Polyhydroxylated GdDTPA-derivatives as high relaxivity magnetic resonance imaging contrast agents

Article abstract of DOI:10.1039/c5ra15071j

The search for new MRI agents endowed with high relaxivity at the magnetic field strength of clinical scanners (1.5-3 T) is still receiving great attention from researchers involved in the development of new probes. Such Gd(iii) complexes should combine a

Full text of DOI:10.1039/c5ra15071j

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Polyhydroxylated GdDTPA-derivatives as high
relaxivity magnetic resonance imaging contrast
agents†
Cite this: RSC Adv., 2015, 5, 74734
a
b
c
c
c
*
*
Lorenzo Tei, Alessandro Barge, Matteo Galli, Roberta Pinalli, Luciano Lattuada,
Eliana Gianoliod and Silvio Aimed
The search for new MRI agents endowed with high relaxivity at the magnetic field strength of clinical
scanners (1.5–3 T) is still receiving great attention from researchers involved in the development of new
probes. Such Gd(III) complexes should combine a fast inner-sphere water exchange rate (kex) with an
enhanced contribution from water molecules in the second and outer coordination spheres. In the
present work, dendrimeric-like structures were synthesized by coupling diethylenetriaminepentaacetic
acid (DTPA) and its mono-methylphosphonic derivative (P-DTPA) with two differently branched, highly
hydrophilic, gluconyl moieties. A 1H and 17O NMR relaxometric study on the corresponding Gd(III)
complexes reveals that the Gd-P-DTPA-polyol complex displays very high relaxivities (around
20 mMꢀ1 sꢀ1 at 298 K) over the 0.5–3 T range of field strengths as a result of a fast kex and the presence
of a strong second sphere contribution.
Received 29th July 2015
Accepted 28th August 2015
DOI: 10.1039/c5ra15071j
www.rsc.org/advances
relaxivity over an extended range of magnetic eld strength.5
High-relaxivity CAs permit the use of lower doses in routine
Introduction
clinical applications and are the candidates of choice in
molecular imaging procedures aimed at detecting low-
concentration targets. Optimizing the three contributions that
determine the observed relaxation rates of water protons,
namely inner-, second- and outer-coordination sphere, could
lead to highly efficient CAs.
The use of Gd(III)-chelates as contrast enhancing agents (CA) for
Magnetic Resonance Imaging (MRI) has developed consider-
able research activity aimed at the design of effective, specic
and safe CAs.1 The efficacy of a contrast agent is expressed by its
relaxivity, r1, which is the enhancement of the longitudinal
proton relaxation rate induced by the paramagnetic agent at
1 mM concentration. Theory predicts high relaxivity for a Gd(III)
complex when the rate of water exchange between the inner
sphere and the bulk solvent (kex ¼ 1/sM) is around 106–107 sꢀ1
and when the molecular reorientational time and electron spin
relaxation time are similar and long at the selected eld
strength.1,2 Nowadays, clinical MR imaging is increasingly
moving to higher elds since the number of installed 3 Tesla
scanners is steadily growing worldwide.3 Higher elds improve
the signal-to-noise ratio (SNR) and provide higher spatial reso-
lution and/or reduced acquisition times.4 Considering this
trend, there is the need of contrast agents endowed with a high
One route followed in the search for higher relaxivities relied
on the lengthening of the molecular reorientational time
through the formation of polymers or of covalent and non-
covalent conjugates between the paramagnetic chelate and
slowly moving substrates6 (dendrimers,7 proteins,8 carbohy-
drates9) and the formation of supramolecular adducts like
micelles or liposomes.10 In most cases, these systems display
maximum r1 values at 20–40 MHz (i.e. 0.5–1 T) but their relax-
ation enhancement ability decreases quickly at higher elds.
Therefore, medium size molecular weight Gd-based
compounds have been proposed in order to obtain good relax-
ivity values over the 0.5–3 T magnetic eld range. Examples of
these systems are based either on oligomeric (2–8 units) Gd(III)
complexes4,11 or on monomeric Gd-complexes with the Gd3+ ion
at the baricentre of the macromolecule with a well-structured
second sphere of hydration.12 Similar symmetric structures
based on DOTA (DOTA ¼ 1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid) but with higher molecular weights
(5–7 kDa) were reported to have a relaxivity drop at magnetic
elds higher than 1 T (40 MHz).13 Moreover, their dimensions
allow only a slow diffusion in the extravascular space.
a
`
Dipartimento di Scienze e Innovazione Tecnologica, Universita del Piemonte Orientale
“Amedeo Avogadro”, Viale T. Michel 11, 15121 Alessandria, Italy. E-mail: lorenzo.tei@
uniupo.it
b
`
Dipartimento di Scienza e Tecnologia del Farmaco, Universita di Torino, via P. Giuria
9, 10125 Torino, Italy
cBracco Imaging SpA, Bracco Research Centre, via Ribes 5, 10010 Colleretto Giacosa,
TO, Italy. E-mail: luciano.lattuada@bracco.com
dDepartment of Molecular Biotechnology and Health Sciences, Molecular Imaging
`
Center, Universita di Torino, Via Nizza 52, 10126, Torino, Italy
† Electronic supplementary information (ESI) available: 1H and 13C NMR spectra
of novel compounds. See DOI: 10.1039/c5ra15071j
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Due to their pharmacokinetic prole, the diagnostic appli- incorporate glucose and galactose/mannose moieties were
cations of this type of contrast agents are different not only from developed with the intent to enhance targeting in vivo.16 Others
the already marketed non-specic agents that freely and quickly groups engineered complexes in which the Gd(III) ion lied at the
distribute into the extracellular space, but also from blood pool barycentre of the macromolecular structure surrounded by a
agents that mainly distribute into the blood system. In light of well-dened second hydration sphere to obtain higher relaxiv-
these considerations, there still is the need for improved non- ities.12,13 Furthermore, a water-solubilizing polyhydroxylic den-
specic contrast agents able to combine high relaxivity with dron was incorporated to improve the poor water solubility of the
rapid extravasation, as required for angiographic- and parent complex.17 Our work started with the synthesis of two
perfusion-based diagnostic applications.
diamides of DTPA (L1 and L2) bearing differently branched
We have faced this problem by synthesizing new Gd(III) polygluconyl groups on the outer surface. The assessment of the
complexes based on the structure of DTPA functionalized with relaxometric properties of their Gd-complexes allowed us to
highly hydrophilic substituents in order to remarkably increase determine the best hydrophilic pendant group for the attain-
the second sphere contribution to the relaxivity by creating a ment of a large second sphere contribution to relaxivity. Two
structured network of water molecules localised around the examples of DTPA-bisamides bearing two acetylglucose units
polyhydroxyl groups. Ligands L1 and L2 are DTPA-bisamides and a dendrimeric glycosidic cluster with six acetylgluconamides
bearing two differently branched gluconyl moieties and L3 is at the periphery were already reported with the aim to create new
based on a DTPA derivative in which the central arm has been blood pool CAs more than to evaluate the effect of the second
replaced by a methyl phosphonate moiety (P-DTPA) and two sphere water molecules surrounding the Gd(III) centre.18 As we
outer acetate moieties bear each the triply gluconyl substituted were aware that DTPA-bisamides are characterized by a very slow
polyol. The choice of a P-DTPA derivative relies on the published water exchange rate (kex) that “quenches” any putative inner
observation that its Gd(III) complex shows an optimal value of sphere relaxivity gain, our next task dealt with the introduction
sM (about 88 ns).14 Moreover, phosphonate moieties have been of two polyhydroxy-containing moieties on P-DTPA ligand.
shown to yield good second sphere contributions to the
Bifunctional derivatives of DTPA have been reported either
observed relaxivity due to hydrogen bonded water molecules with the remote functional group on the central acetate arm or
localised between the phosphonate group and the Gd.15 In the on the diethylenetriamine backbone.19 Only one example of
present work, we report on the synthesis of L1–L3 ligands and
on a 1H and 17O NMR relaxometric study on the corresponding
Gd(III) complexes.
