Synthesis and characterization of monophosphinic acid DOTA derivative: A smart tool with functionalities for multimodal imaging

Article abstract of DOI:10.1016/j.bmc.2017.06.008

A new facile synthetic strategy was developed to prepare bifunctional monophosphinic acid Ln-DOTA derivatives, Gd-DO2AGAP^NBn and Gd- DO2AGAP^ABn. The relaxivities of the Gd-complexes are enhanced compared to Gd-DOTA. Monophosphinic ac

Full text of DOI:10.1016/j.bmc.2017.06.008

Accepted Manuscript
Synthesis and characterization of monophosphinic acid DOTA derivative: A
smart tool with functionalities for multimodal imaging
Satya Narayana Murthy Chilla, Ondrej Zemek, Jan Kotek, Sébastien Boutry,
Lionel Larbanoix, Coralie Sclavons, Luce Vander Elst, Ivan Lukes, Robert N.
Muller, Sophie Laurent
PII:
S0968-0896(17)30227-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.06.008
BMC 13791
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 February 2017
30 May 2017
8 June 2017
Please cite this article as: Chilla, S.N.M., Zemek, O., Kotek, J., Boutry, S., Larbanoix, L., Sclavons, C., Elst, L.V.,
Lukes, I., Muller, R.N., Laurent, S., Synthesis and characterization of monophosphinic acid DOTA derivative: A
smart tool with functionalities for multimodal imaging, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.06.008
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Synthesis and characterization of
monophosphinic acid DOTA derivative: A
smart tool with functionalities for
multimodal imaging
Leave this area blank for abstract info.
Satya Narayana Murthy Chilla a,  Ondrej Zemek,b Jan Kotek,b Sébastien Boutry a,c, Lionel Larbanoix a,c, Coralie
Sclavons a,c Luce Vander Elst,a,c Ivan Lukes,b Robert N. Muller, a,c and Sophie Laurent a,c

a.Department of General, Organic and Biomedical Chemistry, NMR and Molecular Imaging Laboratory,
University of Mons, Avenue Maistriau, 19, Mendeleïev Building, 7000 Mons, Belgium. E-mail:
satya.chilla@umons.ac.be, Fax: +3265373533, Tel: +3265373518
b.Department of Inorganic Chemistry, Universita Karlova, Hlavova 2030, 128 40, Prague 2, Czech Republic. E-
mail: modrej@natur.cuni.cz; Fax: +420-221951253; Tel: +420-221951261
c.Centre for Microscopy and Molecular Imaging (CMMI), Rue Adrienne Bolland, 8, 6041, Charleroi- Gosselies.

Bioorganic & Medicinal Chemistry
Synthesis and characterization of monophosphinic acid DOTA derivatives: A smart
tool with functionalities for multimodal imaging
Satya Narayana Murthy Chilla a,  Ondrej Zemek,b Jan Kotek,b Sébastien Boutry a,c, Lionel Larbanoix a,c,
Coralie Sclavons a,c Luce Vander Elst,a,c Ivan Lukes,b Robert N. Muller, a,c and Sophie Laurent a,c
a.Department of General, Organic and Biomedical Chemistry, NMR and Molecular Imaging Laboratory, University of Mons, Avenue Maistriau, 19,
Mendeleïev Building, 7000 Mons, Belgium. E-mail: satya.chilla@umons.ac.be, Fax: +3265373533, Tel: +3265373518
b.Department of Inorganic Chemistry, Universita Karlova, Hlavova 2030, 128 40, Prague 2, Czech Republic. E-mail: modrej@natur.cuni.cz; Fax: +420-
221951253; Tel: +420-221951261
c.Centre for Microscopy and Molecular Imaging (CMMI), Rue Adrienne Bolland, 8, 6041, Charleroi-Gosselies
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A new facile synthetic strategy was developed to prepare bifunctional monophosphinic acid Ln-DOTA
derivatives, Gd-DO2AGAPNBn and Gd- DO2AGAPABn. The relaxivities of the Gd-complexes are
enhanced compared to Gd-DOTA. Monophosphinic acid arm of these Gd-complexes affords
enhancement of inner sphere water exchange rate due to its steric bulkiness. The different
functionalities of DO2AGAPNBn were appended in trans positions and are designed to conjugate
identical or different vectors according to the potential applications. The conjugation of Gd-
DO2AGAPABn with E3 peptide known to target apoptosis was successfully performed and in vivo MRI
allowed cell death detection in a mouse model.
Available online
Keywords:
Contrast agents
Macrocyclic ligands
Peptide conjugation
Gadolinium complex
Relaxivity
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Magnetic resonance imaging (MRI) is an effective tool in
biomedical research for understanding biological processes in
living organisms as well as in clinical diagnosis due to its
excellent spatial resolution, its non-invasiveness and its limitless
tissue penetration. The resolution and tissue specificity can be
further enhanced by paramagnetic lanthanide complexes due to
their exceptional magnetic properties.1 Nowadays, MRI contrast
agents (CA) can provide detailed information at sub-cellular level
resolution.2 In multimodal imaging a single multimodal probe
could be sufficient to address the same pharmacokinetics and co-
localization of the signal in each modality, allowing the
correlation of the information obtained by the different methods
and avoiding multiple dose injections of agents.3 However, the
sensitivity of each modality can be different by several orders of
magnitude and obtaining a single molecule efficient in each
imaging modality is not trivial.
residence time, τM) and slow molecular tumbling rates
characterized by the rotational correlation time τR.4, 5 Most of the
commercially used MRI contrast agents are low-molecular-
weight compounds and generally have too fast molecular
tumbling, i.e. short τR. In general, they have also a relatively slow
310
water exchange rate4 (e.g. τM of Gd-DOTA = 122ns, (DOTA =
7
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid).6,
It
has been reported that increasing the negative charge of the
complex and bulkiness around central metal atom facilitates the
fast exchange of the coordinated water molecule due to sterical
reasons.4 Extensive research about the relationship between water
exchange rates and solution structures have been related to the
structural isomers of lanthanide(III) DOTA complexes. It is well-
known that DOTA-like ligands wrap around a lanthanide(III) ion
yielding two coordination geometries, namely square
antiprismatic (SA) and twisted square-antiprismatic (TSA).4, 5, 8 It
was observed that the TSA isomers had 10 to 100 times faster
exchange rate of the coordinated water molecule.9 In general,
increased steric repulsion around the paramagnetic center favors
the TSA isomers.2
Gadolinium(III) complexes of polyaminopolycarboxylates are
widely used as CA in MRI.4 The efficacy of a paramagnetic CA
is assessed by its relaxivity (r1). Relaxivity enhancement can be
a—ch—iev—ed by fast water exchange rate (usually expressed as water
 Corresponding author. Tel.: +32-65373516; fax: +32-65373484; e-mail: satya.chilla@umons.ac.be & sophie.laurent@umons.ac.be

