Synthesis of a bifunctional monophosphinic acid DOTA analogue ligand and its lanthanide(III) complexes. A gadolinium(III) complex endowed with an optimal water exchange rate for MRI applications

Article abstract of DOI:10.1039/b410103k

A new bifunctional octa-coordinating ligand containing an aminobenzyl moiety, DO3AP^ABn (H₄DO3AP^ABn = 1,4,7,10-tetraazacyclododecane-4,7,10-triacetic-1-{methyl[(4-aminophenyl)methyl] phosphinic acid}), has been synthesized. Its lanthanide(III) complexes contain one water molecule in the first coordination sphere. The high-resolution ¹H and ³¹P spectra of [Eu(H₂O) (DO3AP ^ABn)- show that the twisted square-antiprismatic form of the complexes is more abundant in respect to the corresponding Eu(III)-DOTA complex. The ¹H NMRD and variable-temperature ¹⁷O relaxation measurements of [Gd(H₂O)(DO3AP^ABn)]^- show that the water residence time is short (²⁹⁸T_M = 16 ns) and falls into the optimal range predicted by theory for the attainment of high relaxivities once this complex would be endowed by a slow tumbling rate. The relaxivity (²⁹⁸r₁ = 6.7 mM^-1 s^-1 at 10 MHz) is higher than expected as a consequence of a significant contribution from the second hydration sphere. These results prompt the use of [Gd(H ₂O)(DO3AP^ABn)]^- as a building block for the set-up of highly efficient macromolecular MRI contrast agents.

Full text of DOI:10.1039/b410103k

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A R T I C L E
Synthesis of a bifunctional monophosphinic acid DOTA analogue
ligand and its lanthanide(III) complexes. A gadolinium(III) complex
endowed with an optimal water exchange rate for MRI applications†
Jakub Rudovsky´,a Jan Kotek,a Petr Hermann,*a Ivan Lukesˇ,a Valentina Mainerob and
Silvio Aimeb
a Department of Inorganic Chemistry, Universita Karlova, Hlavova 2030, 128 40, Prague 2,
Czech Republic. E-mail: petrh@natur.cuni.cz; Fax: +420-221951253; Tel: +420-221951263
b Dipartimento di Chimica I. F. M., Universita` di Torino, Via P. Giuria 7, I-10125, Torino, Italy
Received 5th July 2004, Accepted 12th October 2004
First published as an Advance Article on the web 18th November 2004
A new bifunctional octa-coordinating ligand containing an aminobenzyl moiety, DO3APABn (H4DO3APABn
=
1,4,7,10-tetraazacyclododecane-4,7,10-triacetic-1-{methyl[(4-aminophenyl)methyl]phosphinic acid}), has been
synthesized. Its lanthanide(III) complexes contain one water molecule in the first coordination sphere. The
high-resolution 1H and 31P spectra of [Eu(H2O) (DO3APABn)]− show that the twisted square-antiprismatic form of the
complexes is more abundant in respect to the corresponding Eu(III)–DOTA complex. The 1H NMRD and
variable-temperature 17O relaxation measurements of [Gd(H2O)(DO3APABn)]− show that the water residence time is
short (298sM = 16 ns) and falls into the optimal range predicted by theory for the attainment of high relaxivities once
this complex would be endowed by a slow tumbling rate. The relaxivity (298r1 = 6.7 mM−1 s−1 at 10 MHz) is higher
than expected as a consequence of a significant contribution from the second hydration sphere. These results prompt
the use of [Gd(H2O)(DO3APABn)]− as a building block for the set-up of highly efficient macromolecular MRI contrast
agents.
in the case of [Gd(H2O)(TRITA)]− (H4TRITA = 1,4,7,10-
Introduction
tetraazacyclotridecane-1,4,7,10-tetraacetic acid).6 Further in-
Gadolinium(III) complexes of polyaminopolycarboxylates are
sights into the relationship between water exchange rates and
widely used as contrast agents (CA) in magnetic resonance
solution structures have been gained from in-depth investi-
imaging (MRI).1 The efficacy of a paramagnetic CA is primarily
assessed by its relaxivity (r1) that reports the T1 relaxation
enhancement of (tissue) water protons of the paramagnetic
complex at 1 mM concentration. The search for systems
endowed with high relaxivity continues to be an important task.
