Gd(DOTAlaP): Exploring the boundaries of fast water exchange in gadolinium-based magnetic resonance imaging contrast agents

Article abstract of DOI:10.1021/ic5008928

Here, we describe the synthesis of the single amino acid chelator DOTAlaP and four of its derivatives. The corresponding gadolinium(III) complexes were investigated for their kinetic inertness, relaxometric properties at a range of fields and temperatures, water exchange rate, and interaction with human serum albumin (HSA). Derivatives with one inner-sphere water (q = 1) were determined to have a mean water residency time between 8 and 6 ns in phoshate-buffered saline at 37 °C. The corresponding europium complexes were also formed and used to obtain information on the hydration number of the corresponding coordination complexes. Two complexes capable of binding HSA were also synthesized, of which one, Gd(5b), contains no inner-sphere water, while the other derivative, Gd(4b), is a mixture of ca. 15% q =1 and 85% q = 0. In the presence of HSA, the latter displayed a very short mean water residency time (τ_M³¹⁰ = 2.4 ns) and enhanced relaxivity at intermediate and high fields. The kinetic inertness of Gd(4b) with respect to complex dissociation was decreased compared to its DOTAla analogue but still 100-fold more inert than [Gd(BOPTA)(H₂O)]^2-. Magnetic resonance imaging in mice showed that Gd(4b) was able to provide 38% better vessel to muscle contrast compared to the clinically used HSA binding agent MS-325.

Full text of DOI:10.1021/ic5008928

Article
pubs.acs.org/IC
Open Access on 06/12/2015
Gd(DOTAlaP): Exploring the Boundaries of Fast Water Exchange in
Gadolinium-Based Magnetic Resonance Imaging Contrast Agents
Eszter Boros,† Shima Karimi,‡ Nathaniel Kenton,† Lothar Helm,‡ and Peter Caravan*,†
†The Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard
Medical School, 149 Thirteenth Street, Suite 2301, Charlestown, Massachusetts 02129, United States
‡Laboratoire de Chimie Inorganique et Bioinorganique, Ecole Polytechnique Fed
́ ́
erale de Lausanne, EPFL-BCH, CH-1015 Lausanne,
Switzerland
S
* Supporting Information
ABSTRACT: Here, we describe the synthesis of the single amino acid chelator
DOTAlaP and four of its derivatives. The corresponding gadolinium(III) complexes
were investigated for their kinetic inertness, relaxometric properties at a range of
fields and temperatures, water exchange rate, and interaction with human serum
albumin (HSA). Derivatives with one inner-sphere water (q = 1) were determined to
have a mean water residency time between 8 and 6 ns in phoshate-buffered saline at
37 °C. The corresponding europium complexes were also formed and used to obtain
information on the hydration number of the corresponding coordination complexes.
Two complexes capable of binding HSA were also synthesized, of which one,
Gd(5b), contains no inner-sphere water, while the other derivative, Gd(4b), is a
mixture of ca. 15% q =1 and 85% q = 0. In the presence of HSA, the latter displayed a
310
very short mean water residency time (τM = 2.4 ns) and enhanced relaxivity at
intermediate and high fields. The kinetic inertness of Gd(4b) with respect to complex
dissociation was decreased compared to its DOTAla analogue but still 100-fold more inert than [Gd(BOPTA)(H2O)]2−.
Magnetic resonance imaging in mice showed that Gd(4b) was able to provide 38% better vessel to muscle contrast compared to
the clinically used HSA binding agent MS-325.
protein binding also serves to direct the contrast agent to
specific pathologies, e.g., thrombosis, as well as enhance the
relaxivity.8,9
INTRODUCTION
■
Magnetic resonance imaging (MRI) is a powerful noninvasive
imaging technique, providing high-resolution anatomical
images with excellent soft-tissue contrast. A large fraction of
MRI studies utilize a contrast agent, e.g., more than half the
clinical studies performed at our institution make use of a
gadolinium(III)-based contrast agent. These contrast agents are
simple, water-soluble, ternary gadolinium(III) complexes with
gadolinium(III) coordinated by an octadentate poly-
(aminocarboxylato) ligand and an aqua coligand.1,2
Gadolinium(III) is advantageous for use in MRI contrast
However, the increase in the relaxivity due to protein binding
is greatest by far at low magnetic fields (≤1.5 T) and falls off
precipitously at higher fields. Clinically, 3 T MRI is becoming
common, and the major equipment vendors all sell 7 T
scanners. For small-animal imaging, even higher field strengths
are common. At high fields (≥3 T), the proton Larmor
frequency is much faster than the tumbling rate of protein-
bound complexes but still much slower than the tumbling rate
of discrete monomeric complexes. We10−13 and others14−19
have reported multimeric contrast agents with increased high-
field relaxivity due to a tumbling rate intermediate between that
of protein-bound agents and small molecules. One specific
approach utilized the single amino acid chelate Gd-
(DOTAla).10,20 Multiple Gd(DOTAla) moieties could be
incorporated into oligopeptides of defined structure and size,
resulting in relaxivities tuned for high-field applications. The
modular nature of Gd(DOTAla) also lends itself to the design
of serum albumin-targeted contrast agents.20
7
agents because of its large electron spin number (S = /2) and
long electronic relaxation time, which result in efficient nuclear
relaxation of nearby solvent water molecules. Contrast agents
are characterized by their relaxivity, defined as the change in the
solvent relaxation rate (1/T1 or 1/T2) normalized to the
concentration of the contrast agent (units of mM−1 s−1). First-
generation clinical contrast agents are monomeric gadolinium-
(III) complexes based on a [Gd(DOTA)(H2O)]− or [Gd-
(DTPA)(H2O)]2− (DTPA = diethylenetriaminepentaacetic
acid) framework and have relaxivities in the range 3−5 mM−1
s−1.3,4 Second-generation contrast agents such as gadofosveset
(MS-325, Ablavar) used reversible protein binding to increase
the relaxivity.5 Protein binding slows the rotational tumbling
rate of the complex, and as this rate approaches the proton
Larmor frequency, nuclear relaxation is enhanced.6,7 Specific
Here, we report on the synthesis and properties of a modified
derivative of the DOTAla ligand, DOTAlaP. We sought to
Received: April 19, 2014
Published: June 12, 2014
© 2014 American Chemical Society
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Scheme 1. Synthetic Scheme for Reported Chelators 3, 4a, 4b, 5a, and 5b
improve the properties of the DOTAla ligand by replacing one
of the acetate moieties by methylenephosphonate. The
increased charge of the phosphonate is expected to increase
the solubility of the resultant gadolinium(III) complexes.