In the present work, we designed a suitable ligand for
different modalities, the complexes of which can exhibit
favorable characteristics for both optical imaging and MRI
techniques. This system consists of a kinetically inert
macrocyclic ligand covalently linked to an aromatic
phosphinate moiety. Through this approach, complexes
suited for magnetic and optical imaging and with identical
bio distributions could be used in MRI and optical imaging
techniques and should enable better interpretation of in vivo
molecular
imaging
experiments.10
We
prepared
DO2AGAPNBn (scheme 1), a DOTA derivative with two
different arms: p-nitro benzyl phosphinic acid and
pentanedioic acid arms in trans-position. Consequently, the
nitro group will serve as good precursor of amine function
Scheme-1: Synthesis of monophosphinic acid derivatives and their conjugations with peptide and chromophore rhodamine isothiocyanate

with rhodamine isothiocyanate depicted the availability of
aniline group of complex for further conjugations. The
complex was purified by reverse phase chromatography and
the corresponding high resolution mass confirms the
formation of thiourea. (Scheme 1, see SI fig. S1).
9
for further conjugation of the probe. The hexapeptide E3
was previously selected as a phosphatidylserine (PS)-
specific peptide using the phage-display method.11 PS is a
membrane phospholipid that is externalized by cells
undergoing apoptosis, which is a natural genetically
programmed cell death process occurring in several normal
2.2. Inner sphere hydration number (q) determination
(eg.
embryonic
development),
pathologic
(eg.
neurodegeneration) or therapeutic (eg. anti-cancer treatment)
conditions.12 Apoptosis can be induced in thymus by intra-
peritoneal injection of the synthetic glucocorticoid
dexamethasone (DEX).13 This cell death model was set up in
mice for preliminary MRI testing of the here-presented
compounds, conjugated or not to the E3 peptide.
A previous method for the determination of q by
17O chemical shifts18 used the dysprosium complexes,
mainly because the contact contribution to its induced shift,
is predominant as compared with its pseudo-contact shift
and because Dy3+ causes a relatively small broadening of the
17O signal. However, despite the quite large broadening of
the 17O signal induced by Gd3+, this ion is better suited for
such measurements since it induces only contact shift and no
pseudo-contact shift. As previously reported,19 we compared
the 17O chemical shift induced by the new Gd complex with
well described Gd complexes, such as Gd–DOTA (Dotarem)
and Gd–DTPA (Magnevist), for which q = 1 has been
validated. The value obtained for Gd-DO2AGAPNBn was
estimated to be 0.74.
2. Results and discussion
2.1. Synthesis, complexation and conjugation of contrast
agents
The controlled synthesis of ligand DO2AGAPNBn
6 was
obtained by a multi-step synthetic route starting from prepa-
ration of two pendant arms (Scheme 1). The key
intermediates 4-nitrobenzylphosphinic acid14 and dibenzyl
2-bromodipentanoate15 were obtained according to literature
2.3. 17O and 1H NMRD measurements
procedures. The trans-DO2A
3 was synthesized by selective
The variable temperature dependence of the reduced 17O
transverse relaxation rates (1/T2r) (Fig. 1) is typical of a water
residence time lower than 100 ns at 310 K.20 The observed τM
values of Gd-DO2AGAPNBn 8 and its derivatives (Table 1) show
improvement compared to Gd-DOTA and to other phosphinate
based DO3A complexes.14(table 1).
protection of nitrogen atoms N-1 and N-7 with Boc-O-
succidimyl and further alkylation and deprotection as
17
previously described.16,
Trialkylated product
4
was
2-
obtained
by
alkylating
3
with
dibenzyl
bromopentanedioate in acetonitrile and sodium acetate used
as a base. At this stage, we tried several bases for alkylation.
Among them sodium acetate allowed to obtain a majority of
trialkylated product even though we could not avoid the
formation of minor tetraalkylated product. The second arm
introduction was envisaged (after hydrogenolysis of
compound
with formaldehyde and 4-nitrobenzylphosphinic acid in 6N
HCl, leading to the macrocyclic ligand
(DO2AGAPNBn).
As the secondary amine of has low reactivity, a large
excess of precursor and formaldehyde was used and in
addition, a long reaction time was required. Ligand
DO2AGAPNBn
was isolated by ion exchange
4 to intermediate 5) by Mannich type reaction
1e+6
6
5
6
NBn
Gd-DO2AGAP
chromatography in good yields and the structure was
ABn
Gd-DO2AGAP
1
confirmed by H, 13C, 31P NMR and high-resolution mass
1e+5
NBn
Gd-DO2AGAP
- E3 Pep
spectrometry. CComplexation of the ligand
6
DO2AGAPNBn
with Gd3+ or Tb3+ ions was performed under standard
procedure. High-resolution mass spectrometry reveals peak
corresponding to the molecular ion with the correct isotopic
pattern for each complex. In order to prepare multimodal
contrast agent, the conjugation efficiency of carboxylic acid
0.0029 0.0030 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 0.0037
1/T (k -1
)
Figure 1: Temperature dependence of the reduced transverse
relaxation rate of 17O at 11.75 T of Gd-DO2AGAPNBn 8(28.13 mM),
Gd-DO2AGAPABn 9 (20.12 mM) and Gd-DO2AGAPNBn-E3Pep 11
(26.032 mM)
of the Gd–DO2AGAPNBn
8 complex was tested by using
peptide-coupling reaction with a small hexapeptide under
peptidic coupling methods at room temperature by using
DEPBT and N-methyl morpholine (NMM) as a base. The
compound 11 was obtained with good yield and purified by
reverse phase chromatography. Our trials for conversion of
Proton relaxivity measurements in water revealed that the
Gd-DO2AGAPNBn 8 and its derivatives are stable in the pH range
extending from 3 to 8. The magnetic field dependence of the
proton longitudinal water proton relaxivities (r1 NMRD profile)
measured at 310 K shows higher relaxivities for our new
complexes as compared to those of the parent compound Gd–
DOTA.20 At 60 MHz and 310 K, the relaxivities of Gd–
nitro group of the compound
hydrogenation were not successful. The ligand
treated under hydrogenolysis to obtain
DO2AGAPABn
conditions similar to those used to complex
8 to amino group by catalytic
6
was thus
and Gd-
DO2AGAPNBn 8, Gd–DO2AGAPABn 9 and Gd–DO2AGAPNBn
–
7
E3Pep 11 are enhanced respectively by 39% (r1 = 4.93 mM–1s–1),
9
was obtained after complexation in
. Conjugation
6

