pH-dependent modulation of relaxivity and luminescence in macrocyclic gadolinium and europium complexes based on reversible intramolecular sulfonamide ligation

Article abstract of DOI:10.1021/ja0103647

A series of macrocyclic Eu, Gd, and Tb complexes has been prepared in which the intramolecular ligation of a β-arylsulfonamide nitrogen is rendered pH-dependent, giving rise to changes in the hydration state, q, at the lanthanide center. In complexes base

Full text of DOI:10.1021/ja0103647

J. Am. Chem. Soc. 2001, 123, 7601-7609
7601
pH-Dependent Modulation of Relaxivity and Luminescence in
Macrocyclic Gadolinium and Europium Complexes Based on
Reversible Intramolecular Sulfonamide Ligation
Mark P. Lowe,† David Parker,*,† Ofer Reany,† Silvio Aime,‡ Mauro Botta,§
Giancarlo Castellano,†,‡ Eliana Gianolio,‡ and Roberto Pagliarin|
Contribution from the Department of Chemistry, UniVersity of Durham, South Road,
Durham, DH1 3LE, U.K., Dipartimento di Chimica IFM, UniVersita` degli Studi di Torino, Via P. Giuria,
10125 Torino, Italy, Dipartimento di Scienze e` Tecnologie AVanzate, UniVersita` del Piemonte-Orientale,
Corso Borsalino 54, 10131 Alessandria, Italy, and Dipartimento di Chimica Organica e` Industriale,
UniVersita` di Milano, Viale Venezian 21, 20133 Milano, Italy
ReceiVed February 9, 2001
Abstract: A series of macrocyclic Eu, Gd, and Tb complexes has been prepared in which the intramolecular
ligation of a â-arylsulfonamide nitrogen is rendered pH-dependent, giving rise to changes in the hydration
state, q, at the lanthanide center. In complexes based on DO3A, variation of the p-substituent in the
arylsulfonamide moiety determines the apparent protonation constant log KMLH with values of 5.7, 6.4, and
6.7 for the -CF3, -Me, and -OMe substituents, respectively. Introduction of three â-carboxyalkyl substituents,
R to three ring nitrogens, inhibits displacement of the bound water by added protein and also suppresses
intermolecular binding by endogenous anions (lactate, HCO3-). Measurements of the pH dependence of the
form and intensity of the Eu complexes revealed that intramolecular carboxylate coordination occurred
competitively. This was reduced either by enhancing the electron density at the sulfonamide nitrogen or by
enlarging the chelate ring from 7-8. Amplification of the relaxivity changes in the pH range 8-5 occurred on
protein binding, and over the pH range 7.4-6.8 a 48% change in relaxivity was defined for [Gd‚3a] (298 K,
65.6 MHz) in 50% human serum solution.
Magnetic resonance imaging (MRI) is an established clinical
modality for defining changes in tissue anatomy that may arise
from disease or injury. In the late 1980s, a series of extracellular
gadolinium contrast agents emerged which allowed contrast to
be enhanced between tissues containing differing water con-
centrations.1,2 A second generation of paramagnetic contrast
agents is now beginning to emerge, in which the relaxivity of
the contrast agent is a defined function of a particular biochemi-
cal variable. Thus, Ca2+-dependent gadolinium complexes have
been devised for application in vitro to problems in develop-
mental biology.3 A series of EuII (f 7) complexes has also been
reported which may be expected to exhibit pO2 or time-
dependent relaxivity4salthough this has yet to be proven in an
in vivo model. Attention has also turned to pH-responsive
agents. Such work has been stimulated by the observation that
the extracellular pH of tumors (ca. 6.8-6.9) is more acidic than
both tumor intracellular pH (7.2) and normal extracellular tissue
(ca. 7.4). Although this acidity has been ascribed to overex-
pression of lactate5 it may be related to a change in the
equilibrium position of the Na+/H+ exchanger.6 Such acidity
has important consequences: it may stimulate metastasis7 and
it may enhance the uptake of weakly acidic chemotherapeutic
drugs.
The implementation of a pH-dependent contrast agent for use
in vivo requires that several criteria are satisfied. In addition to
being nontoxic (hence, kinetically stable with regard to the
biological half-life) and relatively cheap, the agent should exhibit
pH-dependent relaxivity modulation in the applied field range
of 20-80 MHzscorresponding to the field strengths of most
commercial MRI instruments. However, as the intensity of the
image will be directly proportional to the local contrast agent
concentration, either the distribution of the complex in the tissue
must be determined or a difference image must be obtained,
for example following sequential administration of two contrast
agents with different relaxivity/pH profiles but identical (or very
similar) tissue biodistributions.7 Evidently, the practicalities
associated with the application of pH-dependent contrast agents
require that a closely related series of well-tolerated complexes
be examined in detail.
† University of Durham.
‡ Universita` degli Studi di Torino.
§ Universita` del Piemonte-Orientale.
| Universita` di Milano.
(1) The Chemistry of Contrast Agents in Medical Magnetic Resonance
Imaging; Merbach, A. E., Toth, E., Eds.; Wiley: New York, 2001.
(2) (a) Caravan, P.; Ellison, J.; McMurry, T. J.; Lauffer, R. B. Chem.
ReV. 1999, 99, 2293. (b) Aime, S.; Botta, M.; Fasano, M.; Terreno, M.
Chem. Soc. Rev., 1998, 27, 19. (c) Lauffer, R. B. Chem. ReV., 1987, 87,
901.
The different ways in which the relaxivity of a given contrast
agent may be modulated can be appreciated by consideration
(3) Li, W. H.; Fraser, S. E.; Meade, T. J. J. Am. Chem. Soc., 1999, 121,
1413.
(4) (a) Burai, L.; Toth, E.; Seibig, S.; Scopellitti, R.; Merbach, A. E.
Chem. Eur. J. 2000, 6, 3761 (b) For related work on pO2-dependent MnII/III
complexes see: Aime, S.; Botta, M.; Gianolio, E.; Terreno, E. Angew.
Chem., Int. Ed. 2000, 39, 747.
(5) Newell, K.; Franchi, A.; Ponyssegur, J.; Tannock, I. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 1127.
(6) Mclean, J. A.; Roscoe, J.; Jorgensen, N. K.; Gorin, F. A.; Cala, P.
M. Am. J. Physiol. 2000, 278, C676.
(7) Beauregard, D. A.; Parker, D.; Brindle, K. M. Proc. Int. Soc. Magn.
Reson. Med. 1998, 6, 53.
10.1021/ja0103647 CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/17/2001

7602 J. Am. Chem. Soc., Vol. 123, No. 31, 2001
Lowe et al.
of the equations defining the dependence of the water proton
relaxation rate, R1. The paramagnetic contribution (eq 1) is made
up of outer-sphere, second-sphere and inner-sphere terms, of
which the inner-sphere is usually the most significant.1,2
Scheme 1
SS
R1p ) R1pOS + R1pIS + R1p
(1)
(2)
cq
1
IS
mide nitrogen to a zinc center fall in the pH range 5-7.5.15
With this in mind, we sought to append a sulfonamide nitrogen
â to a ligand nitrogen donor and hence allow a variation in the
coordination environment of the Ln center as a function of pH
(Scheme 1). Control over the facility of complex protonation
may be exerted by variation of the N electron density which,
in turn, is determined by the nature of the substituent R′. Because
europium complexes exhibit a metal-based emission which is
particularly sensitive to coordination environment, both Eu and
Gd complexes of a series of sulfonamido-ligands have been
prepared and the pH dependence of their emission spectra and
relaxivity behavior has been defined, in the presence and absence
of serum or added protein.
R1p
)
[
]
55.6 T1m + τm
The inner sphere contribution is a function of four variables
(eq 2): the concentration of the complex, c; the number of
directly bound water molecules, q; the exchange lifetime of the
coordinated water molecule, τm; the longitudinal water proton
relaxation time, T1m. Gadolinium complexes in which the bound
water proton exchange rate is a function of pH have been defined
recently,8 in which deprotonation of the coordinated water proton
is assisted by a high charge density at the lanthanide center.9
Labilization of the water molecule itself may be rendered pH-
dependent in cases where water exchange is a function of the
nature of proximate anionic groups which, in turn, define the
structure and dynamics of the second sphere of hydration.8a,b,10
Within the complex series of terms that control the value of
Results and Discussion
The initial target complexes, prepared in order to prove the
concept, were the Eu and Gd complexes of the DO3A-derived16
ligands 1a-1c. It was appreciated at the outset that, when the
T
1m, the pH dependence of the rotational correlation, time τr,
may be picked out as an example. Conjugates of gadolinium
complexes have been devised in which a large pH-dependent
change in the conformation and molecular volume of a
11
macromolecule occurs, thereby perturbing τr and hence T1m
.