Results and discussion
Ligands design
The introduction of polyhydroxylic or glycosylic groups at the
periphery of a Gd(III) complex has been considered previously
with different purposes. In some reports, CAs structures that
Scheme 1 Synthesis of the DTPA-bisamide L1.
Scheme 2 Synthesis of the DTPA-bisamide L2.
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route, aer orthogonal deprotection of two carboxy benzylester
groups, we were able to attach an amino-polyol molecule on
both sides of the P-DTPA derivative. In particular, L-aspartic acid
4-benzyl ester 10 was esteried, using t-butyl acetate in the
presence of catalytic perchloric acid, into L-aspartic acid 1-tert-
butyl 4-benzyl diester 11, which was directly reacted with one
equivalent of t-butyl bromoacetate under Rapoport's two-phase
conditions (i.e.: MeCN/pH 8 phosphate buffer) to yield, aer
chromatographic purication, the mono-alkylated compound
12 in 76.5% yield. Triester 12 was then further alkylated with
2-bromoethyl-triuoromethansulfonate in order to obtain in
good yield the bromoethyl component 13 to be used for the
construction of the P-DTPA derivative (Scheme 4).
Scheme 3 Synthesis of the fully acetylated aminopolyol 9; (i) Boc2O,
TMAOH, DMF, 48h; (ii) Ac2O, Py, 90 ꢁC, 24 h.
The other component was the aminomethylphosphonic acid
di-tert-butyl ester 19 (Scheme 5) obtained in two steps by Man-
nich reaction of dibenzylamine with paraformaldehyde and tert-
butyl phosphite, followed by hydrogenolysis of the benzyl group
at atmospheric pressure with 10% Pd/C catalyst. The Rapoport's
conditions were also used for the alkylation of 19 with two
equivalents of 13 (no excess was used) to get the P-DTPA deriva-
tive 14 in 42.4% yield aer column purication. Cleavage of the
benzyl ester protections of 14 by hydrogenolysis yielded the
diacid pentaester 15. This compound was then coupled to the
amino-protected polyol 9 in DMF at room temperature for 48 h by
using HATU (O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyl-
uroniumhexauoro phosphate) as coupling reagent and
functionalization of
a
lateral acetate pendant arm was
bis-
described20 and to the best of our knowledge
a
functionalized DTPA-like agent has never been reported.
Thus, the bifunctional chelating ligand 15 (Scheme 4) was
designed having four carboxylic and the phosphonic groups
tert-butyl protected and two free carboxylic groups able to react
with amino functionalized moieties. Then, the ligand L3 could
be prepared by reacting the bifunctional agent 15 with two
dendronized polygluconyl groups with the aim to increase the
amount of hydroxyl groups and to create a symmetric Gd(III)
complex with lateral hydrophilic moieties and a central phos-
phonate group.
Synthesis
The polyol functionalised DTPA bisamide L1 (Scheme 1) was
synthesized in one step with an overall 61% yield starting from
DTPA bis-anhydride 4 21 and N,N0-(iminodi-2,1-ethanediyl)bis-D-
gluconamide 3, previously obtained by amidation of diethyl-
enetriamine 1 with d-gluconolactone 2 (Scheme 1). Similarly,
the DTPA bisamide L2 was synthesised by reaction of DTPA bis-
anhydride 4 with the tris-gluconoyl amide derivative 6 obtained
by reaction between pentaerythrityl tetramine 5 22 and d-gluco-
nolactone (Scheme 2). Aiming to use this amino-functionalised
polyol for further syntheses, we decided to prepare the fully
acetylated derivative 9 (Scheme 3). Thus, the amino group of 6
was protected with (Boc)2O and tetramethylammonium
hydroxide in DMF to give 7. Then, all the hydroxyl groups were
acetylated with Ac2O in pyridine to give 8 and, nally, 9 was
obtained by removing the Boc protection with TFA in CH2Cl2.
The synthetic procedure to obtain L3, depicted in Scheme 4,
relies on the strategy published by Williams and Rapoport who
reported the N-bisalkylation of p-nitrophenylalanine benzyl
ester with different bromoethylamines as a direct method for
the construction of protected DTPA analogues.23 Moreover,
following this procedure, modied amino acids have been
bisalkylated with [N-(bromoethyl)amino]diacetic acid t-butyl
ester in order to obtain functionalized DTPA pentaesters as
versatile intermediates for the preparation of conjugates of
metal complexes.19,24 We exploited this idea in a different way by
synthesising a bromoethyl derivative of protected aspartic acid
and reacting it with an aminomethyl phosphonate. By this Scheme 4 Synthesis of L3.
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free Gd3+ ion was quantied by Orange Xylenol UV method as
lower than 0.2% in all complexes.25 The exact concentration of
Gd3+ ions was determined by measurement of the bulk
magnetic-susceptibility shis of the tBuOH signal. The relax-
ivity (r1) values at 20 MHz and 298 K are 5.0 mMꢀ1 sꢀ1 for GdL1,
4.3 mMꢀ1 sꢀ1 for GdL2 and 19.2 mMꢀ1 sꢀ1 for GdL3. In Fig. 1,
the relaxivities of the three Gd-chelates are plotted as a function
of their molecular weight and compared to those reported in the
literature for monomeric Gd-complexes with one coordinated
water molecule at T ¼ 298 K and B0 ¼ 0.5 T. While the r1 values
for the two GdDTPA-bisamides GdL1 and GdL2 are signicantly
lower than the values expected on the basis of their molecular
weight, the r1 of the Gd(III) complex with the monophosphonic
DTPA derivative L3, is markedly higher than expected for a
molecule of such molecular weight. Noteworthy, the relaxivity of
GdL3 is more than four times higher than that of the parent
GdDTPA (r1 ¼ 4.7 mMꢀ1 sꢀ1). A more detailed relaxometric
characterization was carried out through the registration of the
17O NMR-R2p proles as a function of temperature (for an
accurate determination of the exchange lifetime (sM) of the
coordinated water molecule – Fig. 2) and of the 1H-NMRD
proles over the range of frequencies from 0.01 to 120 MHz
(Fig. 3A and B).
Scheme 5 Synthesis of aminomethylphosphonic acid di-tert-butyl
ester 19.