It is even more important in view of applications in the field of
gations on the structural isomers of lanthanide(III) DOTA
complexes. It is known that DOTA-like ligands wrap around
a lanthanide(III) ion yielding two coordination geometries,
namely M (SA, square-antiprismatic) and m (TSA, twisted
square-antiprismatic).1,7 It was found that the m (TSA) isomers
display 10 to 100 times faster exchange of the coordinated
molecular imaging where the low concentration of the targeting
water molecule, both in amide and acid derivatives.8,9 This
sites has to be compensated with an improved sensitivity of the
effect was ascribed to an increased steric repulsion around the
imaging probes.2 It was recognized early on that a significant
water-binding site in the case of m (TSA) isomers. Both the
increase of relaxivity can be expected when the reorientational
motion (represented by the rotational correlation time sR) of
the gadolinium(III) chelates is slowed down upon conjugation
to suitable high-molecular weight synthons. However, such an
[Gd(HDOTP)]4− (H8DOTP = 1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetrakis(methylphosphonic acid)) and complexes of
the phosphinic acid analogs are characterized by a m-type
structure.10,11 Interestingly, the bulkiness of the phosphorus acid
expected enhancement of relaxivity is often “quenched” by
groups in the complexes causes the total expulsion of the water
the occurrence of an exceedingly long exchange lifetime of
molecule from the inner coordination sphere of the central
the coordinated water (water residence time sM).1,3 It has been
lanthanide(III) ion. On the basis of these findings, we surmise
suggested that, at the imaging field of 0.5 T (corresponding
that the design of a DOTA-like system endowed with a fast
to hydrogen Larmor resonance frequency of 20 MHz), an
exchange of coordinated water can be pursued through a fine
optimal value for 298sM is between 10 to 30 ns.1,4 Most of
modulation of the steric constrain around the water-binding
the currently available macrocyclic systems display a residence
site.6,12,13
lifetime too long for the attainment of high relaxivities (e.g.
In this work, we show that the replacement of an acetic
arm with a methylphosphinic acid moiety on DOTA structure
destabilizes the coordinated water molecule yielding its exchange
rate in the region of the optimal values for MRI applications.
298sM of [Gd(H2O)(DOTA)]− has been reported to be 243 ns
at 298 K; H4DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetracetic acid5) when their sR would have been slowed down to
the nanosecond range.
As it has been shown that the exchange of the co-
Moreover, the methylphosphinic acid arm is functionalized
with a p-aminophenyl moiety that represents a site for further
ordinated water in [Gd(H2O)(DOTA)]− and similar com-
conjugation, for instance to a macromolecular system.
plexes occurs through
a a possi-
dissociative pathway,1
ble route to accelerate water exchange rate can be envis-
aged by destabilizing the ground-state nonacoordinated struc-
ture. This approach has been shown to work successfully
Results and discussion
Syntheses
The target ligand, DO3APABn (H4DO3APABn = 1,4,7,10-tetra-
azacyclododecane-4,7,10-triacetic-1-{methyl[(4-aminophenyl)-
methyl)]phosphinic acid}), was obtained by a multi-step
† Electronic supplementary information (ESI) available: additional fig-
ures, equations. See http://www.rsc.org/suppdata/ob/b4/b410103k/.
1 1 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 1 2 – 1 1 7
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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Scheme 1
synthesis starting from hypophosphorous acid and H3DO3A
5 (Scheme 1). The key phosphorus-containing intermediate 2
was obtained according to literature procedure.14,15 Arbuzov
reaction of compound 2 and 4-nitrobenzyl bromide produced a
complex reaction mixture as the by-product Me3SiBr is able to
remove the ester group as well as the P–H bond protecting the
diethoxymethyl group from the intermediate 3. After hydrolysis
of this mixture and subsequent purification by extraction and
recrystallization, the pure phosphonic acid 4 was isolated.
Mannich reaction between the acid 4 and H3DO3A 5 led to the
macrocyclic intermediate 6. As the last nitrogen atom of the
cyclen ring has a low reactivity, a large excess of phosphinic acid
4 and formaldehyde, as well as a long reaction time, was used.