Finally, if a change in the hydration state of a gadolinium
complex is rendered pH-dependent, then R1p may be varied
significantly. Such a variation may be amplified if the complex
is noncovalently associated with a macromolecule, provided that
τm remains relatively fast. These PRE effects are usually largest
for field strengths in the range 10 f 100 MHz. Therefore, if a
pH-dependent agent is sought for application at higher fields
(e.g., at 300 or 400 MHz and hence higher spatial resolution),
then it is more appropriate to seek a system which does not
bind strongly to an endogenous macromolecule. Preliminary
examples of lanthanide complexes possessing pH dependence
of the hydration state have begun to appear,12 including cases
where reversible binding of hydrogen carbonate allows switching
between differently hydrated complexes over the pH range
5-8.9,13,14
sulfonamide nitrogen was not bound to the Gd ion, then binding
of either endogenous anions12,14 or of protein (e.g. via chelation
of amide carbonyl or carboxylate groups) might displace the
bound water and hence suppress the change in relaxivity.17
Therefore, the related series of ligands 2a-2c and 3a,3b were
also prepared. In each case, the nature of the p-substituent in
We report herein examples of gadolinium complexes exhibit-
ing pH-dependent relaxivity by virtue of a switch in hydration
state, associated with the on/off ligation of a sulfonamide
nitrogen donor.12 Early work by Kimura established that the
protonation constants associated with the binding of a sulfona-
(8) (a)Aime, S.; Barge, A.; Botta, M.; Parker, D.; de Sousa, A. S. J. Am.
Chem. Soc. 1997, 119, 4767. (b) Aime, S.; Barge, A.; Bruce, J. I.; Botta,
M.; Howard, J. A. K.; Moloney, J. M.; Parker, D.; de Sousa, A. S.; Woods,
M. J. Am. Chem. Soc. 1999, 121, 5672. (c) Hall, J.; Haner, R.; Aime, S.;
Botta, M.; Faulkner, S.; Parker, D.; de Sousa, A. S. New J. Chem. 1998,
22, 627.
(9) Note that the pH-dependent labilization of exchangeable protons that
are close to the paramagnetic center may constitute a large contribution to
the second-sphere term, R1pSS; for examples see ref 8b and Aime, S.; Barge,
A.; Botta, M.; Howard, J. A. K.; Kataky, R.; Lowe, M. P.; Moloney, J. M.;
Parker, D.; de Sousa, A. S.; Chem. Commun. 1999, 1047.
(10) Zhang, S. R.; Wu, K. C.; Sherry, A. D. Angew. Chem., Int. Ed.
1999, 38, 3192.
(11) Aime, S.; Botta, M.; Crich, S. G.; Giovenzana, G.; Palmisano, G.;
Sisti, M. Chem. Commun. 1999, 1577.
(12) Parker, D.; Lowe, M. P. Chem. Commun. 2000, 707.
(13) Aime, S.; Batsanov, A. S.; Botta, M.; Howard, J. A. K.; Lowe, M.
P.; Parker, D. New J. Chem. 1999, 23, 669.
(14) Bruce, J. I.; Dickins, R. S.; Govenlock, L. J.; Gunnlaugsson, T.;
Lopinski, S.; Lowe, M. P.; Parker, D.; Peacock, R. D.; Perry, J. J. B.; Aime,
S.; Botta, M. J. Am. Chem. Soc. 2000, 122, 9674.
the arylsulfonamide was considered likely to determine the
sensitivity of the derived lanthanide complex to protonation.
The presence of the carboxyalkyl substituents had earlier been
demonstrated to inhibit intermolecular anion binding at a
coordinatively unsaturated lanthanide center.14
(15) (a) Koike, T.; Kimura, E.; Nakamura, I.; Hashimoto, Y.; Shiro, M.
J. Am. Chem. Soc. 1992, 114, 7338. (b) Koike, T.; Kimura, E. Chem. Soc.
ReV. 1998, 27, 179.
(16) DO3A is 1,4,7-tris(carboxyethyl)-1,4,7,10-tetraazacyclododecane;
the tris[R-carboxyethyl]substituted ligand 2 is given the acronym gDO3A,
and the homologue 3, is aDO3A.
(17) Protein binding to Gd DO3A derivatives produces q ) 0 species:
Aime, S.; Gianolio, E.; Terreno, E.; Pagliarin, R.; Giovenzana, G. B.; Lowe,
M. P.; Sisti, M.; Palmisano, G.; Parker, D.; Botta, M. J. Biol. Inorg. Chem.
2000, 5, 488.

pH-Dependent Contrast Agents
J. Am. Chem. Soc., Vol. 123, No. 31, 2001 7603
The synthesis of the desired sulfonamide-functionalized
ligands relied upon the established ring-opening reaction of
N-sulfonylaziridines with secondary amines.18 For example,
Figure 1. Europium emission spectra for [Eu‚1b] at pH 10 (more
intense) and pH 4, revealing the changes in the hypersensitive ∆J ) 2
(ca. 616 nm) and ∆J ) 4 (e.g., 682 nm) transitions (λexc 270 nm; 295
K, H2O, I ) 0.1 M NaCl).
reaction of equimolar quantities of N-p-methoxyphenylsulfonyl-
aziridine 7c (prepared by treatment of the corresponding
sulfonate ester, 8c with base) with 1,4,7-tris(tert-butoxycarbon-
ylmethyl)-1,4,7,10-tetraazacyclododecane, 4, in hot acetonitrile
followed by deprotection with CF3CO2H afforded the desired
ligand 1c. In the case of the ligands 2a-2c and 3a,3b, the
products were isolated as a mixture of the RRR/SSS, RSR/SRS,
and RRS/SSR stereoisomers. Lanthanide complexes were
prepared in aqueous solution following reaction of the ligand
with hydrated LnCl3 at pH 6 (18 h, 90 °C). The purification of
[Ln‚1], [Ln‚2], and [Ln‚3] involved extraction of the lyophilized
complex into 5-40% MeOH/CH2Cl2 at pH 5, The purified
complexes were rather hygroscopic, but each gave a satisfactory
high-resolution electrospray mass spectrum (negative ion mode).
Analysis of the 1H NMR spectrum of [Eu‚1c] in basic media
(277 K, pD 10, 300 MHz) revealed resonances for the most
shifted ring axial protons at 37.5, 30.7, 27.9, and 18.9 ppm.
Such valuessand the shift pattern of the remaining shifted ligand
protonssare similar to those observed for monoamide tricar-
boxylate19 complexes of europium and to similar neutral
monopyridyltriacetate complexes reported more recently.13 They
are consistent with a mono-capped square-antiprismatic coor-
dination environment about the lanthanide center. The proton
NMR spectra for [Eu‚1] recorded in more acidic media were
much less well-defined: exchange broadened spectra were
observed (300 MHz, pD 4) over the temperature range 30° to
-5 °C and the form of the spectra closely resembled those
defined for N-alkylated complexes of DO3A17 in which
cooperative arm rotation occurs at a relatively fast rate on the
NMR time-scale. The 1H NMR spectra of the Eu complexes of
2 and 3 were more complex, owing to the presence of the three
stereoisomeric species (RRS, RRR, RSR and their enantiomers).