Gadolinium(III) complexes with phosphonate-containing
ligands often yield higher relaxivities than complexes of the
analogous carboxylate ligand due to an effect on water in the
second coordination sphere, and this second-sphere effect
persists at high fields.21 We also expected that inner-sphere
water exchange should be faster for the phosphonate-
substituted ligand. For gadolinium(III) complexes undergoing
water exchange via a dissociative mechanism, substitution of a
carboxylate with the bulkier phosphonate donor group results
in faster water exchange as the energy difference between the 8-
coordinate transition state and the 9-coordinate ground state
becomes smaller. On the other hand, this steric crowding can
also result in an 8-coordinate ground state with an inner-sphere
water ligand eliminated, which would result in lower relaxivity.
In this study, we sought to address the following questions:
(i) How does substitution of one of the acetate donors in
DOTAla for methylenephosphonate impact the hydration
number and water exchange kinetics and, in turn, the relaxivity?
(ii) How does phosphonate substitution affect kinetic inertness
with respect to gadolinium(III) dissociation? (iii) What is the
effect of amine versus carboxylate functionalization of the
propionamine arm of DOTAlaP on the hydration number,
water exchange kinetics, relaxivity, and gadolinium(III)
dissociation kinetics? (iv) Are any of these properties altered
when a Gd(DOTAlaP) derivative is bound to serum albumin?
(v) Does phosphonate substitution confer enhanced high-field
relaxivity properties and, if so, do these result in an increased in
vivo MRI signal?
propionamine arm. The gadolinium(III) complexes were
prepared and characterized with respect to variable-field,
variable-temperature relaxivity, water exchange kinetics, albu-
min binding studies, and gadolinium(III) dissociation kinetics.
Europium(III) complexes were also synthesized, and lumines-
cence lifetime measurements were performed to quantify the
hydration number of these complexes.
RESULTS
■
Synthesis. We synthesized DOTAlaP using a reaction
pathway similar to that of DOTAla. Benzyl 2-[[(benzyloxy)-
carbonyl]amino]-3-(1,4,7,10-tetraazacyclododecan-1-yl)-
propanoate represents an intermediate common to both
DOTAla and DOTAlaP, so we used the previously established
synthetic steps10 to furnish this first intermediate (Scheme 1).
In order to introduce a protected phophonate arm onto the
monoderivatized cyclen macrocycle, tri-tert-butyl phosphite was
synthesized according to ref 22 and appended using a Mannich-
type reaction. The reaction was monitored for disappearance of
the monofunctionalized macrocycle using liquid chromatog-
raphy−mass spectrometry (LC−MS), followed by filtration and
aqueous workup of the filtrate. The introduction of these two
chelator arms at this stage is key: because of their steric
hindrance, it is not possible to incorporate them efficiently at a
later stage. The crude intermediate containing the difunction-
alized macrocycle was then redissolved in acetonitrile and
subjected to alkylation with tert-butyl bromoacetate, followed
by preparative high-performance liquid chromatography
(HPLC) to purify the protected intermediate compound 1.
Orthogonal protection group chemistry provided us with a free
and ready-to-couple carboxylate and primary amine on
compound 2 after hydrogenation-mediated removal of the
carboxybenzyl (Cbz) and benzyl protecting groups of 1. The
overall number of steps starting from Cbz-NH-Ser-OH-OBn is
5, and compound 2 is obtained with an approximate 10%
overall yield based on the protected serine. The lower yield is
To address these questions, we synthesized the DOTAlaP
ligand by altering the previously established DOTAla syn-
thesis.10 Additionally, we prepared four DOTAlaP derivatives
substituted at either the amine or carboxylate of the
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Figure 1. Temperature dependence of the 17O NMR (11.7 T) reduced transverse relaxation rates of Gd(3) (6.73 mM, left) and Gd(5a) in PBS
(4.16 mM, right). The solid line represents a fit to the data to determine the mean water residency time τM.
Table 1. Summary of Experimentally Obtained Water Exchange Rates, Activation Enthalpies, and Mean Water Residency Time
at 310 K for Gd(3) and Gd(5a) in Comparison with Previously Reported Gadolinium(III) Complexes
Gd(3)
Gd(5a)
Gd(DOTAla)-OH28
Gd(DOTAla)-amide10
Gd(DOTA)29
Gd(DO3AP)30
298
k
ex
× 106 (s−1
)
97
3
103
2
23.9 1.8
6.4 0.1
61
39
8.6
29
42
17
4.6
49
71
76
4.1
ΔH⧧ (kJ mol−1
)
13.5 1.1
8.1 0.3
310τM (ns)
97
due to the formation of a greater number of side products
compared to the DOTAla synthesis. One single phosphonate
species (1,7-functionalized with respect to the aminopropa-
strength.24 On the other hand, Gd(3) and Gd(5a) had much
higher maximum r2 values consistent with an inner-sphere
O
water ligand (18 and 16 mM−1 s−1, respectively). Figure 1
shows the reduced transverse relaxation rate R2r as a function of
the reciprocal temperature. The crossover to exchange-limited
R2r occurs at a low temperature for both complexes, which
indicates the predicted, very fast water exchange kinetics. We
used a four-parameter model described previously25 to fit the
data and obtained water residency times at 37 °C of 310τM = 8.1
ns for Gd(3) and 310τM = 6.4 ns for Gd(5a); see Table 1. While
these water residency times are considerably shorter than those
seen for [Gd(DOTA)(H2O)]− and [Gd(DOTAla)(H2O)]−,
they are similar to the water residency time for [Gd(DO3AP)-
(H2O)]2−, a close structural analogue of DOTAlaP (Table 1).