Acyclic impurities were removed with strong cation exchange
resin. An extensive chromatography on weak cation exchange
resin gave the pure macrocycle 6. Hydrogenation of 6 with
Pd/C catalyst produced the target ligand isolated as stable
trihydrate (according to elemental and thermal analyses).
Two procedures for preparation of lanthanide(III) complexes
were used. In one method, the aqueous solution of the
lanthanide(III) chloride was mixed with 10% molar excess of
DO3APABn. The solution pH was adjusted to 7 with 1.5 M KOH
and the mixture was heated to 70 ◦C for a few minutes. No free
lanthanide ions were present as assessed by the xylenol orange
test. In the latter method, a slight excess of LnCl3 was added to
the solution of DO3APABn followed by pH adjustment to 7. After
pH stabilization, the mixture was heated at 50 ◦C overnight. The
complexes were purified on Amberlite CG50 with water elution.
The HCl released by the reaction was present in the first fraction,
the pure complex was somehow delayed on column and eluted
in later fractions. Any cations were taken up by the column.
The purified complexes were characterized by 1H and 31P NMR
spectroscopies.
useful as, from the number and relative intensity of the observed
resonances, one can quickly determine the number and relative
abundance of the species eventually present. As it is not possible
to acquire the high-resolution NMR spectra of the gadolin-
ium(III) complex (excessively broad lines), its solution structure
Solution structures of lanthanide(III) DO3APABn complexes
First of all, the occurrence of a coordinated water molecule
was assessed by measuring the water 17O NMR chemical shift
upon titration of 50 mM solution of DO3APABn with Dy3+
ions. The method relies on the fact that the dysprosium(III)
induced shift is dependent upon the number of water molecules
present in the inner coordination sphere of the paramagnetic ion.
As reported some years ago, one coordinated water molecule
is responsible for the dysprosium(III) induced 17O shift about
−40 ppm mM−1.16 For the dysprosium(III)-DO3APABn system,
a value of −39 ppm mM−1 at 298 K was found. This is fully
consistent with the presence of one water molecule in the inner
coordination sphere of the lanthanide(III) ion.
Next, we assessed the solution structures of lanthanide(III)
1
complexes of DO3APABn by means of high-resolution H and
Fig.
1
Variable temperature 31P NMR spectra of [Eu(H2O)
31P NMR spectroscopy. The 31P NMR spectra are particularly
(DO3APABn)]− at pH 7: (a) 298 K, (b) 320 K and (c) 345 K.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 1 2 – 1 1 7
1 1 3

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can be surmised by investigating the europium(III) complex.
The rt 31P NMR spectrum of [Eu(H2O)(DO3APABn)]− displays
three resonances at 92.0, 86.6 and 79.5 ppm, respectively, in
the relative intensity ratio of ca. 1 : 4 : 5 (Fig. 1a). As the
temperature is increased, the two signals with higher dP broaden
and collapse in a single resonance (TC ca. 320 K, Fig. 1b). Then,
at higher temperature, a new dynamic process leading to the
coalescence of the two remaining resonances takes place (TC
ca. 345 K). The high-temperature limiting spectrum therefore
consists of a single 31P resonance to indicate the occurrence
of a fast izomerization process (Fig. 1c). For full details refer
to the supplementary information†. To exclude the possibility
of complex decomposition this experiment was performed
repeatedly on the same sample.