In the case of [Eu‚2c], a sample enriched in the RRR/RRS
species (ca. 70/30) was prepared following partial chromato-
graphic separation of the precursor esters. In this case, the NMR
spectra recorded at pD 4 and 10 were similar in general form
to those obtained with [Eu‚1], except that at lower pD the
spectrum exhibited reduced line-broadening consistent with
some degree of inhibition of arm rotation caused by substitution
R to the ring nitrogen. Such effects have been defined in detail
recently in each of the stereoisomeric tetra {R-(carboxyalkyl)}
series of complexes.20
Table 1. Radiative Rate Constants k (ms-1, (10%) and Derived
Hydration States, q, for Decay of Europium Luminescence at
Limiting pH/pD Values (295 K, I ) 0.1 M NaCl)
pH/pD 4
pH/pD 10
complex
kH O
kD O
qEu
kH O
kD O
qEu
2
2
2
2
[Eu‚1a]
[Eu‚1b]
[Eu‚1c]
[Eu‚2c]
[Eu‚3a]
2.35
2.27
2.32
2.21
2.44
0.76
0.70
0.66
0.65
0.75
1.6
1.6
1.7
1.6
1.7
1.09
1.20
1.43
1.05
0.94
0.74
0.80
0.93
0.72
0.65
0.1
0.2
0.3
0.1
0.05
a Values of q were estimated using the equation: qEu ) 1.2[(kH O
-
2
k
D O) -0.25] which allows for the contribution of unbound water
2
molecules.22
Infrared spectra of lyophilized samples (pH 4 and 10) were
recorded in methanol solution and, in the solid state, in a KBr
matrix and compared to values for the free ligand. Under both
sets of conditions, strong sulfur-oxygen stretching bonds were
observed at 1329 (asymmetric) and 1159 (symmetric) cm-1. The
lack of variation of these stretching frequencies (ligand versus
complex, solid-state versus solution) suggests that the sulfonyl
oxygen does not participate in metal binding. In contrast, in a
recent report of the intramolecular ligation of a dansyl sulfon-
amide group at a cationic yttrium center, N-O chelation was
revealed with well-defined shifts in the sulfonyl-stretching
frequencies.21 Further information about the nature of the
lanthanide coordination environment as a function of pH was
provided by examination of the Eu emission spectrum following
direct (λexc 397 nm) or indirect (λexc 270 nm) excitation.
Profound changes in the form and relative intensity of the
hypersensitive ∆J ) 2 and ∆J ) 4 transitions were observed
(Figure 1) as a function of pH for [Eu‚1]; similar spectra were
recorded at the acidic (pH 3) and basic (pH 10) limits with
[Eu‚2] and [Eu‚3]. Spectra recorded at pH 7 f 10 were much
more intense than those measured in more acidic media.
Independent measurements of the radiative rate constants for
decay of the Eu excited state were undertaken in H2O and D2O
at pH/pD 4 and 10 (Table 1). This allowed an estimation of the
hydration state at Eu.22 In each case q was near zero in more
alkaline media and was greater than 1 (1.6-1.7) in the acidic
limit. The more intense emission observed at higher pH was
(18) Martin, A. E.; Ford, T. M.; Bulkowski, J. E. J. Org. Chem. 1982,
47, 412; Chandrasekhar, S.; McAuley, A. J. Chem. Soc., Dalton Trans.
1992, 2967; Howard, C. C.; Marckwald, W. Chem. Ber. 1899, 32, 2036.
(19) Monoamide-triacetate DOTA-Ln complexes typically reveal >90%
of the square-antiprismatic isomer in solution; this isomer undergoes water
exchange more slowly than the twisted square-antiprismatic isomer in which
the faster exchange is ascribed to a later transition state for water interchange
(independent of complex charge or structure)20: Aime, S.; Anelli, P. L.;
Botta, M.; Fedeli, F.; Grandi, M. P.; Padi, P.; Uggeri, F. Inorg. Chem. 1992,
31, 2422.
(21) The chelation of a sulfonamide dansyl group has been defined at a
tripositive Y center: Aoki, S., Kawatani, H., Goto, T., Kimura, E., Shiro,
M. J. Am. Chem. Soc. 2001, 123, 1123; in analogous anionic Eu and Tb
complexes with a lower charge demand at the metal, the sulfonamide acts
as a monodentate N donor: Lowe, M. P., Parker, D. Inorg. Chim. Acta
2001, 317, 163.
(20) 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.
(22) Beeby, A.; Clarkson, I. M.; Dickins, R. S.; Faulkner, S.; Parker,
D.; Royle, L.; de Sousa, A. S.; Williams, J. A. G.; Woods, M. J. Chem.
Soc., Perkin Trans. 2 1999, 493.

7604 J. Am. Chem. Soc., Vol. 123, No. 31, 2001
Lowe et al.
Figure 2. pH Dependence of the europium luminescence at 612 nm
for [Eu‚1](I/Io, ∆J ) 2; 295 K; I ) 0.1M NaCl); the line shows the fit
to the experimental data points for the estimated protonation constants.
Figure 3. Correlation of the pH-dependent relaxivity of [Gd‚1b] [open
circles] (20 MHz, 298 K) and the luminescence behavior of [Eu‚1b]
[filled circles] (298 K, I/Io at 612 nm).
quantified in terms of the overall quantum yield for sensitized
emission with [Eu‚2c] (λexc 270 nm): at pH 10, æem ) 0.4%,
and at pH 4, æem ) 0.1%.23 The relatively low values obtained
here reflect the competitive deactivation of the intermediate aryl
singlet excited state caused by charge transfer to the EuIII center.
With [Eu‚2a] at pH 10, a higher emission quantum yield of
0.8% was measured, reflecting the partial suppression of this
deactivating process as the p-CF3 substituent renders the aryl
singlet less readily oxidized.
The pH variation of the change in the intensity of the ∆J )
2 transition at 612 nm for [Eu‚1] revealed a sigmoidal curve
(Figure 2) characteristic of a simple two-state equilibrium
process. Analysis by iterative least-squares fitting afforded
apparent protonation constants (log KMLH) of 5.7, 6.4, and 6.7
for the p-CF3-, Me-, and OMe-substituted sulfonamide Eu
complexes. This variation is in accord with the increase in
electron density at the nitrogen.
Similar results ((0.05 log KMLH) were obtained by analyzing
the intensity changes at 682 nm which are also likely to be
associated with sulfonamide nitrogen coordination to the eu-
ropium center. Taken together with the observed pH-dependent
NMR and IR spectra, the behavior of the simple DO3A
derivatives is consistent with a binding equilibrium between a
q ) 0, nitrogen-bound species, and a q > 1 species which
predominates in acidic media (Figure 2 and Scheme 1).24
The relaxivity of [Gd‚1] was recorded as a function of pH in
the range 4-10 (20 MHz, 298 K). At low pH, the limiting
relaxivity was of the order of 7.5 mM-1 s-1 and fell to a value
of 2.0 mM-1 s-1 at pH 10. Such behavior (Figure 3) mirrors
the pH dependence of luminescence emission found with the
analogous Eu complexes. Such a comparison highlights the
utility of examining the Eu luminescence behavior in assessing
the likely relaxivity profile. Examination of the properties of
these lanthanide DO3A complexes served to vindicate the
appropriateness of the pH-dependence of the on/off ligation
mechanism involving the sulfonamide nitrogen. This determines
the local hydration at the lanthanide. However, prior work had
established that N-alkylated Gd-DO3A complexes are not good
candidates for use in vivo. They are not sufficiently stable with
respect to metal dissociation1,2 to allow their safe use. Further-
more, noncovalent protein bindingsfacilitated by interaction of
the aryl moiety to serum albuminshas been shown to lead to
displacement of the metal-bound waters, reducing any relaxivity
change.17 Therefore the [Ln‚2]n- and [Ln‚3]n- complexes were
examined, as such R-N-alkylated complexes exhibit greater
kinetic and thermodynamic stability with respect to metal ion
dissociation.25 In addition, the peripheral carboxyalkyl substit-
uents have been shown to inhibit binding of endogenous anions,
present in serum, such as lactate (2.3 mM), HCO3- (ca. 27 mM),
and HPO4 (ca. 0.9 mM).14
2-
pH Dependence of Luminescence and Relaxivity for
[Ln‚2]n- and [Ln‚3]n-. The variation of luminescence emission
intensity with pH for europium and terbium complexes was
examined in 0.1 M NaCl solution and in a simulated extracel-
lular ionic background, that is a solution containing additionally
2.3 mM lactate, 0.13 mM citrate, 30 mM HCO3-, and 0.9 mM
phosphate. For the terbium complexes, although some slight
changes in spectral form were evident at the two limiting pH
values (i.e., pH 3 and 10), differences were masked by the
spectral complexity inherent in terbium emission spectra,
deriving from the multiplicity of allowed transitions from the
5D4 excited-state manifold. Such complexity is absent in the
corresponding Eu emission spectra. In addition the use of direct
excitation (λexc 397 nm) averts any complications that arise from
differing efficiencies of sensitization for chemically distinct
lanthanide species.
With each of the [Eu‚2] complexes, three distinctively
different europium emission spectra were observed (Figures 4
and 5). In the pH range 6-9, sulfonamide N ligation was
characterized by large changes in the form and intensity of the
hypersensitive ∆J ) 2 (e.g., 614 nm) and ∆J ) 4 (e.g., 682
nm) manifolds, with an apparent log KMLH around 6.7 for
[Eu‚2c] and 5.7 for [Eu‚2a]. Further changes were observed in
the pH range 6-3, associated with an increase in emission
intensity at 617 and 594 nm. For the ∆J ) 2 band, a maximum
in the emission intensity was observed around pH 5 for [Eu‚2c],
[Eu‚2b], and [Eu‚2a] (Figure 5). The spectrum recorded at
intermediate pH (inset to Figure 4), represents a chemically
distinct species, as it is not just a superposition of the spectra
recorded at pH 10 (sulfonamide N-bound) and at pH 3. In more
acidic media, (pH < 4), the spectra resembled those observed
for the complex [Eu‚1], where the carboxyalkyl side chain is
not present. The observed pH/intensity changes were also found
to be independent of the composition of the salt background.