The coordination spheres of Gd(3) and Gd(5a) differ in that
Gd(3) contains an anionic propionate oxygen donor and
Gd(5a) contains a neutral propionamide oxygen donor but
their water exchange kinetics are similar. The propionate to
propionamide substitution does not result in different water
exchange kinetics in other gadolinium(III) complexes.10,26 This
is in contrast to the acetate to acetamide substitution, which is
well established to reduce the water exchange rate.27
Interestingly, substitution at the propionamine [Gd(4a) and
Gd(4b)] gives a ligand with the same donor set as 3, but these
peripheral modifications now result in a gadolinium complex
with no inner-sphere water ligand. Similarly, changing the
amide substituent from Gd(5a) to Gd(5b) results in the latter
complex having no inner-sphere water ligand. For complexes
with exceedingly fast water exchange kinetics, these results
indicate that seemingly minor modifications to the periphery of
the ligand can alter the hydration number of the complex.
Relaxivity Determination at 20 and 60 MHz in PBS.
Determination of the relaxivity of the gadolinium(III)
complexes also gives further indication of their hydration
state. The longitudinal proton relaxivity (r1) was measured in
pH 7.4 PBS at 37 °C at two Larmor frequencies, 20 and 60
MHz. For small molecules with q > 0, the relaxivity is
dominated by the short rotational correlation time at these
1
noate arm) is obtained and isolated, as indicated to us by H
NMR. Compound 2 also represents the last common
intermediate for the synthesis of compounds 3−5b, which all
are furnished by activation of the carboxylate with 1-
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxide hexafluorophosphate (HATU) in the
presence of diisopropylethylamine (DIPEA) as a base, followed
by overnight amide coupling and subsequent HPLC
purification. The coupled, purified products still carry tert-
butyl ester protective groups on the acetate groups and the
phosphonate. These esters are then removed using an acid-
catalyzed deprotection reaction with trifluoroacetic acid in
dichloromethane, yielding the final ligands without the
formation of side products.
Complexes were formed under standard conditions by
mixing the ligand with an aqueous solution of the lanthanide
trichloride salt. The pH of the solution was adjusted to 7 by the
dropwise addition of a 0.1 M solution of NaOH. Complexation
was followed by HPLC in order to confirm that >95% of ligand
was complexed. We also used the xylenol orange test to further
confirm that no free lanthanide was present.23
Water Exchange Rates in Phosphate-Buffered Saline
(PBS). According to our hypothesis, Gd(DOTAlaP) derivatives
should display water exchange rates that exceed the previously
reported water exchange rates of Gd(DOTAla) derivatives. We
determined the water exchange rates of the gadolinium(III)
complexes by variable-temperature measurement of the trans-
verse relaxation time of H217O in the presence and absence of
each gadolinium(III) complex at 11.7 T. In the case of Gd(4a),
Gd(4b), and Gd(5b), we observed only minor changes in the
transverse relaxation rate when the complexes were added,
indicating a lack of an inner-sphere water ligand for these
complexes. For instance, the maximum 17O relaxivity, r2 , was
O
2.5 mM−1 s−1 for these three complexes, while the maximum
O
r2 is typically >15 mM−1 s−1 for q = 1 complexes at this field
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frequencies and the hydration state.20,31 Additionally, phos-
phonate-containing gadolinium(III) chelates have been re-
ported to result in increased second-sphere relaxivity.30,32 If all
of the complexes were q = 1, then we would expect the
relaxivity to increase proportionally with the molecular weight,
ranking them as follows: Gd(3) < Gd(4a) ≅ Gd(5a) < Gd(4b)
≅ Gd(5b). In fact, only Gd(3) and Gd(5a) behave as expected
with a relatively high, but for q = 1 phosphonato complexes
typical, relaxivity.30,31,33 On the other hand, Gd(4a), Gd(4b),
and Gd(5b) show relaxivities below 4 mM−1 s−1, which is more
typical for q = 0 complexes with elevated outer-sphere relaxivity
(Table 2).34 These relaxivity measurements are consistent with
the 17O NMR measurements, which suggests the lack of an
inner-sphere water ligand for Gd(4a), Gd(4b), and Gd(5b).
complexes. The hydration numbers calculated are proportional
to the obtained relaxivity values in PBS at 20 and 60 MHz, with
Eu(3) and Eu(5a) providing q values above 1, while the other
complexes show lower q values. Figure S1 (Supporting
Information) shows the correlation of the 20 MHz relaxivity
values of the gadolinium(III) complex and the apparent
hydration number of the corresponding europium(III)
complex.
Interaction of Ibuprofen-Derived Complexes with
Human Serum Albumin (HSA). We had previously
conjugated ibuprofen to DOTAla and found that a 0.1 mM
solution of its gadolinium(III) complex [compound Gd(6),
Figure 2] was 70% bound to 4.5% (w/v) HSA, and this
complex also displayed high relaxivity (at low fields) in the
presence of HSA.20,25 To understand how the phosphonate
modification affects protein binding and relaxivity for a slow
tumbling system, we synthesized the ibuprofen conjugates
Gd(4b) and Gd(5b) shown in Figure 2.