N,N-diisopropyl ethylamine as base. Product 16, together with a
small amount of the mono-substituted derivative, was collected
by precipitation in cold water. Due to difficulties met during the
purication procedures, it was decided to use this mixture
without separating the mono-functionalized impurity. De-
acetylation of the polyalcohol was afforded by bubbling
ammonia in a refrigerated methanol solution of 16. This reaction
was completed only aer 9 days and aer bubbling more
ammonia every two days. In order to obtain the nal ligand L3,
the last step was the cleavage of the t-butyl esters by reaction with
a 50% solution of triuoroacetic acid in CH2Cl2 and precipitation
by addition of diethyl ether. In order to get pure L3 a simple
procedure was followed: the triuoroacetate salt obtained aer t-
butyl ester deprotection was redissolved in water, brought to pH
7, lyophilized and recovered with methanol. Ligand L3 was thus
obtained as a white solid in 30% yield by ltration of the MeOH
solution. On the other hand, evaporation of the solvent give rise
to an oil which resulted to be the mono-polyol derivative present
in about 28% of the total raw material.
The analysis of the 17O NMR experimental data yielded the
sM values reported in Table 1. The value found for GdL3 (93 ns)
is very similar to that already reported for related GdP-DTPA
derivatives14 and very close to the optimal value for the
achievement of high relaxivity.1,2 On the contrary, very long sM
values (5.6 ms and 7.6 ms, for GdL1 and GdL2 respectively) were
Relaxivity of the Gd(III) complexes
The Gd(III) complexes GdL1, GdL2 and GdL3 were prepared by found for the two bisamides derivatives. This slow water
1H NMR relaxometric titration with a stock solution of GdCl3 at exchange rate, which is invariably found in GdDTPA-bisa-
pH 6.5 monitoring the change in the longitudinal water proton mides,26 is responsible for the low relaxivity shown by GdL1 and
relaxation rate (R1) at 20 MHz and 298 K as a function of the GdL2. The proton relaxivity data measured as a function of the
concentration of Gd3+. The slope of the straight line obtained magnetic eld strength at 298 K and neutral pH (Fig. 3A and B)
corresponds to the relaxivity (r1) of the complex. The residual were well interpolated with the set of values calculated on the
basis of the classical Solomon-Bloembergen-Morgan theory by
xing the sM values to those obtained by 17O NMR-R2p analysis.
The main parameters obtained from the tting of experimental
to calculated data are reported in Table 1. As expected, a
Fig. 1 Relaxivities (r1) values, measured at 298 K and 20 MHz, of GdL1,
GdL2 and GdL3 as a function of their molecular weight compared to
those reported for other q ¼ 1 complexes. Values for Gd-DOTA,
Gd-DTPA and Gd-HPDO3A come from ref. 2, for Gd-4 and Gd-5 from Fig. 2 Temperature dependence of the paramagnetic contribution to
ref. 12a, for Gd-6b, 6c, 6d and 6e from ref. 12b and for P760 and P792 the 17O transverse relaxation rate of water for GdL1, GdL2 and GdL3
from ref. 13.
(10 mM) measured at 2.1 T and pH 7.
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Fig. 3 1/T1 NMRD profiles of GdL1, GdL2 (A) and GdL3 (B) recorded at 298 K and pH 7. The data refer to a 1 mM concentration of the para-
magnetic complexes. The solid curves through the data points were calculated with the parameters reported in Table 1, whereas the dotted lines
in (B) refer to the different contributions to the overall relaxivity.
Table 1 Best-fit parameters obtained from 1H-NMRD at 298 K and 17O-R2p versus T analysis shown in Fig. 2 and 3a
D2 (ꢂ1019 sꢀ2
)
sV (ps)
sR (ps)
sM (ms)
q
qss
rss (A)
s
ss (ps)
˚
Gd-L1
Gd-L2
Gd-L3
2.9 ꢃ 0.57
2.1 ꢃ 0.57
2.2 ꢃ 0.13
22.1 ꢃ 1.8
25.0 ꢃ 1.5
34.2 ꢃ 0.10
305 ꢃ 12.8
470 ꢃ 68.1
560 ꢃ 10.8
5.6 ꢃ 1.2
1
1
1
0
0
—
—
3.7
—
—
7.6 ꢃ 0.6
0.093 ꢃ 0.003
3.5 ꢃ 0.4
93 ꢃ 0.15
a
On carrying out the tting procedure, some parameters were xed to reasonable values: rGd-H (distance between Gd and protons of the innersphere
˚
˚
water molecule) ¼ 3.1 A; a (distance of minimum approach of solvent water molecules to Gd ion) ¼ 4 A; D (solvent diffusion coefficient) ¼ 2.24 ꢂ
10ꢀ5 cm2 sꢀ1. rss (distance between Gd and protons of the second sphere water molecules) ¼ 3.7 A. [D ] Squared mean transient zero-eld splitting
2
˚
(ZFS) energy. [sV] Correlation time for the collision-related modulation of the ZFS Hamiltonian. [sR] Reorientational correlation time. [sM] Exchange
lifetime of the coordinated water molecule. [q] Number of inner-sphere water molecules. [qss] Number of second-sphere water molecules. [sss]
correlation time associated with the motions (reorientation or exchange) of the second-sphere water molecules.
progressive increase in the reorientational correlation time (sR)
occurs upon increasing the molecular weight.
Experimental
General
In the case of GdL3 (Fig. 3B), a good tting of the experi-
mental data was possible only by taking into account a contri-
bution from second sphere water molecules.27 3.5 second
sphere water molecules (qss) were estimated to be located at an
All reagents and solvents were obtained from commercial
suppliers and directly used without further purication. N,N0-Bis-
[2-(2,6-dioxo-4-morpholinyl)ethyl]glycine (DTPA-bis-anhydride)
4,21 pentaerythrityl tetramine tetrahydrochloride 5,22 tert-butyl
phosphite28 and 2-bromoethyl triuoromethanesulfonate29 were
synthesized as reported in literature. TLC was performed on
Merck silica gel 60 TLC plates F254 and visualized by using UV
(254 nm) or 1% KMnO4 in 1 N NaOH. Column chromatography
was performed by using silica gel 60 (70–230 mesh) while ash
chromatography was carried out on silica gel 60 (230–400 mesh).
The 1H, 13C and 31P spectra were recorded on a Bruker Avance 400
instrument. Chemical shis are reported in d relative to an
internal standard of residual chloroform (d 7.27 for 1H NMR and
77.16 for 13C NMR). ESI MS spectra on novel compounds were
acquired on a high resolving power mass spectrometer LTQ
Orbitrap (Thermo Scientic, Rodano, Italy), equipped with an
atmospheric pressure interface and an ESI ion source. Other
mass spectra were recorded with a ThermoFinnigan TSQ700
triple-quadrupole instrument equipped with an electrospray
ionization source. Analytical HPLC was performed on a Merck
KGaA apparatus with the following method: stationary phase:
Lichrospher RP-Select B 5 mm, 250 ꢂ 4 mm column packed by
Merck KGaA; mobile phase: eluent A ¼ 0.01 M KH2PO4 and
0.017 M H3PO4 in H2O, eluent B ¼ MeCN, gradient elution: t ¼
3+
˚
average distance of 3.7 A from the Gd ion with an overall
correlation time (sss) of 93 ps (Table 1). The increase in the
correlation time associated with the collision-related modula-
tion of the zero-eld splitting Hamiltonian (sV) observed in the
case of GdL2 and GdL3, is likely related to the occurrence of a
reduced rate of solvent collisions between the coordinated and
bulk water molecules. Tentatively, one may relate the observed
behavior to the enhanced shielding of the poly-hydroxylic con-
taining moieties with respect to GdL1.