Taken together, such behavior may be rationalized in terms
of the formation of an intermediate carboxylate-bound species,
involving intramolecular ligation of one â-carboxyethyl sub-
(23) For [Tb‚2c], at pH 10 æem ) 3.5% (λexc 270 nm) and at pH æem
)
0.17%; in this case the combined effect of the proximity of the chromophore
(enhancing the efficiency of intramolecular energy transfer) and the absence
of quenching of the Tb 5D4 excited state by OH oscillators serve to enhance
the emission quantum yield for the N-bound complex.
(24) Kang, S. I.; Ranganathan, R. S.; Emswiler, J. E.; Kumar, K.;
Gougoutas, J. Z.; Malley, M. F.; Tweedle, M. F. Inorg. Chem. 1993, 32,
2912.
(25) Details of emission spectral changes and individual wavelength
variations for selected Eu complexes are given in the Supporting Informa-
tion.

pH-Dependent Contrast Agents
J. Am. Chem. Soc., Vol. 123, No. 31, 2001 7605
Figure 6. Correlation of the pH-dependence of relaxivity for [Gd‚2c]
[open circles] (298 K, 20 MHz) with emission intensity changes for
[Eu‚2c] [filled circles] (295 K, I ) 0.1 M NaCl, λexc 397 nm, Iem 614
nm).
Figure 4. Changes in the europium emission spectrum for [Eu‚2c] at
pH 9.93, (line) 5.49 (__ __ __) and 3.00 (- - -) following direct excitation
of the Eu3+ ion at 397 nm, revealing the presence of three chemically
distinct species.
Figure 7. Variation of relaxivity (298 K, 20 MHz, I ) 0.1 MNaCl)
with pH for [Gd‚1a] [triangles], [Gd‚2a] [squares], and [Gd‚3a]
[circles], highlighting the competition between sulfonamide N and side-
chain carboxylate binding.
carboxylate ligation may occur even at pH > 7, leading to a
flatter relaxivity/pH profile (Figure 7). In comparison with
[Gd‚1a], the anionic complex [Gd‚2a] reveals a steadily
increasing relaxivity value from pH 9 to 5 as the mean hydration
state increases. In more acidic media protonation of the ligating
carboxylate occurs leading to a preponderance of a q ) 2
species, associated with the higher relaxivity value of ca. 8.5
observed below pH 3.
Figure 5. Spectral emission intensity changes with pH for [Eu‚2c]
characterizing sulfonamide nitrogen binding (614 nm; left) and in-
tramolecular carboxylate participation (617 nm; right) (295 K, I ) 0.1M
NaCl); very similar profiles were observed in a simulated extracellular
ionic background.
Intramolecular carboxylate participation may be suppressed
either by reducing the nucleophilicity of the carboxylate oxygen
or by enlarging the chelate ring by adding spacer groups to the
pendant arm. An example of the latter class is provided by the
complexes of ligand 3, with an additional methylene group in
the substituents R to the ring N. The variation of Eu emission
intensity with pH for [Eu‚3a] and [Eu‚3b] revealed significant
differences compared to the “gDO3A” analogues. The pH
change in emission intensity at 614 and 682 nm (N-binding)
for the p-CF3 substituted complex revealed an apparent pro-
tonation constant, log KMLH, of 6.1, and the corresponding value
for the p-OMe example was 7.0. Little change in the form of
these pH profiles was observed in a competitive anion back-
ground. Examination of the intensity variation for [Eu‚3b] at
617 nmscharacterizing carboxylate ligationsrevealed no de-
fined intensity maximum at pH 5. The change in the emission
intensity change at 617 nm, between pH 7.4 and pH 5, differed
by 60 and 250% compared to [Eu‚2a] and [Eu‚2c] respectively.
The pH dependence of the relaxivity of [Gd‚3a] (20 MHz,
298 K) was simpler in form than for [Gd‚2a]. An increase from
r1p ) 2.7 mM-1 s-1 at pH 9 (q ) 0) to 10.9 mM-1 s-1 at pH
5. Such a relaxivity value suggests that a q ) 2 complex
predominates in solution below pH 5. This variation was also
examined at 65.6 MHz in a competitive anion background, and
the observed change (Figure 9) was similar with limiting values
of 2.5 mM-1 s-1 (pH 8.5) and 8.7 mM-1 s-1 (pH 5).
stituent, generating a seven-ring chelate.26 Protonation of the
carboxylate oxygen in the complex occurs significantly below
pH 4, leading to the same nine-coordinate “diaqua”-europium
species observed for [Eu‚1].
Support for this hypothesis was provided by examining the
pH-dependence of the relaxivity for the corresponding gado-
linium complexes and comparing with the pH-dependent
changes found in the Eu series. Correlation of the pH/relaxivity
changes for [Gd‚2c] with the pH dependence of the emission
intensity profile for [Eu‚2c] reveal a near mirror-image relation-
ship (Figure 6). Such behavior is consistent with the idea that
increases in r1p are directly proportional to the hydration state
q, while the Eu emission intensity decreases in direct proportion
to it. Moreover, the behavior in the range 9-5 supports the
idea that carboxylate ligation and sulfonamide N binding are
in competition. Therefore, by decreasing the donor ability of
either the sulfonamide N or the carboxylate O, the position of
the equilibrium distribution of complexes should be perturbed.
A simple example is provided by the p-CF3 complex [Gd‚2a]:
the lesser nucleophilicity of the sulfonamide N leads to enhanced
competition from the carboxylate oxygen at higher pH. Thus,
(26) The pH dependence of the emission spectral profile for [Eu‚2c] was
unchanged when using a stereoisomeric mixture containing a 87/13 mixture
of RRR/RRS isomers (compared to the 70/30 ratio used elsewhere).

7606 J. Am. Chem. Soc., Vol. 123, No. 31, 2001
Lowe et al.
Figure 8. Dependence of the relaxivity of [Gd‚2a] (circles) and [Gd‚2c]
(squares) in the absence (open) and presence (filled) of 1.5 mM serum
albumin (20 MHz, 298 K).
Figure 9. Variation of the relaxivity of [Gd‚3a] (65.6 MHz, 298 K,)
with pH in a simulated extracellular anion background (open circles,
0.5 mM complex) and in the presence of human serum solution, diluted
by a half (filled circles, 0.7 mM complex). Very similar behavior was
observed with an 0.2 mM solution of complex in 100% serum, with
an increase of 37% in relaxivity between pH 7.4 and pH 6.8.
Independent measurement of q on the analogous Eu complexes
using luminescence methods (Table 1) gave values of 0.1 for
[Eu‚3a] in the N-bound form at pH 8.5, and q ) 1.7 at pH 4.
Similar values ((0.2) were recorded for [Eu‚3b].
6.8 was only 7%, whereas in the human serum solution the
enhancement of relaxivity was 15%. The corresponding percent-
age enhancements for [Gd‚3a], measured under identical condi-
tions, were 35 and 48% respectively (Figure 9). With [Gd‚3b],
the relaxivity value in the anionic background changed by 19%
and in serum by 20% between pH 7.4 and 6.8. Such
behaviorswith a significant percentage change over the physi-
ologically important pH change at a clinically relevant magnetic
field strengthsaugurs well for the development of such pH-
responsive contrast agents for their application in devising pH
maps in MR images. The different slopes of relaxivity/pH for
structurally similar complexesstuned by the nature of the
sulfonamide group or the ring N “sidearm” substituentssuggests
that it may be feasible to produce ratios of intensity maps to
eliminate the concentration dependence of the primary MR
“intensity” images.
The NMRD profile for [Gd‚3a] was recorded at pH 5 (298
K) and was parametrized in terms of τm ) 35 ns (a value
measured independently by standard VT 17O NMR methods),
τr ) 1.86 × 10-10 s, τv ) 2.1 × 10-11 s, q ) 1.7, and ∆2 )
4.5 × 10-19 s-2. Taken together, these NMRD, luminescence
and relaxivity measurements are consistent with a significantly
reduced degree of intramolecular carboxylate ligation in [Ln‚3]
which is apparently not compromised by intermolecular anion
binding.