Compared to Gd(6), the affinity of Gd(4b) for HSA was
increased 3-fold (Kd = 260 and 80 μM, respectively). The
reason for the higher percentage of bound complex in the case
of Gd(4b) is likely the double negative charge, which may
provide a stronger interaction with the lysine-rich, positively
charged binding pocket of HSA. On the other hand, the
zwitterionic Gd(5b) has an affinity to HSA similar to that of
Gd(6).
Table 2. Measured Relaxivities (mM−1 s−1) in PBS at 37 °C,
1
at H Larmor Frequencies of 20 and 60 MHz
Gd(3)
Gd(4a)
Gd(4b)
Gd(5a)
Gd(5b)
20 MHz
60 MHz
6.4
6.1
3.7
3.5
3.9
3.8
5.3
4.1
3.2
3.0
Determination of the Hydration Numbers Using
Europium(III) Analogues. The hydration number of a
gadolinium(III) complex can be estimated using a surrogate
complex with the luminescent lanthanide ions Eu3+ or Tb3+.
This is done by measuring the rate constants for luminescence
decay in D2O and H2O solutions. An empirical expression
originally reported by Horrocks and Sudnick relates these rate
constants to q (Table 3).35 It is then assumed that
We also measured the relaxivity for these complexes in a
4.5% HSA solution (Table 4). Surprisingly, we observed very
Table 4. Summary of Percent Bound Probe Gd(4b), Gd(5b),
and Gd(6)20 in 4.5% (w/v) HSA as Well as Corresponding
Observed Relaxivity Data in PBS and 4.5% (w/v) HSA at 37
°C
Table 3. Decay Constants [s−1] for the Luminescence
Lifetime of Europium Complexes in H2O and D2O Reported
Here, as Well as the Calculated q Values According to the
Modified Horrocks Equation39
Gd(4b)/ Gd(4b)/ Gd(5b)/ Gd(5b)/ Gd(6)/ Gd(6)/
PBS
HSA
PBS
HSA
PBS
HSA
% bound
to HSA
88
70
70
Eu(3)
Eu(4a)
Eu(4b)
Eu(5a)
Eu(5b)
a
k(D2O)
k(H2O)
q
0.55
2.14
1.4
0.51
1.31
0.5
0.59
1.77
1
0.52
1.76
1.1
ND
20 MHz
(mM−1
3.9
3.8
19
14
3.2
3.0
9
5.6
4.8
37
22
a
ND
s−1
)
a
ND
a
60 MHz
(mM−1
7.8
ND = not determined. Because of the decreased solubility of Eu(5b)
compared to the other europium complexes described here, we were
not able to generate a solution with high enough complex
concentration required for this measurement.
s−1
)
different relaxivity behaviors for these two complexes. At 20
MHz, the relaxivity of Gd(4b) increased over 5-fold when HSA
was added, whereas for Gd(5b), the increase was less than 3-
fold. Such a difference could not be accounted for by the
difference in the fraction bound to albumin between the two
complexes. In the absence of albumin, the relaxivity of Gd(4b)
is about 25% higher than that of Gd(5b), but in the presence of
albumin, the relaxivity of Gd(4b) is 120% and 80% higher than
gadolinium(III) has the same hydration number based on the
similar ionic radii of these ions and their propensity for forming
isostructural complexes. However, application of this method to
phosphonate-containing complexes typically results in an
overestimation of q by 0.3−0.6.36−38 With this consideration
in mind, we find that the q values obtained mirror the
relaxometric behavior of the corresponding gadolinium
Figure 2. Structures of ibuprofen conjugates Gd(4b) and Gd(5b) reported here, as well as previously reported compound Gd(6).
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Figure 3. Relaxivity of HSA-bound Gd(5b) (filled circles) and Gd(4b) (open circles) at pH 7.4, at (A) 10 °C, (B) 25 °C, (C) 37 °C, and (D) 50 °C.
The difference between the solid and open circles is the inner-sphere contribution to the Gd(4b) relaxivity, and the solid line is the fit to the data
described in the text.
that of Gd(5b) at 20 and 60 MHz, respectively (Table 4). In
order to better understand this phenomenon, we performed
additional r1 measurements at different temperatures and
additional field strengths.
contribution from this inner-sphere water (eq 3). We then
calculated this difference at each temperature and field and
simultaneously fit the resultant data set using Solomon−
Bloembergen−Morgan theory40 (Figure 3 and eqs 2−8). While
the HSA-bound relaxivity of Gd(4b) was higher than expected,
it was not as high as one would anticipate for a complex with
one coordinated water, and so we left the hydration number q
as a temperature-dependent adjustable parameter defined by an
equilibrium constant. We found that this entire data set could
be reasonably well described (Figure 3) by five adjustable
parameters: 310q (mole fraction of q = 1 species at 310 K), ΔH°
(enthalpy change for equilibrium between q = 0 and 1 species),
310τM, ΔH⧧ (activation energy for water exchange), and Δ2
[square of the trace of zero-field-splitting (ZFS) tensor; eqs
3−8].
Nuclear Magnetic Relaxation Dispersion (NMRD) of
Gd(4b) and Gd(5b). We acquired relaxivity data for Gd(4b)
and Gd(5b) in PBS and 4.5% (w/v) HSA at 20, 30, 60, 100,
200, and 400 MHz and at 10, 25, 37, and 50 °C (Tables S1−S4
in the Supporting Information, SI). We also measured the
fraction (fb) of Gd(4b) and Gd(5b) bound to HSA at each of
these temperatures (Table S5 in the SI). Because we know the
fraction bound to HSA for each complex and we measure the
relaxivity of the unbound complex independently, we can
calculate the relaxivity due to the HSA-bound species (r1bound
for both Gd(4b) and Gd(5b) (eq 1).