In summary, one may conclude that the high relaxivity
shown by GdL3 is the result of the occurrence of three positive
conditions: (i) an array of second sphere water molecules linked
through hydrogen bond to the phosphonate moiety, (ii) a fast
water exchange rate of the coordinated water molecule and (iii)
the increased molecular weight (sR ¼ 560 ns) determined by the
introduction of the poly-hydroxylic substituents on the surface
of the metal complex. Noteworthy, the relaxivity values for
GdL3, measured at the Larmor frequencies more used on clin-
ical scanners (40, 60, 120 MHz corresponding to 1, 1.5 and 3 T,
respectively), are somehow constant, being 20.8 mMꢀ1 sꢀ1 at
40 and 60 MHz and 19.1 mMꢀ1 sꢀ1 at 120 MHz.
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0 min (5% B), t ¼ 30 min (80%B); T ¼ 45 ꢁC; ow rate: 1 bis-anhydride)21 4 (0.19 g; 0.54 mmol) was added to a solution of
mL minꢀ1; UV detection: 210 nm.
6 (0.8 g; 1.20 mmol) in DMSO (5 mL) and stirred at 50 ꢁC for 8 h.
Synthesis of N,N0-(iminodi-2,1-ethanediyl)bis-D-gluconamide Aer cooling at room temperature, the reaction mixture was
(3). Diethylenetriamine 1 (1 g, 9.7 mmol) was added dropwise to diluted with CH2Cl2 (50 mL); the precipitate was ltered,
a suspension of d-gluconolactone 2 (3.6 g, 20.3 mmol) in DMF washed with CH2Cl2 (200 mL) then lyophilized to remove the
(10 mL). The reaction mixture was stirred at room temperature residual DMSO. L2 was obtained as white solid (0.64 g;
for 1.5 h. The precipitate was ltered, washed with CH2Cl2 0.38 mmol), yield 70%. 1H NMR (300 MHz, D2O) d 4.18 (m, 4H),
(500 mL), to remove DMF and the excess of d-gluconolactone, 3.94 (d, J ¼ 3.2 Hz, 2H), 3.86–3.73 (m, 6H), 3.65–3.54 (m, 8H),
and dried to obtain 3 as a white solid (4.06 g, 8.83 mmol), yield 3.51 (m, 8H), 3.45–3.32 (m, 8H), 3.06–2.77 (m, 6H), 2.51 (s, 18H),
91%; mp 142 ꢁC (dec.); Rf 0.12 (eluent CHCl3/MeOH/25% 2.29 (m, 6H), 0.78 (t, J ¼ 7.2 Hz, 4H). 13C NMR (75 MHz, D2O, pH
NH4OH 30 : 60 : 20); 1H NMR (600 MHz, D2O) d 4.24 (d, J ¼ ¼ 10) d 179.4, 173.7, 168.1, 74.8, 74.7, 73.9, 71.9, 71.4, 70.9, 70.3,
5.0 Hz, 2H), 4.00 (d, J ¼ 5.0 Hz, 2H), 3.72 (d, J ¼ 16.9 Hz, 2H), 63.0, 45.2, 38.5, 9.9. ESI-HRMS (m/z): 1690.6211 (M + H+) (calcd
3.66 (m, 4H), 3.55 (dd, J ¼ 11.0, 4.4 Hz, 3H), 3.31 (dt, J ¼ 12.1, 1690.6865).
9.2 Hz, 4H), 2.71 (t, J ¼ 9.2 Hz, 4H); 13C NMR (151 MHz, D2O) d
Synthesis of (N-[[2,2-bis[D-gluconoylamino)methyl]-3-tert-
175.08 (CO), 73.81 (C-2), 72.50 (C-3), 71.45 (C-4), 70.70 (C-5), butoxycarbonylamino]propyl]-D-gluconamide (7). Compound 6
62.96 (C-6), 47.33 (CH2N), 38.45 (CH2N).
(30.0 g, 45.0 mmol) was dissolved in DMF (400 mL) and a
Synthesis of N,N-bis[2-[(carboxymethyl)[2-[bis[2-[(D-gluconoyl) solution of (Boc)2O (17.2 g; 65.2 mmol) in DMF (100 mL) was
amino]ethyl]amino]-2-oxoethyl]amino] ethyl]glycine (L1). N,N0- slowly added dropwise. Then, tetramethylammonium
Bis-[2-(2,6-dioxo-4-morpholinyl)ethyl]glycine
(DTPA-bis-anhy- hydroxide (8.15 g, 45.0 mmol) was added and the mixture was
dride)21 4 (0.19 g; 0.54 mmol) was added to a solution of 3 (0.5 g; stirred at room temperature for 2 days. The solution was
1.09 mmol) in DMSO (5 mL) and the yellow solution was stirred concentrated at reduced pressure and the residue was treated
at 50 ꢁC for 8 h. Aer cooling at room temperature, the reaction with CH2Cl2 to give a precipitate which was ltered and washed
mixture was diluted with CH2Cl2 (50 mL); the precipitate was with H2O (100 mL) and CH3OH (100 mL). The solid so obtained
ltered, washed with CH2Cl2 (200 mL) then lyophilized to remove was dried to give 7 (25 g, 32.6 mmol), yield: 72%. 1H NMR
the residual DMSO. L1 was obtained as white solid (0.42 g; (600 MHz, D2O) d 4.35 (d, J ¼ 2.7 Hz, 3H), 4.07 (t, J ¼ 3.1 Hz, 3H),
ꢁ
0.33 mmol), yield 61%; mp 128–130 C Rf 0.30 (eluent MeOH/ 3.75 (m, 6H), 3.69 (m, 3H), 3.61 (dd, J ¼ 11.8, 6.4 Hz, 3H), 3.12 (s,
25% NH4OH 7 : 3); HPLC: retention time 2.1 min, 98% (area%); 15H), 3.02 (m, 6H), 2.85 (m, 2H), 1.41 (s, 9H); 13C NMR
1H NMR (600 MHz, D2O, 333 K) d 4.70–4.58 (m, 10H), 4.37 (s, 4H), (151 MHz, D2O) d 175.79 (CO), 158.81 (CO), 81.88 (C), 74.06
4.18 (s, 4H), 4.12–4.05 (m, 12H), 3.98–3.85 (m, 12H), 3.78 (m, (C-2), 72.94 (C-3), 71.52 (C-4), 70.69 (C-5), 62.97 (C-6), 55.67,
12H), 3.49 (m, 4H); 13C NMR (151 MHz, D2O, 333 K) d 175.68, 45.52 (C), 39.94 (CH2N), 38.72 (CH2N), 28.07 (CH3). ESI-HRMS
175.36, 174.90, 171.11, 167.40 (CO), 74.21 (C-2), 74.15 (C-20), 73.97 (m/z): 789.3003 (M + Na+) (calcd 789.3229).