Behavior in the Presence of Protein and Serum. In the
“field” range 20-100 MHz, the noncovalent interaction of a
gadolinium complex undergoing relatively fast water exchange,
for example, kex ) 2 × 107 s-1, with a more slowly tumbling
macromolecule may lead to pronounced relaxivity enhance-
ments, provided that the bound water is not displaced. Such an
effect has been established1,2 for several anionic gadolinium
complexes bearing an aryl residue. Typically such anionic
complexes form a complex with serum albumin with an apparent
Experimental Section
Reagents and Solvents. Acetonitrile was dried over calcium hydride
before use. Water and H2O refer to high purity water with conductivity
e0.04 µS cm-1, obtained from the PURITE purification system.
1,4,7,10-Tetraazacyclododecane is commercially available (Strem) and
was used as received.
affinity constant of the order of 104 M-1 1,2
.
Relaxivity measurements at 20 MHz, probing the interaction
of [Gd‚2a] and [Gd‚2c] with human serum albumin (1.5 mM),
revealed modest but significant enhancements (Figure 8). An
increase in the slope of the relaxivity/pH plot was observed over
the range 7.5-6.5. This change was more marked for the p-OMe
derivative [Gd‚2c]. Such behavior may be related to an enhanced
binding affinity of the complex with the unbound arylsulfon-
amide. At lower pH, the sulfonamide N is not coordinated, and
the aryl moiety is more sterically accessible to interact with
the protein. When the sulfonamide N is bound to Gd, the
structure of the complex is much more compact, and the aryl
group possesses much less conformational freedom to interact
with a protein binding pocket. The enhancement of relaxivity
in the presence of albumin was less pronounced around pH 7
for the p-CF3 analogue, [Gd‚2a] (Figure 8). In this case, the
greater extent of intramolecular carboxylate binding in this pH
range reduces slightly the PRE effect. With [Gd‚3a], the
relaxivity enhancement in the presence of serum albumin was
larger still. An r1p value of 19.4 mM-1 s-1 was measured in
the presence of 1.5 mM serum albumin at pH 5 compared to
13 mM-1 s-1 for [Gd‚2a].
Chromatography. Column chromatography was carried out using
“gravity” silica (Merck). Cation exchange chromatography was per-
formed using Dowex 50W 50 × 4-200 strong ion-exchange resin, which
had been pretreated with 3 M HCl.
1
Spectroscopy. H NMR spectra were recorded at 65.6 MHz on a
1.53 T magnet connected to a Varian VXR400 console, at 199.99 MHz
on Varian Mercury-200 and at 299.91 MHz on Varian Unity-300. 13
C
NMR were recorded on the Varian Mercury-200 at 50.2 MHz and a
Varian Unity-300 at 75.4 MHz. Mass spectra were recorded using a
VG Platform II electrospray mass spectrometer with methanol or water
as a carrier solvent. Accurate masses were determined at the EPSRC
National MS Service at Swansea; isotope calculations for Gd and Eu
complexes refer to 153Eu and 158Gd. Luminescence measurements for
europium and terbium complexes were recorded using an Instrument
SA Fluorolog 3-11 using DataMax for Windows v2.1. Second-order
diffraction effects were obviated by using a 375 nm cutoff filter.
pH measurements were made using a Jenway 3320 pH meter (fitted
with a BDH Glass + combination electrode-microsample) calibrated
with pH 4, 7, and 10 buffer solutions.
(27) Measurements of the pH dependence (pH 4-9) of the Eu emission
spectrum for [Eu‚3b] were undertaken in 0.1 M NaCl, in a simulated clinical
anion background and in 50% aqueous human serum solution. A very similar
spectral form and relative emission intensity was observed at a given pH
value, in each case (λexc ) 397 nm). Such behavior strongly suggests that
the Eu coordination environment is very similar in each of these situations,
supporting the conclusions drawn from the pH dependence of Gd relaxivity.
Measurements of relaxivity for [Gd‚3a] and [Gd‚3b] were
also undertaken at 65.6 MHz, examining the pH range 10-4
in a clinical anion background and in 50% human serum
solution.27 In a simulated extracellular “anion” background, the
percentage change in relaxivity for [Gd‚2c] between pH 7.4 and

pH-Dependent Contrast Agents
J. Am. Chem. Soc., Vol. 123, No. 31, 2001 7607
The longitudinal water proton relaxation rates were measured either
using a Stelar Spinmaster (Mede, Pavia, Italy) spectrometer, operating
at 0.47 T or a modified Varian VXR instrument tuned to 65.6 MHz
(1H), by means of the standard inversion-recovery technique (16
experiments, 2 scans). A typical 90° pulse width was 3.5 µs, and the
reproducibility of the T1 data was (0.5%. The temperature was
controlled with a Stelar VTC-91 air-flow heater equipped with a
copper-constantan thermocouple (uncertainty (0.1 °C). The concen-
tration of the complex was typically 0.1-0.4 mM in deionized water.
The proton 1/T1 NMRD profiles were measured for 1-2 mM
solutions over a continuum of magnetic field strength from 0.00024-
0.28 T (corresponding to 0.01-12 MHz proton Larmor Frequency) on
a Stelar field-cycling relaxometer. The relaxometer works under
complete computer control with an absolute uncertainty in 1/T1 of (1%.
Data points at 0.47 T (20 MHz) and 2.1 T (90 MHz) were added to the
experimental NMRD profiles and were recorded on the Stelar Spin-
master and on a JEOL EX-90 spectrometer, respectively.
reduced pressure and column chromatography on silica [gradient elution;
CH2Cl2 to 4% MeOH-CH2Cl2, Rf ) 0.36 (10% MeOH-CH2Cl2)]
yielded the tert-butyl ester of 1c as a colorless oil (0.14 g, 42%).
1
ESMS + (m/z) ) 728 [M + H]+. H NMR (300 MHz, CDCl3, δ):
7.70 (2H, d, ArH), 6.91 (2H, d, ArH), 6.09 (1H, t, NH), 3.80 (3H, s,
OCH3), 3.37 (2H, s, CH2NSO2), 3.40-2.05 (24H, br, CH2N), 1.44 (18H,
s, C(CH3)3), 1.41 (9H, s, C(CH3)3). 13C NMR (75.4 MHz, CDCl3, δ):
173.0, 172.9 (CO2), 163.1, 130.4, 129.4, 114.5 (Ar), 83.1, 82.6
(C(CH3)3), 56.6, 56.0, 55.9, 53.7, 52.4, 49.9 (CH2N), 54.0-47.0 (br,
CH2N), 28.3, 28.1 (C(CH3)3).
A solution containing the tert-butyl ester (0.10 g, 0.14 mmol) in
80% trifluoroacetic acid-CH2Cl2 (10 mL) was stirred for 5 h. Solvents
were removed under reduced pressure followed by the addition and
then evaporation of CH2Cl2 (3 × 10 mL) and finally ether (10 mL).
The residue was taken up in H2O, filtered, and lyophilized to give a
white solid of the ditrifluoroacetate salt (0.08 g, 77%). ESMS +
1
(m/z) ) 560 [M + H]+. H NMR (300 MHz, D2O, δ): 7.62 (2H, d,
pH Titrations. Luminescence pH titrations were carried out in a
background of constant ionic strength (I ) 0.1 NaCl, 295 K) on
solutions with absorbances of <0.3 at wavelengths gλex to avoid any
errors due to the inner filter effect. Solutions were made basic by
addition of 1 M NaOH and titrated to acidic pH using small aliquots
(typically 0.5 µL) of 1 M or 0.1 M HCl. The luminescence spectra
were recorded at each point (30-40 points per titration). Excitation
wavelengths of 270 nm (sensitization via phenyl chromophore) or 397/
355 nm (direct excitation at Eu/Tb) were used to obtain the lumines-
cence spectra. Excitation and emission slits were 2-5 and 1 nm band-
pass, respectively. Points were recorded at 1 nm intervals with a 0.25
s integration time.
Standard least-squares fitting techniques were used to determine
protonation constants from the luminescence intensity data.
Compounds 4, 5, 7b, and 8b were synthesized by published
procedures.14,18 Compounds 7a, 8a, 7c, 8c, 7d, and 8d were prepared
using the published method.18
ArH), 6.95 (2H, d, ArH), 3.88 (2H, br, CH2NSO2), 3.70 (3H, s, OCH3),
3.60-2.80 (24H, br, CH2N). 13C NMR (75.4 MHz, D2O, δ): 176.7,
172.8 (CO2), 163.1, 129.7, 129.3, 115.0 (Ar), 56.9 (OCH3), 56.2, 55.9,
52.1, 51.3, 50.9, 49.4, 48.7, 38.7 (aliphatic C).