56% (r1 = 5.45 mM–1s–1) and 29% (r1 = 4.28 mM–1s–1) as
compared to Gd–DOTA (r1 = 3.06 mM–1s–1).
Table 1 Parameters obtained from 17O transverse relaxometry and 1H-NMRD of Gd-DO2AGAPNBn 8, Gd-DO2AGAPABn 9, Gd-DO2AGAPNBn
– E3Pep 11 Gd-DO2AGAPABn-Rhod 12 and Gd-DOTA complexes in water at 37 °C.
Parameters
Gd–DO2AGAPNBn(a) Gd–DO2AGAPABn(a)
Gd–DO2AGAPNBn
E3peptide(a) 11
–
Gd-
DO2AGAPABn
Gd–DOTA(a,b)
8
9
-Rhod 12
τM310 [ns]
ΔH≠[kJmol-1]
ΔS≠[ J mol-1K-1]
A/ħ[ 106 rads-1]
B [1020 s-2]
τv298 [ps]
EV[kJ mol-1]
τv310[ps]
τR310 [ps]
20.3 ± 1.4
62.6 ± 0.1
104.0 ± 0.3
-2.90 ± 0.02
16.2 ± 0.4
12.1 ± 0.37
20.0 ± 12.4
13.6 ± 3.0
77.3 ± 3.0
247 ± 22
15.8 ± 0.1
54.0± 0.1
78.5 ± 0.3
-2.8 ± 0.1
16.5 ± 0.5
18.4 ± 0.5
20.0 ± 16.0
24.2 ± 5
10.8 ± 0.4
44.9 ± 0.05
52.2 ± 0.2
-2.82± 0.02
8.2±0.3
16c
-
122 ± 10
50.1 ± 0.2
48.7 ± 0.2
-3.42 ± 0.03
1.94 ± 0.09
11.4 ± 0.5
4.0 ± 0.4
7 ± 1
-
-
-
-
-
13.5 ± 0.6
0.1 ± 13.3
17.2 ± 2.7
115.3 ± 4.0
202 ± 9
25.2±2.1
180±4
90.3 ± 2.4
206 ± 10
53 ± 1
τso [ps]
162±3
404 ± 24
a the following parameters were fixed: (D is the diffusion coefficient = 3 10–9m2s–1, d is the distance of closest approach = 0.36 nm, r is the distance between the proton of the inner-
sphere water molecule and Gd ion = 0.31 nm, q is fixed to 1 and τM was fixed to the value determined by 17O NMR). b ref 21 C M was fixed at the value obtained for Gd-DO2AGAPABn

(16 ns)
The interaction of the phosphinate with the Tb3+ center
could be confirmed by 31P NMR spectroscopy (Fig. 3). For
the complex Tb-DO2AGAPNBn 10, two broad shifted peaks
could be observed at a range of frequencies from 400 to 500
ppm. The direction and magnitude of the shifts are in
agreement with what is observed for lanthanide complexes
where the phosphinate group is in close proximity to the
paramagnetic center. The two peaks corresponding to two
14
12
10
8
isomeric forms SAP and TSAP of 10 are observed at
 468
6
NBn
NBn
ABn
Gd-DO2AGAP
Gd-DO2AGAP
ppm and 413 ppm respectively with a 4:6 ratio. The large