)
r1obs = f r1free + f r1bound
(1)
f
f
kf
[Gd(4b) for q = 0] ⇄ [Gd(4b) for q = 1];
The magnitude and field dependences of these HSA-bound
relaxivities are shown in Figure 3. The high relaxivity of Gd(4b)
could not be well described by a pure second-sphere model,
which assumes some adjustable number of long-lived water
molecules in close proximity to the metal ion. Instead, we
reasoned that the difference in the HSA-bound relaxivities
between Gd(4b) and Gd(5b) was due to the presence of an
inner-sphere water ligand on some fraction of the Gd(4b)
complexes (eq 2). We assumed that the difference in the HSA-
bound relaxivity between Gd(4b) and Gd(5b) was the
kd
Keq = kf /kd = eΔG°/RT = eΔH°/RT−ΔS°/R
(2)
r1IS = r1bound[Gd(4b)] − r1bound[Gd(5b)]
(3)
q/[H2O]
r1IS
=
T
+ τM
(4)
1m
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Table 5. Molecular Parameters for Gd(4b) Bound to HSA Derived from the Simultaneous Fitting of Variable-Temperature
NMRD
a
a
τR (ns)
τv (ps)
Δ2 (s2)
(4 1) × 1018
310τM (ns)
ΔH⧧ (kJ mol−1
28
)
310q
ΔH° (kJ mol−1
)
Gd(4b)
30
20
2.4 0.9
5
0.15 0.02
16.8 2.5
a
The parameter was fixed at this value.
2
2
2
suggests that Gd(4b) is much more thermodynamically stable
than MS-325. In a second experiment, we challenged each
complex with a 100-fold excess of citrate at pH 3 and 37 °C.
With a citrate challenge, we found the opposite behavior:
transchelation proceeded to completion in both cases but over
⎡
⎢
⎣
⎤
γ g μ S(S + 1)
μ
⎛
⎞
⎟
3τC
1
2
H
B
0
e
⎜
⎥
=
2
1 + ω 2τ
6
⎝
⎠
T
15 4π
rGdH
⎦
1m
(5)
(6)
s
v
1
τC
1
1
T
1
τm
=
+
+
τR
1e
1 order of magnitude more rapidly in the case of Gd(4b) (t1/2
9.5 h) than Gd(6) (t1/2 = 110 h) (Figure 4).
=
2
⎡
⎤
⎥
⎦
τv
1 + ω 2τ
4τv
Δ [4S(S + 1) − 3]
1
⎢
⎣
=
+
1 + 4ω 2τv
2
2
T
25
1e
s
v
s
(7)
⧧
⎡
⎤
⎥
⎦
⎛
⎜
⎞
⎟
1
τM
T
ΔH
R
1
T
1
⎢
⎣
= kex
=
−
310.15τm310
⎝
⎠
310.15
(8)
We found that the data were relatively insensitive to the
rotational correlation time as long as this correlation time was
large. This is reasonable because the very short water residency
time should dominate the correlation time for relaxation. In
addition, the data were insensitive to τv, the correlation time for
electronic relaxation, and so we fixed this to 20 ps, in line with
other studies of similar complexes.29 The fitted parameters are
listed in Table 5. The quality of the fit could likely be improved
by the addition of more adjustable parameters, e.g., Lipari−
Szabo-type treatment,31,33,41,42 but the conclusions are unlikely
to change.
The analysis shows that there is a 15% fraction of Gd(4b)
that exists as a q = 1 complex with a short residence time for the
first sphere water molecule. For the equilibrium in eq 2, the two
reactions are the formation and dissociation of the q = 1
complex. In principle, there can also be water exchange on the q
= 1 complex. Because we only observe free water, we cannot
distinguish between these two reactions (dissociation to a
stable q = 0 complex versus water exchange at the q = 1
complex). However, the latter reaction likely must proceed via
the q = 0 state. Therefore, we likely are measuring the
formation and dissociation of water to the gadolinium complex;
i.e., τM, the residence time in the bound state, corresponds to 1/
kd.
Kinetic Inertness. The relatively high relaxivity of Gd(4b)
in the presence of HSA, especially at high fields, prompted us to
further explore the kinetic inertness of this complex with
respect to gadolinium(III) dissociation. Acetate to methyl-
enephosphonate substitution in lanthanide(III) polyaminocar-
boxylates typically results in a more thermodynamically stable
complex (because of the increased basicity of the phospho-
nate), but at the same time, the complex can become more
kinetically labile.43 We measured the half-life of Gd(4b) under
different forcing conditions and compared it directly to the
acetate analogue Gd(6) measured under the same conditions.
In the first experiment, we challenged each complex with 1
equiv of MS-325 ligand [a DTPA derivative of higher
thermodynamic stability than Gd(DOTAla) derivatives] at
pH 3 and 37 °C and monitored transchelation by HPLC.
Under these conditions, we found no measurable decom-
plexation of Gd(4b), while Gd(6) underwent complete
transchelation to MS-325 with a half-life of 115 h. This result
Figure 4. Dechelation of Gd(4b) (filled circles) and Gd(6) (open
circles) at pH 3 and 37 °C in the presence of 10 mM citrate. Solid
lines represent monoexponential fits of the decomplexation reaction.
This is in agreement with the previously reported kinetic data
obtained for acyclic phosphonate complexes compared to their
acetate analogues.44 We hypothesize that the phosphonate arm
promotes the formation of ternary complexes with citrate
molecules, which facilitates decomplexation in the case of
Gd(4b). Although Gd(4b) was more labile than Gd(6), we
note that acyclic complexes like [Gd(DTPA)(H2O)]2− or its
derivatives, such as [Gd(BOPTA)(H2O)]2− (Table S5 in the
SI), are much more labile under these conditions, e.g., reaching
transchelation equilibrium with MS-325 ligand in under 10
min.8,10
In Vivo MRI in Mice with Gd(4b). Because the high field
relaxivity of Gd(4b) was 50% higher than that of the approved
contrast agent MS-325 at 4.7 T, we decided to assess its
relaxation properties in vivo. In addition, both Gd(4b) and MS-
325 showed equivalent affinity to HSA, making this comparison
more direct.5 We therefore performed dynamic MRI in mice
with both compounds.