1-[2′-(4-Trifluoromethylphenylsulfonylamino)ethyl]-4,7,10-tris(car-
boxymethyl)-1,4,7,10-tetraazacyclododecane (1a). The compound was
synthesized by a method similar to that of 1c.
1
tert-Butyl ester of 1a: ESMS + (m/z) ) 766 [M + H]+. H NMR
(300 MHz, CDCl3, δ): 8.05 (2H, d, ArH), 7.68 (2H, d, ArH), 7.22
(1H, t, NH), 3.41 (2H, s, CH2NSO2), 3.50-2.10 (24H, br, CH2N), 1.43
(18H, s, C(CH3)3), 1.40 (9H, s, C(CH3)3). 13C NMR (75.4 MHz, CDCl3,
δ): 177.7, 177.3 (CO2), 147.7, 132.6, 130.8 (Ar), 87.4, 87.0 (C(CH3)3),
61.3, 60.2, 58.0, 53.1, 45.7 (br CH2N), 32.8, 32.6 (C(CH3)3).
1a: ESMS + (m/z) ) 598 [M + H]+. 1H NMR (D2O, δ): 7.88 (2H,
d, ArH), 7.80 (2H, d, ArH), 3.60-2.80 (26H, br, CH2N). 13C NMR
(D2O, δ): 177.0, 171.2 (CO2), 141.9, 127.6, 126.9 (Ar), 56.8, 55.9,
52.0, 51.6, 50.8, 48.2, 48.3, 38.9 (aliphatic C).
2-(4-Trifluoromethylphenylsulfonylamino)ethyl(4-trifluorometh-
ylphenylsulfonate) (8a): 1H NMR (300 MHz, CDCl3, δ) 8.02 (2H, d,
ArH), 7.97 (2H, d, ArH), 7.85 (2H, d, ArH), 7.80 (2H, d, ArH), 5.06
(1H, t, NH), 4.17 (2H, t, CH2O), 3.34 (2H, q, CH2N).
1-[2′-(4-Methylphenylsulfonylamino)ethyl]-4,7,10-tris(carboxy-
methyl)-1,4,7,10-tetraazacyclododecane (1b). The compound was
synthesized by a method similar to that of 1c.
1
tert-Butyl ester of 1b: ESMS + (m/z) ) 712 [M + H]+. H NMR
2-(4-Methoxyphenylsulfonylamino)ethyl(4-methoxyphenylsul-
1
(300 MHz, CDCl3, δ): 7.73 (2H, d, ArH), 7.31 (2H, d, ArH), 3.38
(2H, s, CH2NSO2), 3.42-2.41 (24H, br, CH2N), 2.26 (3H, s, CH3),
1.45 (18H, s, C(CH3)3), 1.42 (9H, s, C(CH3)3).
1b: ESMS + (m/z) ) 544 [M + H]+. 1H NMR (D2O, δ): 7.62 (2H,
d, ArH), 7.33 (2H, d, ArH), 3.60-2.95 (26H, br, CH2N), 2.28 (3H, s,
CH3).
fonate) (8c): H NMR (300 MHz, CDCl3, δ) 7.78 (2H, d, ArH), 7.73
(2H, d, ArH), 6.98 (2H, d, ArH), 6.92 (2H, d, ArH), 5.03 (1H, t, NH),
4.04 (2H, t, CH2O), 3.88 (3H, s, OCH3), 3.86 (3H, s, OCH3), 3.20
(2H, q, CH2N).
2-(Phenylsulfonylamino)ethyl(phenylsulfonate) (8d): 1H NMR
(300 MHz, CDCl3, δ) 7.84 (4H, m, ArH), 7.66 (6H, m, ArH), 5.07
(1H, t, NH), 4.07 (2H, t, CH2O), 3.24 (2H, q, CH2N).
1-[2′-(4-Methoxyphenylsulfonylamino)ethyl]-4,7,10-tris[(3′-car-
boxyl)-1′-carboxypropyl]-1,4,7,10-tetraazacyclododecane (2c). A
solution containing 5 (0.34 g, 0.53 mmol), 7c (0.12 g, 0.56 mmol) and
Na2CO3 (0.06 g, 0.57 mmol) in dry acetonitrile (15 mL) was heated at
reflux for 18 h. The solution was filtered and the solvent removed under
reduced pressure. Column chromatography on silica [gradient elution;
CH2Cl2 to 4% MeOH-CH2Cl2, Rf ) 0.40 (10% MeOH-CH2Cl2)]
yielded the hexamethyl ester of 2c as a colorless oil (0.25 g, 55%).
N-(4-Trifluoromethylphenylsulfonyl)aziridine (7a): 1H NMR (300
MHz, CDCl3, δ) 8.04 (2H, d, ArH), 7.77 (2H, d, ArH), 2.39 (4H, s,
CH2NCH2).
N-(4-Methoxyphenylsulfonyl)aziridine (7c): 1H NMR (300 MHz,
CDCl3, δ) 7.90 (2H, d, ArH), 7.00 (2H, d, ArH), 3.89 (3H, s, CH3O),
2.36 (4H, s, CH2NCH2).
N-(Phenylsulfonyl)aziridine (7d): 1H NMR (300 MHz, CDCl3, δ)
7.96 (2H, m, ArH), 7.60 (3H, m, ArH), 2.40 (4H, s, CH2NCH2).
1,4,7-Tris-[(4′-methoxycarbonyl)-1′-methoxycarbonylbutyl]-1,4,7,10-
tetraazacyclododecane (6). 1,4,7,10-Tetraazacyclododecane (0.50 g,
2.9 mmol), 6 (2.24 g, 8.9 mmol) and K2CO3 (1.0 g, 7.0 mmol) were
heated under argon at 60 °C in dry acetonitrile (15 mL) for 48 h. The
solids were removed by filtration, and the solvent was removed under
reduced pressure. Column chromatography on silica [gradient elution;
CH2Cl2 to 30% THF-CH2Cl2 to 2.5% MeOH-30% THF-67.5%
CH2Cl2, Rf ) 0.30 (70% THF-CH2Cl2)] yielded a pale yellow oil (0.75
g, 38%). ESMS + (m/z) ) 689 [M + H]+. 1H NMR (300 MHz, CDCl3,
δ): 3.65-3.69 (18H, 4 × s, CO2CH3), 3.24-2.80 (4H, br, CH), 2.80-
2.35 (24H, br, NCH2), 1.85-1.60 (16H, br, CH2). 13C NMR (75.4 MHz,
CDCl3, δ): 173.5, 170.9 (CO2), 63.5, 59.0, 51.5, 51.3, 51.0, 46.2, 33.2,
30.0, 21.0 (br, aliphatic C).
1
ESMS + (m/z) ) 860 [M + H]+. H NMR (300 MHz, CDCl3, δ):
7.51 (2H, d, ArH), 6.91 (2H, d, ArH), 3.97 (3H, s, ArOCH3), 3.66
(2H, s, CH2NSO2), 3.61 (18H, s, OCH3), 3.31 (3H, t, CH), 3.21-1.63
(30H, br, CH2N). 13C NMR (75.4 MHz, CDCl3, δ): 176.7, 176.5, 176.1,
175.8 (CO2), 165.7, 133.4, 132.4, 117.5 (Ar), 65.1, 58.7, 56.7, 54.7
(br), 54.3, 53.5 (aliphatic C), 33.8 (CO2CH3).
A suspension of the hexamethyl ester (0.21 g, 0.24 mmol) in 1 M
LiOH (10 mL) was heated at 80 °C for 18 h. The resulting solution
was loaded onto a DOWEX 50W strong acid cation-exchange column
(H+ form), washed with water, and eluted with 12% aqueous ammonia
solution. The solvent was removed under reduced pressure, and the
residue was taken up in water and then lyophilized to give a white
powder of the diaqua-diammonium salt of 2c (0.17 g, 84%). ESMS +
1
(m/z) ) 776 [M + H]+. H NMR (D2O, δ): 7.60 (2H, d, ArH), 6.93
1-[2′-(4-Methoxyphenylsulfonylamino)ethyl]-4,7,10-tris(carboxy-
methyl)-1,4,7,10-tetraazacyclododecane (1c). A solution containing
4 (0.23 g, 0.45 mmol) and 7c (0.10 g, 0.47 mmol) in dry acetonitrile
(25 mL) was heated at reflux for 18 h. The solvent was removed under
(2H, d, ArH), 3.70 (3H, s, OCH3), 3.60-1.50 (35H, br, CH2N, CH).
