srivastava and M. F. Tweedle, Inorg. Chem., 1991, 30,
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8 K. A. Jacobson, B. Fischer and X. Ji, Bioconjugate Chem., 1995, 6,
255–263.
Metallation of N-monoacetyl-Gly-Phe-cystamine-N-triacetyl-
Gly-Phe-OH cyclen (5b). EuCl3·6H2O (32 mg, 0.089 mmol) was
added to a suspension of N-monoacetyl-Gly-Phe-cystamine-N-
triacetyl-Gly-Phe-OH cyclen (5b, 120 mg, 0.089 mmol) in MeOH
(2 mL) and water (4 mL). The pH of the reaction mixture was
adjusted to ca. 9 (2.5 M NaOH solution), the mixture was stirred
for 18 h at rt and was subjected to size exclusion chromatography
as described in the general experimental procedures; fractions
containing the product (the absence of free Eu3+ was confirmed by
negative xylenol orange test17) were combined and were lyophilized
to leave Eu3+ N-monoacetyl-Gly-Phe-cystamine-N-triacetyl-Gly-
Phe-OH cyclen (4b, 87 mg, 65%). Colorless solid; 1H NMR (D2O)
d 24.57 (s); 23.87 (s); 8.45 (s); 7.17–6.84 (m); 6.59 (s); 4.28–4.21 (m);
4.04 (s); 3.74–3.43 (m); 3.20–2.05 (m); 1.47 (s); −3.08 (s); −3.49 to
−3.83 (m); −5.09 (s); −8.92 to −9.19 (m); −9.98 (s); −12.24 (s);
−13.00 to −13.10 (m). HRMS (ESI) m/z: found 1503.4946 [M −
2H]+ (1503.4881 calcd for C64H84N14O15S2Eu).
9 (a) G. Losse and W. Zo¨nnchen, Liebigs Ann. Chem., 1960, 636,
140–143; (b) J. O. Thomas, Tetrahedron Lett., 1967, 8, 335–
336.
10 G. Losse and D. Nadolski, J. Prakt. Chem., 1964, 24, 118–124.
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47, 6625–6637; (b) A. C. Ferrand, D. Imbert, A. S. Chauvin, C. D.
B. Vandevyver and J. C. G. Bu¨nzli, Chem.–Eur. J., 2007, 13, 8678–
8687.
12 V. Boldrini, G. B. Giovenzana, R. Pagliarin, G. Palmisano and M. Sisti,
Tetrahedron Lett., 2000, 41, 6527–6530.
13 Products of peralkylation at nitrogen including some O-alkylations
have not been isolated but their structures were inferred from the MS
(ESI-TOF) analysis.
14 For selected examples see: (a) A. Roigant, I. Gardinier, H. Bernard, J. J.
Yaounac and H. J. Handel, J. Chem. Soc., Chem. Commun., 1995, 1233–
1234; (b) J. I. Bruce, R. S. Dickins, L. J. Govenlock, T. Gunnlaugsson,
S. Lopinski, M. P. Lowe, D. Parker, R. D. Peacock, J. J. B. Perry, S.
Aime and M. Botta, J. Am. Chem. Soc., 2000, 122, 9674–9684; (c) T.
Gunnlaugsson, J. P. Leonard, S. Mulready and M. Nieuwenhuyzen,
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1700.
DTT-mediated reduction of 4b. DTT (43 mg, 0.28 mmol) was
added to a solution of Eu3+ N-monoacetyl-Gly-Phe-cystamine-
N-triacetyl-Gly-Phe-OH cyclen (4b, 42 mg, 0.028 mmol) in
H2O (2 mL). The mixture was stirred for 18 h at rt and
was subjected to size exclusion chromatography as described in
the general experimental procedures; fractions containing the
product were combined and were lyophilized to leave impure
Eu3+ N-monoacetyl-Gly-Phe-NH(CH2)2SH-N-triacetyl-Gly-Phe-
OH cyclen (4a, 24 mg, 60%) containing a significant amount
of Eu3+ DOTAM-Gly-Phe-OH (3). The absence of free Eu3+
was confirmed by negative xylenol orange test.17 Colorless solid;
HRMS (ESI) m/z: found 1428.4727 [M − 2H]+ (1428.4738 calcd
for C62H79N13O15SEu).
15 For selected examples see: (a) W. J. Kruper, P. R. Rudolf and C.
A. Langhoff, J. Org. Chem., 1993, 58, 3869–3876; (b) A. Heppler,
S. Froidevaux, H. R. Ma¨cke, E. Jermann, M. Be´he´, P. Powell and
M. Hennig, Chem.–Eur. J., 1999, 5, 1974–1981; (c) M. Woods, G. E.
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