A catheter was placed in the tail vein so the mouse could be
injected while positioned in the scanner. We performed 3D T1-
weighted imaging before and immediately after injection of
each compound (0.1 mmol kg−1). Immediately after injection,
the blood vessel enhancement observed with Gd(4b) was 38
2% higher than that observed with MS-325 (Figure 5)
consistent with the higher relaxivity of the former.
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Figure 5. Preinjection, as well as 1 min postinjection, coronal images obtained with MS-325 (second from left) and Gd(4b) (right). Gd(4b) shows
visibly better contrast in the vena cava, which can be quantified as 38 2% better contrast (vs muscle). The dose of agent, time point, and image
windowing was the same for both studies. Kidney (ki) and vena cava (vc) are denoted in the far right image.
The NMRD results have profound consequences for the
development of slow tumbling contrast agents for high-field
applications. The variable-temperature data clearly indicate that
the water residency time, τM, is the dominant correlation time
for modulating the relaxivity in this system. Figure 6 shows data
DISCUSSION
■
We have synthesized and evaluated the properties of five
different derivatives of a new phosphonate analogue (DOTA-
laP) of the single amino acid chelate DOTAla. On the basis of
literature examples, we anticipated and observed an increased
relaxivity due to the established second-sphere effect of the
phosphonate. We also expected the phosphonate group to
accelerate water exchange. However, for [Gd(DOTAla)-
(H2O)]− and Gd(DOTAla)-propionamide derivatives, water
exchange is already very fast (310τM = 9 and 17 ns, respectively).
For the Gd(DOTAlaP) complexes investigated here, we found
that further acceleration of water exchange through crowding of
the coordination sphere may instead lead to stabilization of the
q = 0, 8-coordinate state. The five complexes investigated here
comprised two that existed primarily as 9-coordinate q = 1
complexes, while the other three appear to be primarily q = 0,
presumably 8-coordinate complexes. These conclusions are
supported by 17O NMR and 1H relaxivity data on the
gadolinium(III) complexes and by luminescence lifetime
measurements on the europium(III) complexes. The energy
difference between the 8- and 9-coordinate states is quite small,
and peripheral changes to the macrocyclic ligand outside the
inner coordination sphere can alter the coordination number in
a nonpredictable manner. This delicate equilibrium between q
= 0 and rapidly exchanging q = 1 underlines a challenge in the
design of gadolinium(III) complexes with extremely fast water
exchange rates.
Two of the investigated derivatives, Gd(4b) and Gd(5b),
were designed to bind HSA. The relaxivities of these
compounds in the absence of HSA were similar and low,
although r1 for Gd(4b) was about 25% higher than r1 for
Gd(5b). This small increase in the relaxivity could be due to
the presence of a small amount of q = 1 species for Gd(4b).
However, in the presence of HSA, the relaxivity difference
between the complexes is magnified, and this increased
difference seen when the molecular tumbling rate is decreased
is indicative of a small fraction of q = 1 species. Variable-
temperature NMRD allowed us to estimate that about 15% of
Gd(4b) exists as a q = 1 species at 37 °C, at least while it is
bound to albumin. The presence of this q = 1 complex whose
coordinated water has an extremely short residency time results
in remarkably high relaxivities at high field for a complex bound
to HSA. For instance, the relaxivity at 4.7 T was 50% higher
than that for the albumin-targeted approved agent MS-325.
Figure 6. Inner-sphere relaxivity of Gd(4b) bound to HSA at two
different temperatures (10 and 37 °C) and Larmor frequencies.
at two temperatures and two fields to illustrate this point. At
0.47 T (20 MHz), the relaxivity increases dramatically as the
temperature is lowered. At 37 °C, τM at 2.4 ns is too short for
optimal relaxivity, but as the temperature is lowered, τM
increases and the relaxivity goes up. On the other hand, at
higher fields (>2 T), the optimal correlation time is in the 1−3
ns range.45 At 37 °C, a τM of 2.4 ns is ideal for relaxivity at 2.4 T
(100 MHz). However, as the temperature is lowered and τM
increases, the correlation time becomes too long for optimizing
high-field relaxivity. The very short τM provides increased
relaxivity at high fields and offers a strategy to increase the
relaxivity of slow tumbling entities at high magnetic fields. We
note that the data in Figure 6 are for a complex that is only 25%
(at 10 °C) or 15% (at 37 °C) for q = 1. Thus, the relaxivity
gains for a single q = 1 species are 4−6-fold higher.
Because the relaxivity of q = 0.15 Gd(4b) was still clearly
enhanced compared to q = 1 MS-325 at higher fields, we
considered its evaluation for high-field in vivo imaging. For this,
we first tested the kinetic inertness of Gd(4b) under forcing
conditions. We found the somewhat decreased kinetic
inertness, when compared with the all-acetato analogue Gd(6),
is a typical reflection of the results previously reported in
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conjunction of phosphonato compounds.32,43 However, the
complex was still quite inert compared to approved acyclic
chelates.46 This encouraged the in vivo evaluation and cross-
comparison with the approved HSA binding compound MS-
325. The subsequent in vivo imaging experiment at 4.7 T was
found to mirror the in vitro results well because we were able to
obtain better angiographic contrast for Gd(4b) compared to
MS-325. An increased blood signal is clearly found in the
increased relaxivity for this compound at intermediate magnetic
fields. We consider this outcome a promising result that
encourages us to further investigate τM acceleration as a way to
enhance the relaxivity at higher fields because an entirely q = 1
complex with such a fast exchange rate would have 5 times the
relaxivity observed with Gd(4b). However, the somewhat
unpredictable nature of the compound behavior in terms of the
inner-sphere hydration number is a clear challenge.