- Pep
4
proportion of TSAP isomer is in good agreement with the
fast water exchange of the Gd complex.
Gd-DO2AGAP
Gd-DOTA
2
ABn
Gd-DO2AGAP
-Rhod
0
0.01
0.1
1
10
100
1000
proton Larmor frequency (MHz)
Figure 2: 1H-NMRD relaxivity profiles of Gd–DO2AGAPNBn 8, Gd–
DO2AGAPABn 9, Gd–DO2AGAPNBn–E3Pep 11 and Gd–DOTA in
water at 37 °C.
The NMRD profiles were fitted using the inner sphere
and outer sphere theories (Fig. 2). As expected, considering the
molecular weight of Gd-DO2AGAPNBn 8 and its derivatives, the
value of τR is increased as compared to that of Gd-DOTA (Table
1). In addition, the temperature dependence of the proton
longitudinal relaxivity at 20 MHz of complexes 8, 9, 11 and 12
confirms the fast water exchange for all the complexes (see SI
fig. S1). The r1 value of Gd-DO2AGAPABn 9 at 25°C and 20 MHz
(r1=7.2 s-1mM-1) is larger than the value reported for Gd-
DO3APABn at 25°C and 10 MHz (6.7 s-1mM-1)14 as could be
expected from the larger molecular weight of 9. In addition, the
298
M values are in the same range, 16.2 ns for Gd-DO3APABn 14
Figure 3: 31P spectrum of Tb–DO2AGAPNBn 10 (D2O)
and 38 ns for Gd-DO2AGAPABn 9.
2.5. Photophysical measurements
2.4. 31P NMR measurement
The emission experiments were performed in H2O at
25°C. After excitation of Tb-DO2AGAPNBn 10 at 267nm, no f-f

transition of Tb3+ was observed showing no antenna effect
probably because of the distance between the benzyl group and
the central metal ion. In addition, the photo-physical properties of
Gd-DO2AGAPABn-Rhodamine 12 was examined by recording
the excitation and emission spectra of 1mM solution in H2O (see
SI fig. S2). For this complex a strong absorption could be
observed at 556 nm and excitation at this wavelength resulted in
broad emission band centered at 581 nm.
phosphinic moiety that induces a τR increase and a
significant molecular crowding which results in fast water
exchange rates. The complexes were conjugated with small
peptide on carboxylic acid arm or with a chromophore on
amine of the phosphinate arm. Both functionalities were
proved to be enough reactive to obtain multimodal contrast
agent. The 31P NMR of Tb–DO2AGAPNBn 10 complex
showed that the phosphinic acid moiety is coordinated to
metal centre but photophysical studies have not shown
luminescence properties. In vivo experiments showed
promising results in molecular MRI of cell death by
conjugating PS-specific E3 hexapeptide to Gd-
DO2AGAPNBn 8 and targeting PS exposed by thymocytes in
a mouse model of thymus cortex apoptosis induced by
dexamethasone.
3. In vivo results
Thymus is a primary lymphoid organ in which T-
lymphocytes (or
T cells) develop for immune response
capability. It is composed of an outer zone called cortex and an
inner zone called medulla. T cell maturation (CD4+CD8+) from
immature thymocytes occurs during their migration from cortex
to medulla.22 Dexamethasone (DEX) promotes thymocyte
6. Experimental
apoptosis and vessel permeability increase in the cortical region
23
of thymus.13,
Immunohistochemistry of activated caspase-3
6.1. General procedures
demonstrated ongoing apoptotic phenomenon in thymic cortex of
mice 18h after injection of DEX at a dose of 30mg/kg (see SI fig.
S3). MRI showed signal intensity enhancement in thymic region
corresponding to cortex in DEX-treated mice after injection of
Gd-DO2AGAPNBn-E3pep, suggesting specific targeting of PS on
apoptotic thymocytes (see SI fig. S3). Cortex/medulla signal
intensity enhancement ratio was shown to be significantly
increased (p<0.01) in DEX-treated mice after injection of Gd-
DO2AGAPNBn-E3pep, regarding control groups (no DEX
treatment+injection of Gd-DO2AGAPNBn-E3pep or injection of
Gd-DO2AGAPNBn and DEX-treated+injection of Gd-
DO2AGAPNBn) (see SI fig. S3).
The chemicals were purchased from different companies:
Acros, Aldrich, Fluka, Merck, Strem, Chematech, Polypeptide,
TCI and VWR. All reagents were used without further
purification. All solvents were distilled and/or dried prior to use
by standard methodology except for those, which were reagent
grades. Unless and otherwise mentioned, all the reactions were
carried out under a nitrogen atmosphere and the reaction flasks
were pre-dried by heat gun under vacuum. Pure water (18 MΩ
cm–1) was used throughout.1, 2 1H, 13C and 31P NMR spectra were
recorded on Bruker 300 or 500 spectrometers. Chemical shift
1
scale was referenced by following ways: H – internal reference
CDCl3 at 7.27 ppm, MeOH at 3.33 ppm or D2O at 4.75 ppm or
TMS at 0.0 ppm; 13C – internal reference CDCl3 at 77.0 ppm,
MeOH at 50 ppm or TMS at 0.0 ppm; 31P – external reference
85% H3PO4 0.0 ppm and experiments were performed at 25°C.
ESI low-resolution mass spectra (ESI-MS) were recorded on
Waters micromass ZQ system (Waters, Belgium) with full
spectral detection mode in positive and negative ion modes. For
HR-MS all experiments were performed on a Waters QToF 2
mass spectrometer. The analyte solutions (10–5M methanol/water,
80:20) were delivered to the ESI source. Analytical thin layer
chromatography (TLC) was performed on silica gel 60 F254 plates
(E. Merck, Germany) using different mobile phases. The
compounds were visualized by UV254 light and TLC plates were
developed in Iodine chamber. The purification of the compounds
was performed on KP-silica, KP-C18 cartridges from Biotage
over Biotage flash chromatography instrument (Uppsala,
Sweden) and by using silica gel 60 (70-230 mesh) from Merck.
Reversed-phase analytical HPLC was performed in a stainless
steel Xbridge (length 250 mm, internal diameter 4.6 mm, outside
diameter 9.5 mm and particle size 5μm) C18 column. Preparative
HPLC was performed in a stainless steel Xbridge (length 250
mm, internal diameter 41.4 mm, outside diameter 50.8 mm and
particle size 5 μm) C18 column (Varian). The compounds were
purified using a gradient with the mobile phase starting from
95% solvent A (water) and 5% of solvent B (ACN) to 70% B in
10 min, 100% B in 18 min, 100% B isocratic till 24 min and
decreased to 5% B in 28 min. The flow rate generally used for
analytical HPLC was 1 mL/min and for preparative HPLC was
12 mL/min. All the solvents for HPLC were filtered through a
Nylon-66 Millipore filter (0.45 μm) prior to use. Hexapeptide-E3
Figure 3: Percentage of change of the cortex/medulla signal intensity
ratio as a function of time after injection of Gd-DO2AGAPNBn
-
E3pep in DEX-treated mice (n=3, blue line), Gd-DO2AGAPNBn in
DEX-treated mice (n=2, gray line), Gd-DO2AGAPNBn-E3pep in
untreated mice (n=2, orange line), Gd-DO2AGAPNBn in untreated
mice (n=2, yellow line). **: p<0.01 for DEX-treated mice+Gd-
DO2AGAPNBn-E3pep regarding control groups.
4. Conclusion
In summary, a facile synthetic route was developed to
prepare monophosphinic acid DOTA derivatives with two
different functional groups such as carboxylic acid and nitro
or amine. These new ligands DO2AGAPNBn
6
and
were easily complexed with lanthanides.
Their relaxivities are enhanced due to the presence of
DO2AGAPABn
7