13C NMR (D2O, δ): 186.8, 186.1, 185.9, 185.7 (CO2), 167.7, 133.9,
133.8, 119.5 (Ar), 68.7, (br, aliphatic C), 60.4 (OCH3), 55.7, 51.5, 39.4
(br, aliphatic C).

7608 J. Am. Chem. Soc., Vol. 123, No. 31, 2001
Lowe et al.
1-[2′-(4-Trifluoromethylphenylsulfonylamino)ethyl]-4,7,10-tris[(3′-
carboxyl)-1′-carboxypropyl]-1,4,7,10-tetraazacyclododecane (2a).
The compound was synthesized by a method similar to that of 2c.
Hexamethyl ester of 2a: ESMS + (m/z) ) 898 [M + H]+. 1H NMR
(300 MHz, CDCl3, δ): 8.10 (2H, d, ArH), 7.72 (2H, d, ArH), 3.63
(18H, s, OCH3), 3.60-1.70 (35H, br, CH2N). 13C NMR (75.4 MHz,
CDCl3, δ): 173.3, 172.9 (CO2), 149.2, 127.7, 126.1, (Ar), 62.1, 53.5,
51.6, (br, aliphatic C), 30.6 (CO2CH3).
2a: ESMS + (m/z) ) 814 [M + H]+. 1H NMR (300 MHz, D2O, δ):
7.92 (2H, d, ArH), 7.85 (2H, d, ArH), 3.80-1.60 (35H, br, CH2N,
CH). 13C NMR (75.4 MHz, D2O, δ): 181.5, 180.9, (CO2), 146.5, 130.2,
129.4 (Ar), 66.3, 49.1, 33.4, 26.2 (br, aliphatic C).
the salt residues with 5% MeOH-CH2Cl2 and filtered, and the solvent
was removed under reduced pressure. The residue was taken up in water
and lyophilized to give a white powder (14 mg, 90%). HR-ESMS (m/
z): [M - H]- calcd for C23H33N5O9S1Eu1, 708.1211; found, 708.1210.
1H NMR (300 MHz, D2O pD ) 10, 277 K, δ): partial assignment
37.5 (1H, s, ring CH axial), 30.7 (1H, s, ring CH axial), 27.9 (1H, s,
ring CH axial), 18.9 (1H, s, ring CH axial), typical of a square-
antiprismatic geometry about Eu, the remaining resonances span 8.8
to -31.1 ppm. IR (KBr and MeOH cm-1) νSO ) 1329 (asymm), 1159
(symm).
Europium(III) 1-[2′-(4-Trifluoromethylphenylsulfonylamino)ethyl]-
4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane, [Eu‚1a]:
HR-ESMS (m/z): [M - H]- calcd for C23H30F3N5O8S1Eu1, 746.0980;
found, 746.0982. 1H NMR (300 MHz, D2O pD ) 10, 277 K, δ): partial
assignment 36.8 (1H, s, ring CH axial), 30.6 (1H, s, ring CH axial),
28.8 (1H, s, ring CH axial), 20.4 (1H, s, ring CH axial), typical of a
square-antiprismatic geometry about Eu, the remaining resonances span
12.2 to -29.3 ppm.
Europium(III) 1-[2′-(4-Methylphenylsulfonylamino)ethyl]-4,7,10-
tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane, [Eu‚1b]: HR-
ESMS (m/z): [M - H]- calcd for C23H33N5O8S1Eu1, 692.1262; found,
692.1258. 1H NMR (300 MHz, D2O pD ) 10, 277 K, δ): partial
assignment 39.0 (1H, s, ring CH axial), 31.9 (1H, s, ring CH axial),
29.2 (1H, s, ring CH axial), 19.8 (1H, s, ring CH axial), typical of a
square-antiprismatic geometry about Eu, the remaining resonances span
8.9-31.9 ppm.
Europium(III) 1-[2′-(4-Methoxyphenylsulfonylamino)ethyl]-4,7,10-
tris[(3′-carboxyl)-1′-carboxypropyl]-1,4,7,10-tetraazacyclododec-
ane, [Eu‚2c]: HR-ESMS (m/z): [M - H]- calcd for C32H45N5O15S1Eu1,
924.1845; found, 924.1842. 1H NMR (300 MHz, D2O pD ) 10, 298K,
δ): partial assignment 36.7 (1H, s, ring CH axial), 35.7 (1H, s, ring
CH axial), 33.7 (1H, s, ring CH axial), 27.4 (1H, s, ring CH axial),
typical of a square-antiprismatic geometry about Eu, the remaining
resonances span 10.9 to -29.8 ppm.
1-[2′-(Phenylsulfonylamino)ethyl]-4,7,10-tris[(3′-carboxyl)-1′-car-
boxypropyl]-1,4,7,10-tetraazacyclododecane (2b). The compound was
synthesized by a method similar to that of 2c.
1
Hexamethyl ester of 2b: ESMS + (m/z) ) 830 [M + H]+. H
NMR (300 MHz, CDCl3, δ): 7.90 (2H, m, ArH), 7.48 (3H, m, ArH),
3.62 (18H, s, OCH3), 3.60-1.75 (35H, br, CH2N). 13C NMR (75.4 MHz,
CDCl3, δ): 173.2, 172.9 (CO2), 167.4, 132.3, 129.7, 127.1, (Ar), 51.7,
(br, aliphatic C), 30.7 (CO2CH3).
2b: ESMS + (m/z) ) 746 [M + H]+. 1H NMR (300 MHz, D2O, δ):
7.74 (2H, m, ArH), 7.55 (3H, m, ArH), 3.70-1.65 (35H, br, CH2N,
CH). 13C NMR (75.4 MHz, D2O, δ): 183.3, 183.1, (CO2), 140.3, 136.5,
132.4, 129.5 (Ar), 67.1, 54.2, 49.3, 40.5, 37.1, 26.1 (br, aliphatic C).
1-[(2′-(4-Methoxyphenylsulfonaminoethyl]-4,7,10-tris-[(4′-carboxy)-
1′-carboxybutyl]-1,4,7,10-tetraazacyclododecane (3b). A solution of
6 (0.106 g, 0.15 mmol), 7c (0.035 g, 0.16 mmol) and Na2CO3 (0.017
g, 0.16 mmol) in dry acetonitrile (5 mL) was heated at reflux for 48 h.
The solution was filtered and the solvent removed under reduced
pressure. Following column chromatography on silica [gradient elution;
CH2Cl2 to 30% THF-CH2Cl2, Rf ) 0.45 (30% THF-CH2Cl2)] the
hexamethyl ester of 3b was obtained as a pale yellow oil (0.118 g,
85%).. HR-ESMS + (m/z): [M - H]+ calcd for C41H67N5O15S1Na1,
924.4243; found, 924.4252.
A suspension of the hexamethyl ester (0.118 g, 0.13 mmol) in 1 M
LiOH (10 mL) was heated at 80 °C for 18 h. The resulting solution
was loaded onto a DOWEX 50W strong acid cation-exchange column
(H+ form), washed with water, and eluted with 12% aqueous ammonia
solution. The solvent was removed under reduced pressure, and the
residue taken up in water and then lyophilized to give a white powder
of the diaqua-diammonium salt of 3b (0.067 g, 65%). ESMS (m/z) )
817 [M - H]-. 1H NMR (200 MHz, D2O, δ): 7.70 (2H, d, ArH), 7.00
(2H, d, ArH), 3.37 (3H, m, CH), 3.30-2.09 (26H, br, CH2N), 1.90-
1.20 (12H, b, CH2CH2CO2). 13C NMR (50.3 MHz, D2O, δ): 185.4-
184.8 (CO2), 165.7, 131.9, 117.5, (Ar), 66.8-66.0, (br, aliphatic C),
58.4 (OCH3), 54.0-49.6 (br, aliphatic C), 39.8, 32.0, 26.2 (br, aliphatic
C).
1-[(2′-(4-Trifluoromethylphenylsulfonaminoethyl]-4,7,10-tris-[(4′-
carboxy)-1′-carboxybutyl]-1,4,7,10-tetraazacyclododecane (3a). Com-
pound 3a was synthesized in a manner similar to that for 3b.