proton relaxivity of the complex is 6.7 mM−1 s−1 (25 ◦C,
10 MHz, pH 7.0). Such a value is about 15% higher than that of
[Gd(H2O)(DOTA)]− complex under the same conditions.1a
Further information about the determinants of the proton
relaxivity has been acquired by measuring the temperature-
1
dependent water 17O T2r data (Fig. 3) and H NMRD profile
(Fig. 4). The experimental data were treated on the ba-
sis of the Solomon–Bloembergen–Morgan (SBM) theory of
paramagnetic relaxation1,19 implemented in equations used for
multiparametrical fitting (see supplementary information†). The
simultaneous fitting of the 1H NMRD profile and 17O T2r data
have been carried out by fixing some parameters to the values
previously found for related Gd(III) chelates and the relevant
parameters obtained are presented in Table 1. Namely, the
parameters Ev (1 kJ) and Er (16.1 kJ) have been fixed to the values
reported for Gd(III) DOTA complex.5 The value of the hyperfine
coupling constant A / h(−2.98 × 106 rad s−1) was estimated from
contact contribution to lanthanide induced 17O chemical shift
of water obtained in the presence of [Ln(H2O)(DO3APABn)]−
complexes (supplementary information†). The values of rGdH
When the 1H NMR spectrum of [Eu(H2O)(DO3APABn)]−
at 298 K is compared with the corresponding spectrum of
[Eu(H2O)(DOTA)]− and related derivatives,7–9 the presence of
three sets of resonances has been clearly identified. Focusing
1
on the low-field region of the H NMR spectrum (region of
the axial hydrogens of the macrocyclic ring) (Fig. 2), one can
assign the four most-shifted resonances (from 38 to 32 ppm) to
one diastereoisomer with a square-antiprismatic structure (M)
whereas the two sets of resonances, from 28 to 17 ppm, have to
be ascribed to two m-type isomers (twisted square-antiprismatic
structure). The intensity ratio between (m1 plus m2) and M
resonances is about 6 : 4. Thus, we assign the 31P signals at 92.0
and 79.5 ppm to two m-type isomers and the 31P resonance at
86.6 ppm to a M-type species. In principle, due to the prochirality
of the phosphinate moiety, four diastereoisomers would have
been expected. Most likely, the M-type structure displays a large
preference for only one arrangement on the phosphorus atom.
Thus, the dynamic behaviour shown in variable-temperature
31P NMR spectra corresponds first to a m–M izomerization
and then, at higher temperature, to an epimerization process
of the phosphinate moiety. Corresponding variable-temperature
1H NMR spectra parallel the behaviour observed in 31P NMR
spectra.
˚
˚
and rGdO were fixed to the values (rGdH = 3.1 A, rGdO = 2.6 A)
reported in the literature for related complexes.20 A good fit for
the 1H NMRD profiles (Figure 4) has been obtained only upon
introducing a contribution to the overall relaxivity arising from
one water molecule in the second coordination sphere (qss
=
1), in addition to the usual contributions from the inner-sphere
water molecule (q = 1) and from water molecules diffusing in the
proximity of the paramagnetic complex (outer-sphere contribu-
tion). The Gd–H distance (Rss) is the value estimated on the
basis of the X-ray crystal structure of [Nd(H2O)(HDO3AP)]−
(H5DO3AP = 1,4,7,10-tetraazacyclododecane-4,7,10-triacetic-
1-methylphosphonic acid).21 The residence lifetime of the
298
second-sphere water used in the fitting procedure (
s
mss
= 1 ns)
Fig. 3 Variable-temperature 17O NMR transverse relaxation rates
R2r measurements in the presence of [Gd(H2O)(DO3APABn)]− complex
(pH 7.0). The curve shows the best simultaneous fit of experimental
data.
Fig. 2 Low-field region of the 1H NMR spectrum of [Eu(H2O)
(DO3APABn)]− at 298 K (400 MHz).
Interestingly, the m–M isomer ratio for [Eu(H2O)
(DO3APABn)]− complex is much higher than the value found for
the parent [Eu(H2O)(DOTA)]− complex (ca. 0.27).7a It is most
likely that the presence of the bulky phosphinate moiety shifts
the M↔m equilibrium towards the less sterically demanding17
m-type structure.
1H and 17O NMR relaxometry of [Gd(H2O)(DO3APABn)]−
complex
The stability of the complex was qualitatively assessed by
measurement of water proton relaxivity dependence on pH
(see supplementary information†). The value of r1 was constant
over the studied pH region (2–11) showing that the complex
is stable in acidic solution and protonation/deprotonation of
the distant amino group (pKa 4.76, ref. 18) does not have any
effect on relaxometric properties of the complex. The water
Fig. 4 1H NMRD profile of [Gd(H2O)(DO3APABn)]− at pH 7 (25 ◦C:
full circles; 37 ◦C: open circles). The curve represents the best fit of the
data resulted from simultaneous fitting based on SBM equations.1,19
1 1 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 1 2 – 1 1 7

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Table 1 Results from simultaneous fitting of 1H NMRD and variable-
temperature 17O NMR R2r data in presence of [Gd(H2O)(DO3APABn)]−.