was synthesized on Liberty1 automated microwave peptide
synthesizer with synthesis scale of 0.02 to 5 mmol by Fmoc
chemistry. The microwave operating power was 120V/60Hz.
6.2.3. 2-{4,10-Bis-carboxymethyl-7-[hydroxy-(4-nitrobenzyl)
phosphinoy- lmethyl]1,4,7,10 tetraazacyclododec-1-yl}-
pentanedioic acid (6):
6.2. Synthetic procedures of the compounds
10% Pd/C (1.0 g) was added to a solution of 4 (8.0 g,
0.01 mol) in MeOH (100 mL) and the resulting suspension was
shaken for 4h under H2 atmosphere. After filtration of the
suspension over a Celite pad and after evaporation of the solvent
under reduced pressure, 3.2 g of 5 were obtained. The crude
product (3.2 g, 8.13 mmol) was dissolved in 6 N HCl (20 mL).
1.95g (0.065 mol) of paraformaldehyde and 6.5 g of 4-
nitrobenzylphosphinic acid (0.03 mol) were added to the latter
solution and stirred at 50 °C for 2 days. Reaction mixture was
cooled to 0°C and all solvents were evaporated under vacuum.
The product was purified using strong cation exchanger
chromatography (Dowex 50, H+-form, 6×25 cm, elution with
water followed by 5% aq. ammonia). After evaporation of the
6.2.1. Di-tert-butyl 4-[2-(benzyloxy)-2-oxoethyl]-10-[(3-phenyl
propanoyl)oxy]-1,4,7,10-tetraazacyclododecane-1,7-dicarbo-
xylate (2):
To a solution of 1,4,7,10-tetraazacyclododecane free
base 1 (714 mg, 4.067 mmol) in chloroform (35 mL), N-(tert-
Butoxycarbonyloxy) succinimide (8.134 mmol) was added. The
reaction mixture was stirred at room temperature for 28h. Solvent
was removed by rotary evaporation and 30 mL of NaOH 3 M
was added to the remaining residue. The aqueous phase was
extracted with chloroform (3.3 mL). The extracts were combined
and dried (over K2CO3). The solvent was removed by rotary
evaporation and the residue was dried in vacuum for several
hours. 513 mg of the unpurified compound from the preceding
reaction (1.3761 mmol) was solubilized in acetonitrile. 0.67 mL
(2.87 mmol) of benzyl bromoacetate and 0.474g (3.412 mmol)
K2CO3 were added to this solution at room temperature, after
which the mixture was heated under reflux for 8h. The solids
were discarded by filtration over Celite and the solvent was
removed under vacuum. Chromatographic purification (alumina,
98/2 DCM/MeOH) yielded 2 as a slowly solidifying colourless
+
solvents, the product was collected as a yellow oily NH4 -salt.
The mixture was again subjected to anion exchanger (Dowex 5,
Cl– form, 15×3.8 cm elution with water 5% HCl). Evaporation of
the solvents provided HCl salt of ligand. The chloride salt was
dissolved in water, poured onto a column of a strong cation
exchanger (Dowex 50, H+-form, 4×25 cm) and the column was
eluted with 10% aq. pyridine. After evaporation of the excess of
pyridine from the eluate, the Hpy+-salt was dissolved in a
minimum amount of water and subjected to reverse phase flash
chromatography, eluted with water/acetonitrile and yielded 6
1
1
oil (736 mg, 87% over all for two steps). HNMR (500 MHz,
(0.82 g, 30% based on 4). H NMR (500 MHz, D2O) δ (ppm):
CDCl3) δ (ppm): 7.31(m, 10H), 5.11(s, 4H), 3.54(s, 4H), 3.49(m,
8H), 2.86(m, 8H), 1.41 (s, br 18H). 13C (500 MHz, CDCl3) δ
(ppm): 171.48, 157.11, 136.02, 79.33, 66.04, 55.04, 54.51,