(C-3), 73.07 (C-30), 72.72 (C-4), 72.19 (C-40), 71.30 (C-5), 62.56 (C-6),
58.16, 56.80, 56.02 (CH2CO), 53.92, 50.42 (CH2N), 47.41, 46.55 lamino)methyl]-3-tert-butoxycarbonylamino]propyl]-2,3,4,5,6-
(CH2N), 37.49, 36.50 (CH2NH). ESI-HRMS (m/z): 1276.4563 penta-O-acetyl-D-gluconamide (8). To suspension of
(M + H+) (calcd 1276.5379).
compound 7 (10.0 g; 13.0 mmol) in Ac2O (186 mL; 1.97 mol),
Synthesis of (N-[[2,2-bis[2,3,4,5,6-penta-O-acetyl-D-gluconoy-
a
Synthesis of N-[[2,2-bis[(D-gluconoylamino)methyl]-3-amino] pyridine (25.7 g; 326 mmol) was added at room temperature.
ꢁ
propyl]-D-gluconamide (6). Pentaerythrityl tetramine tetrahy- The mixture was then heated to 90 C to dissolve the reagents,
drochloride22 5 (14 g; 0.051 mol) was suspended in a mixture of then cooled at room temperature. Aer 1 day at room temper-
DMF (170 mL) and Et3N (25.4 mL; 0.184 mmol). A solution of ature, the mixture was concentrated at reduce pressure and the
d-gluconolactone 2 (32.7 g; 0.184 mol) in DMF (195 mL) was residue was dissolved in EtOAc (200 mL) and washed with water
slowly added dropwise (1.5 h) at room temperature. The mixture (200 mL) and with 10% aqueous NaHCO3 (200 mL). The organic
was stirred at room temperature for 4 days. The reaction phase was dried (Na2SO4) and evaporated to give 8 (15 g;
1
mixture was ltered and concentrated at reduce pressure. The 10.7 mmol), yield: 82%, H NMR (600 MHz, CDCl3) d 8.21 (s,
residue was treated with MeCN to give a precipitate which was 1H), 7.81 (s, 2H), 6.00 (s, 1H), 5.65 (m, 3H), 5.45 (m, 3H), 5.26
ltered and washed with MeCN. The solid was dried to give 6 as (m, 3H), 5.07 (m, 3H), 4.31 (m, 3H), 4.17 (br m, 3H), 3.37 (m,
a white solid and as hydrochloride salt (30.5 g; 0.046 mmol), 4H), 3.22 (m, 2H), 3.04 (m, 2H), 2.29, 2.10, 2.06 (m, 45H), 1.42 (s,
1
yield 90%; Rf 0.44 (eluent MeOH/25% NH4OH 7 : 3); H NMR 9H). 13C NMR (151 MHz, CDCl3) d 170.84 (CO), 170.11 (CO),
(600 MHz, D2O) d 4.35 (m, 3H), 4.06 (dt, J ¼ 9.5, 3.2 Hz, 3H), 3.75 170.04 (CO), 158.25 (CO), 80.26 (C), 71.50 (C-2), 70.43 (C-3),
(m, 6H), 3.69 (m, 3H), 3.60 (dd, J ¼ 11.8, 6.3 Hz, 3H), 3.21–3.10 70.28 (C-4), 69.23 (C-5), 61.68 (C-6), 39.65, 28.66 (CH3), 21.09
(m, 6H), 3.06–2.96 (m, 2H); 13C NMR (151 MHz, D2O) d 176.55 (CH3), 21.04 (CH3), 20.92 (CH3), 20.64 (CH3); ESI-HRMS (m/z):
(CO), 175.99 (CO), 74.04 (C-2), 73.91 (C-20), 72.95 (C-3), 72.74 1419.4123 (M + Na+) (calcd 1419.4814).
(C-30), 71.52 (C-4), 70.83 (C-5), 70.68 (C-50), 62.96 (C-6), 62.93
(C-60), 44.86 (C), 38.59 (CH2N), 38.47 (CH2N). ESI-HRMS (m/z): lamino)methyl]-3-amino]propyl]-2,3,4,5,6-penta-O-acetyl-D-glu-
667.2175 (M + H+) (calcd 667.2880).
conamide (9). Triuoroacetic acid (3.67 g; 32.0 mmol) was
Synthesis of (N-[[2,2-bis[2,3,4,5,6-penta-O-acetyl-D-gluconoy-
Synthesis of N,N-bis[[[2,2-bis[(D-gluconoylamino)methyl]-3- added to a solution of compound 8 (4.50 g; 3.20 mmol) in
amino]propyl]-D-gluconamide]-2-oxoethyl]amino] ethyl]glycine CH2Cl2 (60 mL). Aer 3 days at room temperature, further TFA
(L2). N,N0-Bis-[2-(2,6-dioxo-4-morpholinyl)ethyl]glycine (DTPA- (1.82 g; 16.0 mmol) was added to the reaction mixture. Aer
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further 4 days at room temperature, the solvents were removed with 5% aq. NaHCO3 (2 ꢂ 250 mL) and H2O (2 ꢂ 200 mL); the
under reduced pressure and the residue was dissolved in new aqueous phases were collected and extracted with EtOAc
CH2Cl2 (7 mL) and TFA (5.49 g; 48.0 mmol). The mixture was (3 ꢂ 100 mL). All organic phases were collected, dried over
stirred at room temperature for 1 day, then evaporated under Na2SO4 and evaporated to obtain 11 (27 g; 0.097 mol) as a pale
reduced pressure. The residue was dissolved in CH2Cl2 (10 mL) yellow oil. Yield 86.6%. TLC (silica gel; eluent: EtOAC; Rf 0,57).
and evaporated several times affording 9 as a white solid and as 1H-NMR (CDCl3) 400 MHz d ¼ 7.31 (m, 5H, Har), 5.14 (s, 2H,
triuoroacetic salt (3.00 g; 2.13 mmol), yield: 67%; Rf 0.59 CH2Ph), 3.68 (t, 1H, J ¼ 6.3 Hz, CHNH2), 2.77 and 2.66 (dd, 2H,
(eluent EtOAc/n-hexane 8 : 2); 1H NMR (400 MHz, CDCl3) d 8.27 J1 ¼ 16.3 Hz, J2 ¼ 4.9 Hz, CH2CO2Bn), 1.76 (bs, 2H, NH2), 1.40 (s,
(s, 1H), 7.78 (s, 2H), 7.34 (s, 2H), 5.60 (m, 3H), 5.46 (m, 3H), 5.24 9H, CCH3). 13C-NMR (CDCl3) 100 MHz d ¼ 173.7 (CO2Bn), 171.5
(d, J ¼ 3.3 Hz, 3H), 5.09 (td, J ¼ 6.0, 3.2 Hz, 3H), 4.34 (m, 3H), (CO2tBu), 136.1 (Car), 128.7 (CHar), 81.8 (CCH3), 66.8 (CH2Ph),
4.16 (dd, J ¼ 12.4, 5.8 Hz, 3H), 3.02 (m, 6H), 2.70 (s, 2H), 2.24, 52.2 (CHNH2), 39.6 (CH2CO2Bn), 28.3 (CCH3). MS (ESI+; MeOH):
2.13, 2.09, 2.07 (s, 45H). 13C NMR (101 MHz, CDCl3) d 170.99 m/z 280.2 (M + H+); 302.2 (M + Na+).