Value for Ev (1 kJ) was adjusted and fixed at [Gd(H2O)(DOTA)]− values
(see text for details)5
complex to the overall tumbling of a slowly moving system often
represents the source of a “quenching” effect on the attainable
relaxivity.1a
Parameter
[Gd(H2O)(DO3APABn)]−a
[Gd(H2O)(DOTA)]−
Experimental
298r1/mmol−1 s−1b
D2/1020 s−2
298sM/ns
6.7
5.7
0.16
244
49.8
77
16.1
11
−3.7
2.38
—
—
—
—
—
The H3DO3A·H2SO4 was a kind gift from Bracco SpA (Milano).
Hypophosphorus acid (50% aqueous solution), HC(OEt)3,
CF3CO2H, 4-nitrobenzyl bromide and HN(SiMe3)2 were ob-
tained from Fluka and 10% Pd on charcoal was from Aldrich.
Lanthanide(III) oxides and chlorides were purchased from
Aldrich, Strem or Alfa. All commercially available reagents were
used as received. Paraformaldehyde was filtered from an aged
aqueous formaldehyde solution and dried in a desiccator over
P2O5. Compound HP(O)(OEt)[CH(OEt)2] (1) was synthesized
according to a literature procedure from crystalline H3PO2.14
Synthesis of compounds 1, 2 and 3 was done under argon
atmosphere. D2O (99.98% D) was received from Chemtrade
(Germany). Tlc was performed on silica-coated aluminium
sheets (Merck (with UV indicator) or Silufol (Kavalier, Czech
0.25 0.01
16.2 0.1
20.6 0.3
88
29
11.2 0.1
−2.89
2.6
3.1
3.6
15
9
DHM/kJ mol−1
298sR/ps
4
1
Er/kJ
298sv/ps
(A / )/106 rad s−1c
˚
R
R
GdO/A
˚
GdH/A
˚
A/A
DHmss/kJ
298
s
/ps
2
rss
˚
Rss/A
3.6
298
s
/ns
1
—
1
—
mss
qd
qss
1
1
i
Republic)) in PrOH–aq. NH3 (25%)–water 7 : 3 : 3 with
a Bold numbers were fixed during the fitting. b Millimolar relaxivity of
[Gd(H2O)(DO3APABn)]− was measured at 25 ◦C and 10 MHz. Corre-
sponding value for [Gd(H2O)(DOTA)]− was adopted from literature.1a
c Value of hyperfine coupling constant was calculated from 17O chemical
shift measurement through the lanthanide series. d Hydration value q
was obtained for Dy(III) induced shift data.
detection using ninhydrin or Draggendorf spray, iodine vapours
or UV irradiation. Elemental analyses were performed at the
Institute of Macromolecular Chemistry of the Czech Academy
of Science (Prague). NMR spectra of organic compounds were
recorded using Varian UNITYINOVA 400 (see Scheme 1 for
labelling of macrocycles). ES/MS spectra were run on the
Bruker ESQUIRE 3000 with ion-trap detection in positive or
negative modes.
is similar to the value previously reported for [Gd(HDOTP)]4−
(1–3 ns).22
The values of the electronic parameters D2 (0.25 × 1020 s−2) and
sv (11 ps) as well as the value of rotation correlation time 298sR
(88 ps) obtained from the best fit are comparable to those found
for the [Gd(H2O)(DOTA)]− complex. A very important result
concerns the water residence lifetime (298sM) of the inner-sphere
water molecule. From variable-temperature 17O NMR transverse
relaxation time measurements, it has been found that 298sM in
[Gd(H2O)(DO3APABn)]− is 16 ns, i.e. a value which is one order
of magnitude lower than that found for [Gd(H2O)(DOTA)]−
(Ethyloxy)[bis(ethyloxy)methyl](trimethylsilyloxy)phosphine
(2)15
Crude 1 (28.6 g, 0.146 mol, purity 94%) and HN(SiMe3)2 (68 ml,
0.32 mol) were mixed and the mixture was heated at 110 ◦C for
6 h. Excess hexamethyldisilazane was distilled off at rt and 4
kPa pressure into cold finger. Fractional distillation at 0.04 kPa
produced 32.2 g (yield 83%, purity 98%) of highly moisture-
sensitive phosphite. dP (161.9 MHz; CDCl3; ext. 85% H3PO4,
(
298sM = 243 ns).5 The value obtained corresponds to an average
◦
◦
1
31P{ H}) 146.8; bp 52–55 C / 0.04 kPa (bp 51 C / 0.013 kPa;
value of water residence lifetime for the two isomers. In principle,
one should have expected that the curve of 17O T2r vs temperature
would reflect the contributions from the two m and the M
isomers. The observation of a curve characterized by a single
component (Fig. 3) would suggest that the M isomer has a sM
value similar to those of m-type isomers. Such a drop in the
value of water residence lifetime for the m as well as M isomer
can be most likely ascribed to the bulkiness of the phosphinate
group, which may cause elongation of the Gd–water bond.