46.68, 28.46. ESI-MS (C36H52N4O8); m/z = 668 [M+H]+
8.12-8.1 (d, J = 10Hz, 2H), 7.43-7.41 (d, J = 10Hz, 2H), 3.75-
3.64 (m, 5H), 3.48-2.85 (m, 20H), 2.63-2.61 (m, 2H), 1.96 (s br ,
2H). 13C NMR (500 MHz, D2O) δ (ppm): 177.3, 174.9, 173.5,
173.2, 146.5, 138.9, 131.1, 130.0, 122.1, 122.0, 66.5, 58.9, 58.2,
54.6, 54.1, 52.7, 52.4, 52.3, 51.1, 50.5, 41.2, 31.3, 30.2, 25.2. 31
P
6.2.2. 2-(4,10-Bis-benzyloxycarbonylmethyl-1,4,7,10 tetraaza -
cyclododec-1-yl)-pentanedioic acid dibenzyl ester (4):
(500 MHz, D2O) δ (ppm): 38.22 (S). HRMS (ESI): calcd for
C25H38N5O12P; [M+H]+ 632.2333; found 632.2334.
Trifluroacetic acid (14 mL) was added to the solution of
2 (10.21 g, 0.015 mol) in dichloromethane (100 mL) and the
mixture was stirred at room temperature for 16 hours. The
reaction mixture was evaporated under reduced pressure. Diethyl
ether was used to precipitate the crude oil. The precipitate was
washed with excess of ether followed by filtration and dried to
obtain 3. This product was directly used in the next step without
further purification. 7.0 g (0.0149 mmol) of deprotected product
and 2.64g (0.03 mmol) of CH3COONa were added to a solution
of 5.8 g (0.015 mol) of dibenzyl 2-bromopentadioate in 100 mL
of ACN and stirred for 48 h. The solid was discarded by filtration
over a pad of Celite and the solvent was removed under reduced
pressure to recover brownish oily crude mixture.
6.2.4.
2-{7-[(4-Aminobenzyl)hydroxyphosphinoylmethyl]-
4,10-bis-carboxymethyl-1,4,7, 10 tetraaza-cyclododec-1-yl}-
pentanedioic acid (7):
10% Pd/C (0.1g) was added to a solution of 6 (0.15g,
0.23 mmol) in EtOH (10 mL) and the resulting suspension was
shaken for 4 h under H2 atmosphere. The suspension was filtered
over a celite pad, and after evaporation of the solvent under
1
reduced pressure 7 was obtained (0.13g, 92%). H NMR (500
MHz, D2O) δ (ppm): 6.82-6.8 (d, J = 10Hz, 2H), 6.33-6.3 (d, J =
10Hz, 2H), 3.75-3.64 (m, 5H), 3.4-2.80 (m, 20H), 2.65-2.61 (m,
2H), 1.96 (s br , 2H). 13C NMR (500 MHz, D2O) δ (ppm): 177.3,
174.9, 173.5, 173.2, 145.5, 138.9,138.3, 129.1, 116.1, 122.0,
66.5, 58.9, 58.2, 54.6, 54.1, 52.7, 52.4, 52.3, 51.1, 50.5, 41.2,
31.3, 30.2, 25.2. 31P (500 MHz, D2O) δ (ppm): 37.22 (s). ESI-MS
(C25H40N5O10P); m/z = 602.6 [M+H]+
Chromatographic
purification
(silica
gel,
98/1.5/0.5
EtOAc/MeOH/TEA) of this mixture yielded 4 as slight yellow
1
viscous oil. (8.1g, 70%) H NMR (300 MHz, CDCl3) δ (ppm):
7.38-7.28 (m, 20H), 5.28-5.04 (m, 8H) 3.6-3.55 (m, 1H), 3.43 (s,
br 4H), 3.1-3.0 (m, 6H), 2.6 (m, 4H), 2.52-2.5 (m, 6H), 2.22-1.97
(m, 4H). 13C NMR (300 MHz, CDCl3) δ (ppm): 177.75, 174.55,
172.8, 170.8, 135.73, 135.4, 128.72, 128.67, 128.59, 128.50,
128.45, 128.25, 128.17, 71.16, 67.16, 67.00, 66.54, 66.5, 59.89,
55.84, 55.21, 50.91, 49.60, 46.37, 30.59, 29.72, 26.18, 22.75,
20.52. ESI-MS (C45H54N4O8); m/z = 779 [M+H]+
6.3. Preparation of Gd3+ complex of DO2AGAPNBn (8)
The complex was obtained by adding portion wise
GdCl3·6H2O to a solution of 6 (0.26 g, 0.69 mmol) in H2O (5
mL), maintaining the pH between 6.2 and 6.7 by addition of 1 N
Pyridine. The final pH of the solution after stirring 24 h was 6.3.
The excess of the free lanthanide was removed as Gd(OH)3