(CO), 170.61 (CO), 170.47 (CO), 72.46 (C-2), 70.07 (C-3), 69.37
Synthesis of N-2-(t-butoxycarbonylmethyl)-L-aspartic acid,
(C-4), 69.07 (C-5), 62.00 (C-6), 45.20 (C), 38.96 (CH2N), 38.49 1-tert-butyl-4-benzylester (12). A mixture of L-aspartic acid, 1-t-
(CH2N), 21.01 (CH3), 20.96 (CH3), 20.72 (CH3), 20.59 (CH3); butyl-4-benzylester 11 (27 g; 0.0967 mol), t-butyl bromoacetate
ESI-HRMS (m/z): 1297.3996 (M + H+) (calcd 1297.4470).
(20.00 g; 0.102 mol), acetonitrile (160 mL) and 2 M phosphate
Synthesis of (dibenzylamino)methylphosphonic acid di-tert- buffer pH 8 (80 mL) was vigorously stirred at room temperature;
butyl ester (18). Paraformaldehyde (2.14 g; 71.2 mmol) was aer 18 h the phases was separated and the organic phase was
added to a solution of dibenzylamine (12.7 g; 64.5 mmol) in evaporated. The residue thus obtained was dissolved in EtOAc
ꢁ
MeCN (130 mL). The suspension was heated to 80 C for 1 h, (300 mL) and washed with H2O (2 ꢂ 150 mL) and brine (2 ꢂ
then the resulting solution was cooled to rt. A solution of tert- 150 mL). Aer drying over Na2SO4, the organic solution was
butyl phosphite (71.2% of purity, 25 g; 71.2 mmol) in MeCN was evaporated to give a crude (34 g) that was puried by ash-
added dropwise over 25 min to the reaction mixture and the chromatography [silica gel; eluent: 4 : 1 n-hexane/EtOAc; Rf
solution was stirred at room temperature for 22 h. The solvent 0,34] to obtain 29.1 g of 1. (Yield: 76.5%) 1H-NMR (CDCl3)
was evaporated under vacuum and 0.1 N HCl (320 mL) was 400 MHz d ¼ 7.32 (m, 5H, Har), 5.14 (s, 2H, CH2Ph), 3.59 (t, 1H,
added to the residue. The suspension was extracted with CH2Cl2 J ¼ 6.3 Hz, CHNH), 3.36 (dd, 2H, J1 ¼ 17.0 Hz, CH2CO2tBu), 2.78
(3 ꢂ 150 mL) and the combined organic phases washed with and 2.71 (dd, 2H, J1 ¼ 16.0 Hz, J2 ¼ 6.4 Hz, CH2CO2Bn), 1.48 and
water (3 ꢂ 150 ml), dried (Na2SO4) and evaporated in vacuo. The 1.44 (s, 18H, CCH3). 13C-NMR (CDCl3) 100 MHz d ¼ 172.3
crude (27.3 g) was puried by chromatography on silica gel (CO2Bn), 171.3, 171.1 (CO2tBu, 2C), 136.1 (Car), 128.9, 128.6
(15 : 85 EtOAc/petroleum ether; Rf 0.2) and the desired product (CHar), 82.1, 81.6 (CCH3), 66.9 (CH2Ph), 58.0 (CHNH), 50.3
was obtained (12.4 g, 30.8 mmol) as a colourless oil. Yield: (CH2CO2tBu), 38.5 (CH2CO2Bn), 28.6, 28.3 (CCH3). ESI-HRMS
1
47.7%. H-NMR (CDCl3) 400 MHz d ¼ 7.43 (d, 4H, J ¼ 7.2 Hz, (m/z): 394.2167 (M + H+) (calcd 394.2230).
Har), 7.32 (t, 4H, J ¼ 7.2 Hz, Har), 7.24 (m, 2H, Har), 3.94 (s, 4H,
Synthesis of N-2-bromoethyl-N-2-(t-butoxycarbonylmethyl)-L-
CH2Ph), 2.79 (d, 2H, J ¼ 10.2 Hz, CH2P), 1.49 (s, 18H, CCH3). aspartic acid, 1-tert-butyl-4-benzylester (13). 2-Bromoethyl tri-
13C-NMR (CDCl3) 100 MHz d ¼ 139.6 (Car), 129.6–128.6–127.3 uoromethanesulfonate29 (34.93 g; 0.136 mol, 1.7 eq.) was
(CHar), 82.4 (CCH3), 59.5 (CH2Ph), 52.5 (CH2P, J ¼ 163 Hz), 30.9 slowly dropped into a solution of compound 12 (31.5 g;
(CCH3); 31P-NMR (CDCl3) 162 MHz d ¼ 19.4. MS (ESI+; MeOH): 0.08 mol) and 2,6-lutidine (27 g; 0.25 mol, 3.1 eq.) in toluene
m/z 404 (M + H+).
(450 mL) stirred at ꢀ15 ꢁC under nitrogen atmosphere. The
Synthesis of aminomethylphosphonic acid di-tert-butyl ester reaction mixture was stirred at room temperature for 22 h then
(19). (Dibenzylamino)methylphosphonic acid di-tert-butyl ester diluted with H2O (200 mL) and extracted with EtOAc (200 mL).
(33.3 g; 82.7 mmol) was dissolved in MeOH (500 mL) and The organic solution was dried over Na2SO4 and evaporated to
hydrogenated at atmospheric pressure on Pd/C 10% (3.3 g). give a crude (33 g) that was puried by chromatography [silica
Aer 2 h the mixture was ltered through Millipore® apparatus gel, eluent: 7 : 3 n-hexane/iPr2O, Rf 0,44] to obtain 13 (24,9 g;
(0.5 mm) and the solution evaporated under vacuum. The crude 0,05 mol) as a pale yellow oil. Yield 62.4%. 1H-NMR (CDCl3)
(18.4 g) was used without any further purication. Quantitative 400 MHz d ¼ 7.37 (m, 5H, Har), 5.15 (s, 2H, CH2Ph), 3.87 (t, 1H,
1
yield. H-NMR (CDCl3) 400 MHz d ¼ 2.81 (d, 2H, J ¼ 10.2 Hz, J ¼ 7.4 Hz, CHN), 3.40, 3.15 (m, 6H, CH2CH2Br and CH2CO2tBu),
CH2P), 1.49 (s, 18H, CCH3). 13C-NMR (CDCl3) 100 MHz d ¼ 2.85 and 2.69 (dd, 2H, J1 ¼ 16.0 Hz, J2 ¼ 7.4 Hz, CH2CO2Bn), 1.46
83.2 (CCH3), 40.6 (CH2P, J ¼ 153 Hz), 29.5 (CCH3); 31P-NMR and 1.44 (s, 18H, CCH3). 13C-NMR (CDCl3) 100 MHz d ¼ 171.0
(CDCl3) 162 MHz d ¼ 21.0. MS (ESI+; MeOH): m/z 224 (M + H+). (CO2Bn and CO2tBu, 3C), 136.1 (Car), 128.9, 128.7 (CHar), 82.4,
Synthesis of L-aspartic acid, 1-tert-butyl-4-benzylester (11). 81.5 (CCH3), 67.0 (CH2Ph), 62.9 (CHN), 56.1, 54.9 (NCH2, 2C),
Perchloric acid (70% aq.; 19.28 g; 0.134 mol) was dropped into a 36.7 (CH2CO2Bn), 30.8 (CH2Br), 28.5 (CCH3). ESI-HRMS (m/z):
suspension of L-aspartic acid 4-(phenylmethyl) ester (25 g; 500.1206 (M + H+) (calcd 500.1648).