MeCN; 5−95% B over 15 min. Method D: solvent A = 10 mM
NH4OAc, solvent B = 10% 10 mM NH4OAc, 90% MeCN; 5−35% B
over 15 min. The synthesis of ligands was carried out as shown in
Scheme 1. Chemicals were supplied by Aldrich Chemical Co., Inc., and
were used without further purification. Solvents (HPLC grade) were
purchased from various commercial suppliers and used as received.
The monoalkylated cyclen precursor to compound 1 was synthesized
as described previously.10 Tri-tert-butyl phosphite was synthesized
according to a procedure reported by Manning et al.22
General Procedure for Amide Couplings (Step To Afford
Products 4a, 4b, 5a, and 5b). Amide coupling followed by
deprotection of the tert-butyl protective groups was done by activation
of the carboxylate with HATU (1.2 equiv) and DIPEA (1.2 equiv) for
5 min in N,N-dimethylformamide (DMF), followed by the addition of
amine (1 equiv) dissolved in DMF and stirring at room temperature
for 18 h. After confirmation of the presence of the amide-coupling
product by LC−MS (method B), the intermediate was isolated by
preparative HPLC (method A), eluting between 11 and 13 min. The
clean fractions containing the desired product were pooled and
lyophilized to afford the intermediate as an off-white powder in 15−
35% yield.
General Deprotection Procedure. Redissolution of intermedi-
ates described above in a 1:1 mixture of dichloromethane and TFA,
followed by stirring for 18 h, afforded the final ligand with quantitative
yield.
General Metal Complexation Procedure (with Lanthanides
Gadolinium, Europium, and Terbium). The ligand was dissolved
in H2O (1 mL). An amount of a stock solution containing LnCl3·
6H2O (0.95 equiv based on the ligand weight) is added to the ligand
solution under monitoring of the pH. The pH is adjusted to 7 using a
0.1 M NaOH solution. The lightly cloudy solution is filtered and
lyophilized to afford the corresponding lanthanide complex as an off-
white powder. The purity of the complex was assessed using LC−MS;
the presence of free lanthanides was excluded using the xylenol orange
test.
Di-tert-butyl 2,2′-[4-[3-(benzyloxy)-2-[[(benzyloxy)-
carbonyl]amino]-3-oxopropyl]-10-[(di-tert-butoxyphosphoryl)-
methyl]-1,4,7,10-tetraazacyclododecane-1,7-diyl]diacetate (1).
2-[[(Benzyloxy)carbonyl]amino]-3-(1,4,7,10-tetraazacyclododecan-1-
yl)propanoate (1.475 g, 3.05 mmol, 1 equiv) and paraformaldehyde
(0.34 g, 11.45 mmol, 3.75 equiv) were dissolved in tetrahydrofuran
and vigorously stirred under a dinitrogen atmosphere for 0.5 h. Tri-
tert-butyl phosphite (1.145 g, 4.5 mmol, 1.5 equiv) was then added,
and the reaction was stirred for 16 h. Reaction monitoring by LC−MS
showed the product as [M + H]+ (m/z 690.4). The reaction mixture
was then filtered, concentrated in vacuo, and redissolved in EtOAc
(200 mL). The organic layer is washed with 50 mL of saturated
Na2CO3 and 50 mL of brine, dried with sodium sulfate, and
concentrated to afford the dialkylated intermediate (1.73 g, 2.5 mmol,
82%), which was used in the subsequent step without further
characterization. The intermediate was dissolved in MeCN (75 mL).
K2CO3 (0.68 g, 4.9 mmol, 2 equiv) was added, and the mixture was
stirred vigorously after the addition of tert-butyl bromoacetate (0.766
g, 0.575 mL, 3.94 mmol, 1.6 equiv). After 16 h, water (70 mL) was
added and most of the MeCN was removed in vacuo. The oily residue
was taken up in EtOAc (80 mL) and washed with water and brine
(100 mL each). The organic layer was separated, dried with sodium
sulfate, and concentrated to afford the crude product, which was
redissolved in MeCN (5 mL) and purified using preparative HPLC
(method A). The product elutes at 12.2 min. Fractions containing the
product are pooled and lyophilized to afford the product as a white
powder (0.43 g, 0.46 mmol, 20% yield). 1H NMR (CDCl3, 500 MHz,
ppm): 9.9 (s, br, 2H), 7.36−7.34 (m, 10H), 5.25−5.16 (m, 4H), 4.3
(m, 1H), 3.35−2.68 (m, 21H), 2.04 (s, 4H), 1.51−1.43 (m, 36H). 13C
NMR (CDCl3, 125.7 MHz, ppm): 175.4, 170.6, 170.3, 135.0, 131.1,
129.4, 128.6−127.9, 81.4, 81.0, 67.7, 57.3−47.4, 30.6, 28.2, 22.1. 31P
NMR (CDCl3, 202.4 MHz, ppm): 17.7. LC−ESI-MS. Calcd for
C47H76N5O11P4: m/z 917.5. Found: m/z 918.6 ([M + H]+). Rt = 7.8
min (method C).
CONCLUSIONS
■
In summary, we have successfully synthesized a mono-
phosphonate analogue of the single amino acid chelator
DOTAla, which we term DOTAlaP. The Gd(DOTAlaP)
derivatives show enhanced second-sphere relaxivity compared
to Gd(DOTAla). In two derivatives, we found increased water
exchange kinetics, and this combination of fast water exchange
and increased second-sphere relaxivity results in improved
relaxometric behavior. We also found, however, that function-
alization of this single amino acid chelate of either the nitrogen
or carbon terminus with different R groups can sometimes yield
gadolinium complexes with no inner-sphere water (q = 0) or a
mixture of q = 0 and 1 complexes. This decrease in the
hydration number appears as a side effect of accelerated water
exchange and does not correlate with a specific R group or
location of functionalization. For one derivative, Gd(4b), where
R was chosen such that the corresponding gadolinium complex
had affinity to HSA, we found that the hydration number was
0.15 in the presence of HSA, i.e., a 6:1 mixture of q = 0 to q = 1.