Another possible contribution to the reduction of 298sM might
arise from the overall arrangement of the second hydration
sphere. A similar decrease in water residence time was reported
for some gadolinium(III) complexes of DO3A (with two water
molecules in the first coordination sphere; H3DO3A = 1,4,7,10-
tetraazacyclododecane-4,7,10-triacetic acid).23 This feature was
explained by the perturbation of the second hydration sphere
due to presence of a distant group able to form hydrogen bonds.
given in ref. 15).
(4-Nitrophenyl)methylphosphinic acid (4)
Solution of 4-nitrobenzyl bromide (26 g, 0.12 mol) in dry CH2Cl2
(150 ml) was slowly dropped (3 h) into solution of the phosphite
2 (30 g, 0.11 mol) in dry CH2Cl2 (100 ml). The mixture was
stirred overnight. Methanol (50 ml) was added, solution was
stirred for 1 h and filtered through a fine frit. The filtrate was
evaporated to dryness in vacuum. The residue was dissolved in
EtOH (150 ml) and aqueous HCl (35%, 150 ml) was added.
The mixture was refluxed overnight, cooled and solvents were
removed in vacuum. To the residue, water (100 ml) was added
and the mixture was slowly neutralized with aqueous KOH
(1 mol dm−3) to ca. pH 10. The solution was extracted with
CHCl3 (3 × 100 ml) and the aqueous phase was acidified with
conc. aqueous HCl with stirring. The resulting suspension was
stirred for 1 h and filtered to give the first crop of a crude product.
The filtrate was evaporated to dryness in vacuum and the residue
(mostly KCl) was extracted with EtOH (3 × 50 ml). The ethanol
was evaporated, the residue was dissolved in water with a base
addition and the second crop of product was obtained upon
acidification as above. Both crops of product were recrystallized
together from boiling water to get 19 g (86%) of 4 as yellow
Conclusions
The overall relaxometric properties of [Gd(H2O)(DO3APABn)]−
makes this complex a very promising candidate for the prepa-
ration of macromolecular systems with very high relaxivities.
The optimal water exchange should allow the exploitation
of long sR values. Moreover [Gd(H2O)(DO3APABn)]− can be
easily conjugated to polymeric systems and the presence of the
aromatic ring should provide a sufficiently rigid spacer to limit
internal motions of the chelate once bound to a macromolecular
substrate. The superimposition of an internal rotation of the
2
crystals. dH (400 MHz, d6-dmso, Me4Si) 3.48 (2 H, d, JPH
1
18.9), 7.42 (1 H, d, JPH 541) 7.57 (2 H, m), 8.23 (2 H, m);
1
dP (161.9 MHz, d6-dmso, ext. 85% H3PO4) 31.1 (dt, JPH 541,
2JPH 19.1)
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 1 2 – 1 1 7
1 1 5

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molar ratio of LnCl3 and ligand in water followed by addition
of KOH to adjust the pH to 7. The reaction mixtures were
briefly heated to 70 ◦C and then stirred at rt overnight. The
1,4,7,10-Tetraazacyclododecane-4,7,10-triacetic-1-{methyl
[(4-nitrophenyl)methyl)]phosphinic acid} (6)
The H3DO3A·H2SO4 (5·H2SO4) (6.4 g, 14.4 mmol) was dissolved
in azeotropic HCl (70 ml) and phosphinic acid 4 (11.6 g,
57.6 mmol) was added. The suspension was heated to 80 ◦C
and paraformaldehyde (3.45 g, 115 mmol) was added in small
portions over 6 h. The mixture was then heated at 110 ◦C for 2
d. Subsequently, it was filtered and the filtrate was evaporated
to dryness in vacuum and co-distilled with water (3 × 150 ml) to
remove excess HCl. The residue was dissolved in water (20 ml)
and poured on top of a Dowex 50 column (3 × 15 cm, H+-form).