0.112 mol) in tert-butyl acetate (515 mL; 3822 mol) stirred at
Synthesis of N,N0-[[[[bis(1,1-dimethylethoxy)phosphinyl]
room temperature. Aer 18 h at room temperature the obtained methyl]imino]di-2,1-ethanediyl]bis[N-[2-(1,1-dimethylethoxy)-2-
clear solution was diluted with H2O (470 mL) and the phases oxoethyl]-L-aspartic acid 1,10-bis(1,1-dimethylethyl) 4,40-
were separated; the aqueous phase was extracted with EtOAc bis(phenylmethyl) ester (14). A solution of bromo-derivative 13
(2 x 235 mL). The organic phases were collected and washed (4.11 g; 8.22 mmol) in acetonitrile (20 mL) was slowly dropped
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into an emulsion of aminomethylphosphonic acid di-tert-butyl
ester 19 (916 mg; 4.11 mmol) in acetonitrile (10 mL) and 2 M
phosphate buffer pH 8 (20 mL) over 8 hours under vigorous
stirring. Aer 22 h, the phases were separated and the aqueous
layer was extracted with EtOAc (2 ꢂ 30 mL). The organic layer
was evaporated. The oily residue was dissolved with EtOAc
(20 mL), and the solution was washed with 1 : 1 water/brine (2 ꢂ
30 mL) and dried over Na2SO4. The crude (4.49 g) was puried
by silica gel chromatography (silica gel; EtOAc/petroleum ether
4 : 6; Rf 0.32). Compound 14 was obtained (1.86 g; 1.75 mmol) as
a yellow oil. Yield 42.4%. 1H-NMR (CDCl3) 400 MHz d ¼ 7.32 (m,
10H, Har), 5.09 (s, 4H, CH2Ph), 3.80 (dd, 2H, J ¼ 3.6 Hz, CHN),
3.35 (s, 4H, CH2CO2tBu), 2.82–2.62 (m, 14H, NCH2CH2N, NCH2P
and CH2CO2Bn), 1.46 and 1.41 (s, 54H, CCH3). 13C-NMR (CDCl3)
100 MHz d ¼ 171.3 (CO2Bn and CO2tBu, 6C), 136.3 (Car), 128.9,
128.6 (CHar), 82.5, 81.8, 81.0 (CCH3), 66.7 (CH2Ph), 62.2 (NCH),
55.0 (CH2CO2tBu), 54.7, 51.2 (NCH2CH2N), 52.2 (CH2P), 36.5
(CH2CO2Bn), 30.9 and 28.5 (CCH3). 31P-NMR (CDCl3) 162 MHz
d ¼ 18.6. ESI-HRMS (m/z): 1062.5503 (M + H+) (calcd 1062.6031).
Synthesis of N,N0-[[[[bis(1,1-dimethylethoxy)phosphinyl]
methyl]imino]di-2,1-ethanediyl]bis[N-[2-(1,1-dimethylethoxy)-2-
oxoethyl]-L-aspartic acid 1,10-bis(1,1-dimethylethyl) ester, (15).
Compound 14 (973 mg; 1.10 mmol) was dissolved in methanol
(50 mL) and hydrogenated at atmospheric pressure on 10%
Pd/C (50 mg). Aer 30 min the reaction mixture was ltered
through Millipore apparatus® (0.5 m lter) and the solution
evaporated under vacuum. The crude (725 mg) was used in the
next step without any further purication. 1H-NMR (CDCl3)
400 MHz dd ¼ 3.89 (m, 2H, CHN), 3.49–3.36 (m, 4H,
CH2CO2tBu), 3.25–2.95 (m, 10H, NCH2CH2N, NCH2P) 2.75
(CH2CO2H), 1.50 and 1.46 (s, 54H, CCH3). 13C-NMR (CDCl3)
100 MHz dd ¼ 173.0 (COOH), 170.8, 170.2 (CO2tBu, 4C), 84.2,
82.7, 82.2 (CCH3), 61.7 (NCH), 54.5 (CH2CO2tBu), 54.0, 50.1
(NCH2CH2N), 51.8 (CH2P, J ¼ 155 Hz), 35.6 (CH2CO2H), 30.9
and 28.5 (CCH3). 31P-NMR (CDCl3) 162 MHz d ¼ 15.0. ESI-HRMS
(m/z): 882.4855 (M + H+) (calcd 882.5092).
Synthesis of compound (17). Ammonia was bubbled for
45 min into a solution of the fully protected ligand 16 (19.48 g)
in methanol (400 mL) refrigerated with an ice bath. Aer 1 h the
ice bath was removed, CaCl2 valve was applied and the solution
was allowed to stir at room temperature for 9 days. Every two
days more ammonia was bubbled for 10–15 min into the solu-
tion. The reaction was followed by TLC (CHCl3/MeOH 9 : 1) and
by MS until disappearance of any peaks related to partially
acetylated product. Then, the solvent was evaporated in vacuum
obtaining 18.1 g of the crude which was used without any
further purication in the next deprotection step. 1H NMR
(400 MHz, D2O) d 4.34 (bs, 6H), 4.06 (bs, 6), 3.82–3.71 (m, 8H),
3.68 (t, J ¼ 6.8 Hz, 4H), 3.59 (m, 4H), 2.94 (bs, 8H), 2.78 (s, 4H),
2.73 (s, 2H), 1.42 (m, 56H). 13C NMR (101 MHz, CDCl3) d 171.9,
171.5, 170.8, 170.7, 169.7, 82.4, 78.0, 70.9, 70.0, 62.4, 46.7, 39.2,
31.7, 31.0, 29.2. ESI-HRMS (m/z): 2179.1102 (M + H+) (calcd
2179.0495).
Synthesis of compound (L3). A round-bottomed ask con-
taining the phosphonic proligand 17 (18.1 g, 8.3 mmol) was
cooled with an ice bath; then a mixture of CH2Cl2/TFA 1 : 1
(100 mL) was added dropwise giving a yellow-brown solution.