precipitate, which appeared at pH 9. The resulting solution was
treated with chelex-100 to remove free Gd3+ ions. The absence of
free Gd3+ ions was confirmed by an Arsenazo III test24 (in 0.1 M
NaAc/HAc buffer solution, pH 5.2). The pH of the supernatant
was decreased to 7 and the solution was freeze-dried. The
complex was dissolved in water, purified by reverse phase flash
chromatography and freeze-dried, yielding Gd–DO2AGAPNBn (8)
as a white powder (450mg, 90%). The compound was purified by
preparative HPLC as mentioned above. HPLC: Rt = 9.5 min,
purity (215nm): 96% HRMS (ESI): calcd for C25H34GdN5O12P;
[M+Na]+ 805.1126; found 805.1090.
6.7. Conjugation of Rhodamine with Gd-DO2AGAPABn (12)
32 mg of rhodamine isothiocyanate (0.072 mmol) and 6
µl of pyridine (0.07 mmol) were added to the aqueous solution of
complex 9 (45 mg, 0.06 mmol) at room temperature. The mixture
was stirred in the dark for 48 h. The mixture was freeze-dried and
subjected to purification by reverse phase flash-chromatography
over C18 silica gel using conditions as mentioned previously and
12 was obtained after 10 min of elution. (17 mg, 20%). ESI-MS
(C54H65GdN8O13PS); m/z = 1274 [M+Na]+
6.8. 17O NMR Measurements and proton NMRD profiles
6.4. Preparation of Gd3+ complex of DO2AGAPABn (9)
17O NMR measurements were performed at 11.75 T on
350L samples contained in 5 mm o.d. tubes on a Bruker Avance
500 spectrometer (Karlsruhe, Germany). Temperature was
regulated by air or nitrogen flow controlled by a Bruker BVT
3200 unit. 17O transverse relaxation times of distilled water (pH
6.5–7) were measured using a CPMG sequence and a subsequent
two-parameter fit of the data points. The 90° and 180° pulse
lengths were 27.5 and 55 s, respectively. The 17O T2 of water in
complex solution was obtained from the line width measurements
or CPMG sequence when linewidth was  80Hz. All spectra were
proton decoupled. The data are presented as the reduced
Gadolinium(III) complex of DO2AGAPABn was
prepared by mixing a 1:1.1 molar ratio of GdCl3 6H2O (0.054g,
.
0.14 mmol) and ligand 7 (0.08g 0.13 mmol) in water followed by
addition of 1N pyridine to adjust the pH to 7. The reaction
mixture was briefly heated to 50 °C and then stirred at room
temperature overnight. The complex was purified on Amberlite
CG-50 with water elution. The solution was tested negatively for
the presence of free lanthanide(III) ions by using arsenazo III test
as an indicator (in 0.1 M NaAc/HAc buffer solution, pH 5.2).
The complex was dissolved in water, purified by reverse phase
flash chromatography and freeze-dried, yielding Gd–
DO2AGAPABn as a pale off white powder (80 mg, 75%). The
concentration of gadolinium(III) ions in solution was determined
by relaxometric measurement. HRMS (ESI): calcd for
C25H37GdN5O10P; [M+Na]+ 775.1530; found 775.1534.
r
p
transverse relaxation rate {1/T2 = 55.55/([Gd-complex] ×q×T2 ),
where: [Gd complex] is the molar concentration of the complex,
p
q is the number of coordinated water molecules and T2 is the
paramagnetic transverse relaxation rate}. The fitting of the
experimental data was performed as described in literature.20,21
The gadolinium concentration was determined by ICP-AES on a
JobinYvon JY 70 instrument (Longjumeau, France) and was
6.5. Preparation of Tb3+ complex of DO2AGAPNBn (10)
1
further confirmed by H relaxometry of a decomplexed sample.
Terbium(III) complex of DO2AGAPNBn was prepared
Proton nuclear magnetic relaxation dispersion (NMRD) profiles
were measured on a Stelar Spinmaster FFC (Mede, Italy) fast
field cycling NMR relaxometer over a magnetic field range from
0.24 mT to 1.0 T. Measurements were performed on 0.6 mL
samples contained in 10 mm o.d. Pyrex tubes. Additional
relaxation rates at 20 and 60 MHz were obtained on a Minispec
mq20 and a Minispec mq60, respectively.
.
by mixing a 1:1.1 molar ratio of TbCl3 6H2O (14.2mg, 0.004
mmol) and ligand 6 (0.022g, 0.004mmol) in water followed by
addition of 1N pyridine to adjust the pH to 7. The reaction
mixture was briefly heated to 50 °C and then stirred at room
temperature overnight. The complex was purified on Amberlite
CG-50 with water elution. The solution was tested for the
absence of free lanthanide(III) ions by using arsenazo III test as
an indicator (in 0.1 M NaAc/HAc buffer solution, pH 5.2). The
complex was dissolved in water, purified by reverse phase flash
chromatography and freeze-dried, yielding Tb–DO2AGAPNBn as
6.9. In vivo MRI and image analysis
Animal experiments were performed under approval of
the ethics committee of the Center for Microscopy and Molecular
Imaging (CMMI protocol number 2012-06; LA1500589).
Apoptosis was induced in thymus of 4-6 weeks old CD-1 female
mice (Charles River (L’Arbresle, France)) by intraperitoneal
injection of 30mg/kg freshly prepared water-soluble
dexamethasone (DEX, Sigma-Aldrich, Belgium, 25mg/ml in
PBS [Phosphate buffered saline] solution). For MRI
observations, mice were anesthetized with isoflurane vaporized at
2-2.5% in O2 à 2 l/min and placed in a warmed bed into the MRI
scanner. Mice respiration was monitored during all experiment
and anesthesia was adapted to 1.5-2% of isoflurane with O2 flow
of 0.4 l/min to maintain 20 respirations per minute. MRI was
performed 16-18h after dexamethasone injection, on a Bruker
Biospec 9.4T (Bruker, Karlsruhe, Germany). MR signal was
detected using a volume coil (40 mm diameter) adapted for
mouse body and connected to the animal bed. Images were
acquired and visualized with the Paravision software (Bruker).
The sequence used for this study was a T1 spin-echo sequence
(RARE): TR : 389.1 ms ; TE : 8.9 ms ; NEX (number of
an
off-white
powder
(20
mg,
72%).
ESI-MS
(C25H33TbN5O12P2Na); m/z = 832.2
6.6. Conjugation of Peptide (E3) with Gd–DO2AGAPNBn (11)
0.2g (0.25 mmol) of 8 was dissolved in 5 mL of DMF
to which 0.21 g (0.28 mmol) of peptide-E3, 89 mg (0.32 mmol)
of DEPBT and 0.1 mL (0.752 mmol) of DIPEA were added. The
reaction mixture was stirred for 4h and diluted with 20 mL of
water; a white precipitate was isolated by filtration and washed
with ethyl acetate (50 mL). The solid was dried under reduced
pressure and purified by semi-preparative HPLC using method A
and obtained after 16 min of retention time. The compound was
freeze-dried, and 11 was obtained as a white solid (0.27 g, 70%).
HRMS (ESI): calcd for C58H93GdN12O24P; [M+H]+ 1528.5398;
found 1528.5422.