ESMS (m/z) ) 854 [M - H]-. 1H NMR (200 MHz, D2O, δ): 7.99
(4H, m, ArH), 3.46 (3H, m, CH), 3.30-2.09 (26H, br, CH2N, CH2CO2),
1.90-1.20 (12H, b, CH2CH2CO2). 13C NMR (50.3 MHz, D2O, δ):
184.7-180.7 (CO2), 144.0, 136.6, 130.2, 129.4, 128.8, (Ar), 68.5-
66.0, (br, aliphatic C), 58.4 (OCH3), 51.2-49.7 (br, aliphatic C), 39.4,
33.0-28.6, 25.8 (br, aliphatic C).
Lanthanide Complexes. All of the complexes were synthesized
using the method outlined for Eu[1c] using the appropriate LnCl3‚6H2O
starting material. For Ln[2a-c] and Ln[3a,b] the complexes were
extracted from the lyophilized salt mixture using 20% MeOH-CH2Cl2
and 40% MeOH-CH2Cl2, respectively. Each complex was rather
hygroscopic and was stored in a desiccator under inert gas.
Europium(III) 1-[2′-(4-Methoxyphenylsulfonylamino)ethyl]-4,7,10-
tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane, [Eu‚1c]. An
aqueous solution (10 mL) containing 1c (13 mg, 22 µmol) and
EuCl3‚6H2O (10 mg, 27 µmol) was adjusted to pH 5.5 using 1 M NaOH
and heated at 90 °C for 18 h. After the solution was cooled, the pH
was raised to 10.0, and the solution was filtered through a Celite plug
to remove the excess europium as Eu(OH)3. The pH was then lowered
to 5.0 and the solution lyophilized. The compound was extracted from
Europium(III) 1-[2′-(4-Trifluoromethylphenylsulfonylamino)ethyl]-
4,7,10-tris[(3′-carboxyl)-1′-carboxypropyl]-1,4,7,10-tetraazacyclodo-
decane, [Eu‚2a]: HR-ESMS (m/z): [M - H]- calcd for C32H42F3N5-
O14S1Eu1, 962.1614; found, 962.1618. 1H NMR (300 MHz, D2O
pD ) 10, 298 K, δ): partial assignment 36.3 (1H, s, ring CH axial),
35.8 (1H, s, ring CH axial), 33.4 (1H, s, ring CH axial), 28.3 (1H, s,
ring CH axial), typical of a square-antiprismatic geometry about Eu,
the remaining resonances span 11.2 to -28.8 ppm.
Europium(III) 1-[2′-(Phenylsulfonylamino)ethyl]-4,7,10-tris[(3′-
carboxyl)-1′-carboxypropyl]-1,4,7,10-tetraazacyclododecane, [Eu‚2b]:
HR-ESMS (m/z): [M - H]- calcd for C31H43N5O14S1Eu1, 894.1740;
found, 894.1737. 1H NMR (300 MHz, D2O pD ) 10, 298K, δ): partial
assignment 36.0 (1H, s, ring CH axial), 35.5 (1H, s, ring CH axial),
33.2 (1H, s, ring CH axial), 27.2 (1H, s, ring CH axial), typical of a
square-antiprismatic geometry about Eu, the remaining resonances span
10.8 to -29.8 ppm.
Europium(III) 1-[(2′-(4-Methoxyphenylsulfonaminoethyl]-4,7,10-
tris-[(4′-carboxy)-1′-carboxybutyl]-1,4,7,10-tetraazacyclododec-
ane, [Eu‚3b]: HR-ESMS (m/z): [M - H]- calcd for C35H51N5O15S1Eu1,
966.2330; found, 966.2315. 1H NMR (D2O pD ) 10, 298K, δ): partial
assignment 40.8 (1H, s, ring CH axial), 32.8 (1H, s, ring CH axial),
31.6 (1H, s, ring CH axial), 28.0 (1H, s, ring CH axial), typical of a
square-antiprismatic geometry about Eu, the remaining resonances span
12.7 to -32.2 ppm.
Europium(III) 1-[(2′-(4-Trifluoromethylphenylsulfonaminoethyl]-
4,7,10-tris-[(4′-carboxy)-1′-carboxybutyl]-1,4,7,10-tetraazacyclodo-
decane, [Eu‚3a]: HR-ESMS (m/z): [M - H]- calcd for C35H48F3N5-
O14S1Eu1, 1004.2083; found, 1004.2081. 1H NMR (D2O pD ) 10, 298
K, δ): partial assignment 40.6 (1H, s, ring CH axial), 33.1 (1H, s, ring
CH axial), 28.6 (2H, s, ring CH axial), typical of a square-antiprismatic
geometry about Eu, the remaining resonances span 14.5 to -28.6 ppm.
Gadolinium(III) 1-[2′-(4-Methoxyphenylsulfonylamino)ethyl]-
4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane, [Gd‚1c]:
HR-ESMS (m/z): [M - H]- calcd for C23H33N5O9S1Gd1, 713.1240;
found, 713.1243.

pH-Dependent Contrast Agents
J. Am. Chem. Soc., Vol. 123, No. 31, 2001 7609
Gadolinium(III) 1-[2′-(4-Trifluoromethylphenylsulfonylamino)-
ethyl]-4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,
[Gd‚1a]:HR-ESMS (m/z): [M - H]- calcd for C23H30F3N5O8S1Gd1,
751.1008; found, 751.1009.
Gadolinium(III) 1-[(2′-(4-Trifluoromethylphenylsulfonaminoethyl]-
4,7,10-tris-[(4′-carboxy)-1′-carboxybutyl]-1,4,7,10-tetraazacyclodo-
decane, [Gd‚3a]: HR-ESMS (m/z): [M - H]- calcd for C35H48F3N5O14-
S1Gd1, 1009.2112; found, 1009.2109.
Gadolinium(III) 1-[2′-(4-Methylphenylsulfonylamino)ethyl]-4,7,10-
tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane, [Gd‚1b]: HR-
ESMS (m/z): [M - H]- calcd for C23H33N5O8S1Gd1, 697.1291; found,
697.1295.
Gadolinium(III) 1-[2′-(4-methoxyphenylsulfonylamino)ethyl]-
4,7,10-tris[(3′-carboxyl)-1′-carboxypropyl]-1,4,7,10-tetraazacyclodo-
decane, [Gd‚2c]: HR-ESMS (m/z): [M + Na - 2H]- calcd for
C32H45N5O15S1Na1Gd1, 951.1694; found, 951.1701.
Gadolinium(III) 1-[2′-(4-Trifluoromethylphenylsulfonylamino)-
ethyl]-4,7,10-tris[(3′-carboxyl)-1′-carboxypropyl]-1,4,7,10-tetraaza-
cyclododecane, [Gd‚2a]: HR-ESMS (m/z): [M - H]- calcd for
C32H42F3N5O14S1Gd1, 967.1642; found, 967.1635.
Gadolinium(III) 1-[2′-(Phenylsulfonylamino)ethyl]-4,7,10-tris[(3′-
carboxyl)-1′-carboxypropyl]-1,4,7,10-tetraazacyclododecane, [Gd‚2b]:
HR-ESMS (m/z): [M - H]- calcd for C31H43N5O14S1Gd1, 899.1768;
found, 899.1761.
Terbium(III) 1-[2′-(4-Methoxyphenylsulfonylamino)ethyl]-4,7,10-
tris[(3′-carboxyl)-1′-carboxypropyl]-1,4,7,10-tetraazacyclododec-
ane, [Tb‚2c]: HR-ESMS (m/z): [M - H]- calcd for C32H45N5O15S1Tb1,
930.1887; found, 930.1883. 1H NMR (65 MHz, D2O pD ) 10, 298 K,
δ): partial assignment -442 (1H, s, ring CH axial), -505 (1H, s, ring
CH axial), -540 (1H, s, ring CH axial), -561 (1H, s, ring CH axial),
typical of a square-antiprismatic geometry about Eu, the remaining
resonances span 630 to -180 ppm.
Acknowledgment. We thank EPSRC and the COST D-18
Action for support, Dr. Kevin Brindle (Cambridge) for formative
discussions, and the reviewers for helpful comments.
Supporting Information Available: Several Figures show-
ing the pH dependence of Eu spectral emission and the relaxivity
of [Gd‚3a], in differing salt backgrounds (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
Gadolinium(III) 1-[(2′-(4-Methoxyphenylsulfonaminoethyl]-4,7,-
10-tris-[(4′-carboxy)-1′-carboxybutyl]-1,4,7,10-tetraazacyclododec-
ane, [Gd‚3b]: HR-ESMS (m/z): [M - H]- calcd for C35H51N5O15S1Gd1,
971.2344; found, 971.2334.
JA0103647