Aer 20 min, the ice bath was removed and the solution was
kept under stirring at room temperature for 16 h. The solvent
was removed and the residue was dissolved in dichloromethane
(5 mL) and evaporated; this procedure was repeated four times.
The crude was treated with Et2O to obtain the precipitation of a
white solid which was ltered, dissolved in water and lyophi-
lized. The slightly yellow solid was then re-dissolved in water,
brought to pH 7 by adding 2 M NaOH and lyophilized again. The
solid obtained was recovered with MeOH and kept under stir-
ring for 1 h. The white solid formed was ltered and dried in
vacuum to obtain 4.6 g (2.50 mmol, 30% yield) of product. The
methanolic solution was dried at the rotary evaporator and in
vacuum to obtain 2.9 g of the mono-derivative as a yellow oil. 1H
NMR (400 MHz, D2O) d 4.39 (bs, 6H), 4.10 (bs, 6H), 3.82–3.76
(m, 8H), 3.76–3.70 (m, 4H), 3.64 (dd, J ¼ 11.6, 6.3 Hz, 4H), 3.32
(s, 4H), 3.07 (s, 8H), 2.96 (s, 2H). 13C NMR (101 MHz, CDCl3) d
171.9, 171.5, 170.8, 170.7, 169.7, 82.4, 78.0, 70.9, 70.0, 62.4, 46.7,
39.2, 31.7, 31.0, 29.2. ESI-HRMS (m/z): 1842.6012 (M + H+) (calcd
1842.6739).
Synthesis of compound (16). A solution of diacid 15 (4.91 g;
5.58 mmol), polyalcohol 9 (17.40 g; 13.38 mmol, 2.4 equivalents)
and HATU (5.08 g; 13.38 mmol) in 50 mL of DMF was brought to
0 ꢁC with an ice bath in nitrogen atmosphere. To this solution,
DIPEA (9.32 mL, 53.52 mmol, 9.6 eq.) was added keeping the
temperature below 5 ꢁC. The mixture was kept under stirring at
room temperature for 48 h monitoring the reaction by TLC
Water proton relaxation measurements
(silica gel; CHCl3/MeOH 9 : 1), then the reaction solution was The longitudinal water proton relaxation rates were measured
poured in ice, stirred for 30 min and ltered. The solid was at 25 ꢁC by using a Stelar Spinmaster (Stelar, Mede, Pavia, Italy)
washed with water, dissolved in chloroform and dried over spectrometer operating at 0.5 T (21.5 MHz Proton Larmor
Na2SO4. Aer ltration and solvent evaporation, the desired Frequency), by mean of the standard inversion-recovery tech-
product was obtained as pale yellow gummy solid (15.3 g, 80% nique. The temperature was controlled with a Stelar VTC-91 air-
1
yield). (Rf 0.52, silica, CHCl3/MeOH 9 : 1). H NMR (400 MHz, ow heater equipped with a copper constantan thermocouple
CDCl3) d 7.86 (sb, 8H), 5.61 (m, 6H), 5.46 (m, 6H), 5.31 (m, 6H), (uncertainty 0.1 ꢁC). The proton 1/T1 NMRD proles were
4.97 (m, 6H), 4.29 (m, 6H), 4.16 (m, 6H), 3.83 (m, 2H, CHN), measured at 25 ꢁC on a fast eld-cycling Stelar relaxometer over
3.60–2.75 (m, 14H, CH2CO2tBu, NCH2CH2N, NCH2P), 2.28 (s, a continuum of magnetic eld strengths from 0.00024 to 0.47 T
16H, CCH2N), 2.07 and 2.02 (m, 90H, CH3COO), 1.54 and 1.46 (s, (corresponding to 0.01–20 MHz proton Larmor frequencies).
54H, CCH3).13C NMR (101 MHz, CDCl3) d 172.8, 170.9, 170.5, The relaxometer operates under computer control with an
169.8, 169.6, 168.7, 81.4, 69.9, 68.9, 61.4, 45.8, 38.4, 30.7, 30.0, absolute uncertainty in 1/T1 of ꢃ1%. Additional data points in
28.3, 28.2, 20.7. ESI-HRMS (m/z): 3439.2986 (M + H+) (calcd the range 21.5–70 MHz were obtained on the Stelar Spinmaster
3439.3665).
spectrometer. The concentration of the solutions used for the
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relaxometric characterization was determined by measuring the
bulk magnetic-susceptibility shis of the tBuOH signal.
7 (a) M. R. Longmire, M. Ogawa, P. L. Choyke and
H. Kobayashi, Wiley Interdiscip. Rev.: Nanomed.
Nanobiotechnol., 2014, 6, 155; (b) A. Bumb, M. W. Brechbiel
and P. Choyke, Acta Radiol., 2010, 51, 751.
17O-NMR measurements
8 P. Caravan, Acc. Chem. Res., 2009, 42, 851.
Variable temperature 17O-NMR measurements were recorded at
2.1 T on a JEOL90 spectrometer, equipped with a 5 mm probe, by
using a D2O external lock. The experimental settings were:
spectral width 9000 Hz, 90ꢁ pulse (12 ms), acquisition time 10 ms,
1024 scans and without sample spinning. Aqueous solutions
containing 2.6% of 17O isotope (Yeda, Israel) were used. The
observed transverse relaxation rates (RO2obs) were calculated from
the signal width at half-height (Dn1/2): RO2obs ¼ pDn1/2. Para-
magnetic contributions to the observed transversal relaxation
rate (R2p) were calculated by subtracting from RO2obs the diamag-
netic contribution measured at each temperature value on pure
water enriched with 2.6% 17O isotope.
9 J. Martinelli, M. Fekete, L. Tei and M. Botta, Chem. Commun.,
2011, 47, 3144.
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`
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Conclusions
The synthesis of three novel DTPA derivatives bearing poly-
hydroxylated pendant arms is reported. While the two DTPA
bisamides L1 and L2 with differently branched polygluconyl
groups were easily synthesised from DTPA bis-anhydride, the
monophosphonic P-DTPA ligand L3 was prepared by a multi-
13 (a) L. Van Der Elst, Y. Raynal, M. Port, P. Tisnes and
R. N. Muller, Eur. J. Inorg. Chem., 2005, 6, 1142; (b) M. Port,
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C. Corot, O. Rousseaux, I. Raynal, L. Devoldere, J. M. Idee
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172.
step procedure which included the synthesis of
a bis-
functionalized DTPA bifunctional ligand via the Rapoport
reaction. The relaxivity of GdL1 and GdL2 were very low because
DTPA-bisamides are characterized by slow water exchange rates
of the coordinated water molecule. On the other hand, the
relaxivity values measured for GdL3 over a wide range of
imaging elds (0.5–3 Tesla) are among the highest till now
reported for monomeric Gd(III) complexes with medium sized
molecular weight (ꢄ2000 Da). This nding makes GdL3 a very
promising system for applications at the currently used
magnetic elds of the clinical MRI scanners.
14 J. Kotek, P. Lebduskova, P. Hermann, L. Vander Elst,
R. N. Muller, C. F. G. C. Geraldes, T. Maschmeyer, I. Lukes
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