The column was washed with water (500 ml), 50% aqueous
EtOH (1500 ml) and aqueous ammonia (5%, 200 ml). The crude
product was eluted with ammonia. The fraction was evaporated
to dryness and the residue was dissolved in a small amount
of water (5 ml). It was chromatographed on Amberlite CG50
column (5 × 20 cm, H+-form) with water elution. Fractions
containing the pure (1H and 31P NMR) product 6 were combined
and evaporated to dryness. The residue was dissolved in water
(5 ml) and the solution was dropped into stirred anhydrous
EtOH (500 ml). The suspension was stirred overnight, filtered
and washed with EtOH (30 ml) and diethylether (30 ml). The
light yellow product was dried at rt in air overnight to yield 4.7 g
(53%) of 6·3H2O. dH (400 MHz; 90 ◦C; tBuOH); 3.04–3.19 (20 H,
m, ring CH2 + CH2–P–CH2); 3.46 (2 H, br s, CH2COOH); 3.63
(4 H, br s, CH2COOH); 7.38 (2 H, m, aryl); 8.08 (2 H, m, aryl); dc
(100.6 MHz; 90 ◦C; tBuOH) 36.6 (1 C, d, JPC 79.4); 46.1 (2 C,br
s); 46.8 (2 C, br s); 47.8 (2 C, br s); 47.9 (2 C, br s); 48.5 (1 C, d, JCP
88.5); 51.3 (1 C, br s); 53.4 (2 C, br s); 120.975 (2 C, s); 127.9 (2 C,
d, JCP 4.2); 140.2 (1 C, d, JCP 8); 143.3 (1 C, d, JCP 3) 168.3 (2 C, br
s); 170.7 (1 C, br s); dP (161.9 MHz; 90 ◦C; ext. 85% H3PO4) 33.8
(br s); m / z (ESI/MS) 560.2 (M + H)+, C22H35N5O10P requires
560.5; 581.1 (M + Na)+, C22H34N5NaO10P requires 581.5; Found:
C, 41.96; H, 7.20; N, 10.76. Calc. for C22H34N5O10P·3H2O: C,
43.07; H, 6.57; N, 11.41%
1
complexes for H and 31P NMR spectroscopy were isolated in
the solid state; the slight excess of LnCl3·xH2O was added to the
solution of DO3APABn followed by pH adjustment to 7. After
the pH stabilization the mixture was heated at 50 ◦C overnight.
The complexes were purified on Amberlite CG50 with water
elution. All solutions were tested negative in the presence of free
lanthanide(III) ions by using xylenol orange as an indicator (in
0.1 M NaAc/HAc buffer solution, pH 5.2). The concentration
of lanthanide(III) ions in complex solutions was determined by
1
measuring the H NMR shift caused by the change of bulk
magnetic susceptibility.24
1H relaxometry
The water proton 1/T1 longitudinal relaxation rates (10 MHz,
25 and 37 ◦C) were measured with a Stelar Spinmaster Spec-
trometer FFC relaxometer (Mede, Pv, Italy; installed at the
Laboratorio Integrato di Metodologie Avanzate, Bioindustry
Park del Canavese (Colleretto Giacosa, Torino, Italy)) on 0.8–
1.2 mM aqueous solution of the complex. The 1H spin–lattice
relaxation times T1 were acquired by the standard inversion
recovery method with typical 90◦ pulse width of 3.5 ls, 16
experiments of 4 scans. The reproducibility of the T1 data was
5%. The temperature was controlled with a Stelar VTC-91 air-
flow heater equipped with a copper–constantan thermocouple
(uncertainty of 0.1 ◦C). The 1/T1 NMRD profiles of water
protons were measured over a continuum of magnetic field
strength from 0.00024 to 0.70 T (corresponding to 0.01–30 MHz
proton Larmor frequency) on the fast field-cycling relaxometer.
The relaxometer operates under complete computer control
with an absolute uncertainty in the 1/T1 values of 1%. The
concentration of the aqueous solutions of the complexes utilized
for the measurements was in the range 1.0–5.0 mM.