averages): 16; acquisition time : 14 minutes 56 seconds; matrix:
344 x 192; slice thickness: 1 mm; FOV : 3.2 x 1.8 cm ; spatial
resolution: 93 x 94 microns; fat saturation. Images were acquired
on mice, treated with dexamethasone (developing apoptosis in
the cortical region of thymus) and on control mice, up to 2 hours
(9 time points) after receiving an intravenous injection of Gd-
DO2AGAPNBn-E3pep or Gd-DO2AGAPNBn (200 micromoles
Gd/kg). Image analysis was performed with the ImageJ software
by measurement of a signal intensity enhancement ratio in
thymus (cortex versus medulla), expressed as a percentage of
change relative to the pre-contrast situation (considered as 100
%). Statistical significance of differences between percentages of
change were tested in Microsoft Excel using Student’s t-test.
3
4
Frullano, L.; Meade, T.; J. Biol. Inorg. Chem., 2007, 12,
939-949;
a) Que, E. L.; and Chang, C. J.; Chem. Soc. Rev. 2010, 39,
51-60; b) Caravan, P.; Chem. Soc. Rev. 2006, 35, 512–523;
c) Hermann, P.; Kotek, J., Kubicek, V.; Lukes, I.; Dalton.
Trans., 2008, 3027–3047
5
6
a) Tóth, É.; Pubanz, D.; Vauthey, S.; Helm, L.; Merbach,
A. E.; Chem. Eur. J., 1996, 2, 1607-1615; b) Caravan, P.;
Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. Chem. Rev.,
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Powell, D. H.; Dhubhghaill,O. M. N.; Pubanz, D. Helm,
L.; Lebedev, Y. S.; Schlaepfer, W.; Merbach, A.; J. Am.
Chem. Soc., 1996, 39, 9333–9346.
6.10. Immunohistochemistry
Mice were sacrificed in accordance with the ethics committee of
CMMI (protocol 2012-06), 18h after intraperitoneal injection of
DEX (30 mg/kg). Thymus was collected and fixed in 4%
formalin during 24h. After PBS washing, tissue was dehydrated
and embedded in paraffin using a Tissue-Tek VIP machine
(Sakura Finetek, Japan). Thymus sections of 5-micron thickness
were cut using a microtome and were automatically processed by
7
8
Chilla, S. N. M.; Laurent, S.; Vander Elst, L.; Muller., R.
N. Tetrahedron, 2014, 70, 5450-5454.
Aime,S.; Botta, M.; Fasano, M.; Paula, M.; Marques, M.;
Geraldes, C. F. G. C.; Pubanz, D.; Merbach, A. E.; Inorg.
Chem., 1997, 36, 2059-2068.
9
a) Dunand, F. A.; Aime, S.; Merbach, A. E.; J. Am. Chem.
Soc., 2000, 122, 1506-1512; b) Woods, M.; Aime, S.;.
Botta, M.; Howard, J. A. K.; Moloney, J. M.; Navet, M.;
Parker, D.; Port, M.; Rousseaux, O.; J. Am. Chem. Soc.,
2000, 122, 9781-9792; c) Woods, M.; Kovacs, Z.; Zhang,
S.; Sherry, A. D.; Angew. Chem. Int. Ed., 2003, 42, 5889 –
5892.
a
Discovery XT machine (Ventana/Roche, Belgium) for
immunohistochemistry of activated (cleaved) caspase-3 enzyme.
Sections were incubated at 37°C for 1h with anti-cleaved
caspase-3 (Asp175) primary antibody (#9661, Cell Signaling,
Netherlands), at dilution 1:100 in Ventana/Roche buffer, and for
24 minutes with secondary antibody (1 :200; biotinylated anti-
rabbit IgG made in goat (#BA-1000, Vector laboratories,
LabConsult, Belgium)). Areas of caspase-3 activation were
revealed by a streptavidin-biotin peroxidase detection system
(Discovery DAB Map (#760-124, Ventana/Roche, Belgique))
allowing for diaminobenzidine precipitation at locations of
antibody binding (brownish coloration) on thymic tissue. After
nuclei coloration with hematoxylin, sections were dehydrated and
mounted for light microscope observation.
10
11
a) Frullano L.; and Meade, T.; J. Biol. Inorg. Chem., 2007,
12, 939-949; b) Bonnet, C. S.; and Toth, E.; C. R. Chim.,
2010, 13, 700–714.
Laumonier, C.; Segers, J.; Laurent, S.; Michel, A.; Coppée,
F.; Belayew, A.; Vander Elst, L.; Muller, R.N.; J. Biomol.
Screen., 2006, 11, 537-545.
Acknowledgements
12
13
Elmore, S.; Toxicol Pathol. 2007, 35, 495-516.
The authors thank to the ARC Programs of the French
Community of Belgium, UIAP VII program of Belspo, the
Région Wallone (Gadolymph project n° 1317980 through
the EuroNanoMed 2013 framework), Holocancer and
a) Brooks, K. J.; Bunce, K.T.; Haase, M.V.; White, A.;
Kumar Changani, K.; Bate, S.T.; Reid, D.G.; Steroids.
2005, 70, 267–272. b) Herold, M. J.; McPherson, K.G.;
Reichardt, H.M.; Cell. Mol. Life Sci. 2006, 63, 60-72.
Interreg programs. The support and sponsorship concerted
14
a) Rudovsky, J.; Cigler, P.; Kotek, J.; Hermann, P.;
Vojtsek, P., Lukes, I.; Peters, J. A.; Vander Elst, L.; Muller,
R. N.; Chem. Eur. J., 2005, 11, 2373 – 2384; b) Rudovsky,
J.; Kotek, J.; Hermann, P.; Lukes, I.; Mainero V.; and
Aime., S.; Org. Biomol. Chem., 2005, 3, 112-117;
by COST Actions TD1004 and EMIL program are
acknowledged. The support from the Grant Agency of the
Czech Republic (Project No. 16-03156S) is acknowledged.
The authors thank the Center for Microscopy and Molecular
Imaging (CMMI, supported by the Wallonia Region). The
DIAPATH (Digital Image Analysis in Pathology) research
unit of CMMI is thanked for immunohistochemical tissue
processing.
15
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