17O relaxation measurements
1,4,7,10-Tetraazacyclododecane-4,7,10-triacetic-1-{methyl
[(4-aminophenyl)methyl)]phosphinic acid} DO3APABn
Variable-temperature 17O NMR relaxation measurement were
performed at 500 MHz Bruker AM-500 (11.7 T, 67.8 MHz)
spectrometer and Bruker VT-1000 temperature control unit was
used to stabilize the temperature. The complex solutions were
enriched by addition of H217O (2.6% Yeda, Israel) to overall
ca. 0.1% 17O concentration. Transversal 17O NMR relaxation
rates were measured by standard Carr–Purcell–Meiboom–Gill
spin echo pulse sequence; 8–10 increments on d2 exponentially
sampled; at = 0.2; d1 = 0.2 (d2 is delay time corresponding to the
time of echo; at is acquisition time; d1 is repetition time). These
data were compared with those calculated from the line-width
at half-height. The deviations were lower that 5%. These NMR
spectra were conducted without frequency lock.
Macrocycle 6·3H2O (2.5 g, 4.1 mmol) was dissolved in water (150
ml). Several drops of azeotropic HCl and 10% Pd/C (0.5 g) were
added. The flask was filled with hydrogen, and the nitroderivative
was hydrogenated at rt and 1 atm for 2 d. The catalyst was
filtered off and the filtrate evaporated to dryness in vacuum.
Chromatography on Amberlite CG50 as for 6 gave fractions of
pure ligand which were combined and water was removed in
vacuum. The residue was dissolved in water (3 ml) and dropped
into stirred anhydrous EtOH (500 ml). The suspension was
stirred overnight, filtered and washed with EtOH (30 ml) and
diethylether (30 ml). The product was dried in air at rt overnight
to yield 2.2 g (91%) of DO3APABn·3H2O. dH (400 MHz: D2O;
◦
90 C; tBuOH) 2.96 (2 H, d, JPH 16, H16), 3.01 (2 H, d, H13,
Data evaluation
JPH 4), 3.05 (4 H, br s, H6), 3.18 (4 H, br m, H3), 3.20 (4 H,
br m, H2), 3.22 (4 H, br m, H5), 3.45 (2 H, br m, H15), 3.65
(4 H, br m, H14), 7.24 (2 H, m, H18), 7.32 (2 H, m, H17); dC
1
The treatment of the H NMR T1 and17O NMR T2 data were
performed with Micromath Scientist fitting routines based on
◦
(100.6 MHz: D2O; 90 C; tBuOH) 39.1 (1 C, d, C16, JCP 73.4)
SBM equations (refer to supplementary information†).1,19
49.1 (2 C, s, C6); 49.6 (2 C, s, C2); 51 (1 C, d, C13, JCP 96.6);
51.2 (2 C, s, C3); 51.4 (2 C, s, C5) 54.4 (1 C, s, 15); 56.7 (2 C, s,
C14); 123.3 (2 C, d, 19, JCP 2.7); 129.1 (1 C, s, C20); 131.6 (2
C, d, C18, JCP 5.3) 135.6 (1 C, d, C17, JCP 7.2); dP (161.9 MHz;
D2O; 90 ◦C; ext. 85% H3PO4) 32.5 (br s); m / z (ESI/MS) 552.2
(M + Na)+, C22H36N5NaO8P requires 552.0; 530.3 (M + H)+,
C22H36N5O8P requires 530.5; 359.3 (M − P(O)(OH)–NO2Bn)+,
C15H27N4O6 requires 359.4; Found: C, 45.70; H, 7.19; N, 11.67.
Calc. for C22H36N5O8P·3H2O: C, 45.28; H, 7.25; H, 12.00%
Acknowledgements
We thank Bracco SpA for a kind gift of H3DO3A·H2SO4.
Support from the Grant Agency of Czech Republic (No.
203/020493 and No. 203/03/0168), the Ministry of Edu-
cation of Czech Republic (CZEJ07/98:1491000111), GAUK
(423/2004/B-CH/PrF) and MUIR (FIRB) is acknowledged.
The work was done in frame of COST D18 Action.
Complex preparation
Lanthanide(III) complexes of DO3APABn for 17O and 1H NMR
relaxometric measurements were prepared by mixing a 1 : 1.1
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O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 1 2 – 1 1 7
1 1 7