The corresponding mean water residency time obtained for the
albumin-bound complex was shorter than the rotational
correlation time, which, in this case, leads to τM becoming
the dominant correlation time for relaxation. Because τM is
considerably shorter than τR, higher inner-sphere relaxivity was
achieved for this complex at intermediate and high magnetic
fields than what can be obtained with the clinically approved
HSA targeting agent MS-325. We have demonstrated the effect
of higher relaxivity also in vivo with higher blood vessel to
muscle contrast for Gd(4b) when compared to MS-325. Future
endeavors will include the identification of fast exchanging
complexes that are exclusively q = 1 in order to maximize the
relaxivity gains at high fields.
EXPERIMENTAL PROCEDURES
■
General Materials and Methods. 1H and 13C NMR spectra were
recorded on a Varian 11.7 T NMR system equipped with a 5-mm
broad-band probe. HPLC purification of intermediates was performed
on a Rainin, Dynamax (Phenomenex C18 column: 250 mm × 21.2
mm, 10 μm) using method A: 0.1% trifluoroacetic acid (TFA) in water
with a gradient of 5−95% (0.1% TFA in MeCN) over 20 min with a
15 mL min−1 flow rate. HPLC purity analyses (both UV and MS
detection) were carried out on an Agilent 1100 system (Phenomenex
Luna C18(2) column: 100 mm × 2 mm, 0.8 mL min−1 flow rate) with
UV detection at 220, 254, and 280 nm and positive-mode electrospray
ionization (ESI+) using the following methods. Method B: solvent A =
water, solvent B = MeCN; 2−60% B over 15 min. Method C: solvent
A = 10 mM NH4OAc, solvent B = 10% 10 mM NH4OAc, 90%
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2-Amino-3-[4,10-bis[2-(tert-butoxy)-2-oxoethyl]-7-[(di-tert-
butoxyphosphor ryl)methyl]-1,4,7,10-tetraazacyclododecan-
1-yl]propanoic Acid (2). Compound 1 (0.43 g, 0.47 mmol) was
dissolved in EtOH (40 mL). Pd/C [10% (w/v), 0.215 g] was added,
and the reaction mixture was purged first with dinitrogen and then
charged with dihydrogen (1 atm). The mixture was then stirred under
a dihydrogen atmosphere for 3 h, after which the reaction was found to
be complete. The Pd/C was removed by filtration, and the solvent was
removed in vacuo to afford the product as a colorless oil (0.32 g, 0.46
mmol, quantitative conversion). 1H NMR (CD3OD, 500 MHz, ppm):
5.15 (s, br, 1H), 4.46−2.48 (m, 24H), 1.63−1.47 (m, 36H). 13C NMR
(CD3OD, 125.7 MHz, ppm): 167.9, 160.5, 86.6, 82.6, 55.2−48.5, 30.6,
29.2, 26.9. 31P NMR (CD3OD, 202.4 MHz, ppm): 16.9. LC−ESI-MS.
Calcd for C32H64N5O9P: m/z 693.4. Found: m/z 694.7 ([M + H]+). Rt
= 5.4 min (method B).
2,2′-[4-(2-Amino-2-carboxyethyl)-10-(phosphonomethyl)-
1,4,7,10-tetraazacyclododecane-1,7-diyl]diacetic Acid (3). 1H
NMR (CD3OD, 500 MHz, ppm): 5.15 (s, br, 2H), 4.26−2.46 (m,
25H). 13C NMR (CD3OD, 125.7 MHz, ppm): 175.1, 169.6, 55.4−
48.9. 31P NMR (CD3OD, 202.4 MHz, ppm): 6.3. LC−ESI-MS. Calcd
for C16H32N5O9P: m/z 469.2 Found: m/z 470.1 ([M + H]+). Rt = 1.1
min (method B).
([M + H]+). Calcd for C31H51EuN7O9P [Eu(5b)]: m/z 849.3. Found:
m/z 850.4 ([M + H]+).
Luminescence. Luminescence lifetime measurements of europium
complexes in H2O and D2O were performed on a Hitachi f-4500
fluorescence spectrophotometer. The concentrations of the samples
were 5−10 mM. For the measurements in D2O, the complexes were
first dissolved in D2O (99.98% D), lyophilized, and dissolved in D2O
again to reduce the amount of residual HDO. Measurements were
taken with the following settings: excitation at 396 nm (Eu) emission
at 616 nm (Eu) 30 replicates, 0.04 ms temporal resolution (0−20 ms),
PMT voltage = 400 V. Lifetimes were obtained from monoexponential
fits of the data using Igor Pro software (version 6.0, Wavemetrics, Lake
Oswego, OR).
17O NMR of Gadolinium Complexes for Determination of τM.
17O NMR measurements of solutions were performed at 11.7 T on
350 μL samples contained in 5 mm standard NMR tubes on a Varian
spectrometer. The temperature was regulated by an air flow controlled
by a Varian VT unit. The H217O transverse relaxation times of a >4
mM solution of all gadolinium complexes (pH 7.4, 10 mM PBS buffer)
were measured using a CPMG sequence. The exact concentration of
the sample was determined by inductively coupled plasma mass
spectrometry (ICP-MS). Reduced relaxation rates, 1/T2r, were
calculated from the difference of 1/T2 between the sample and
water blank and then divided by the mole fraction of coordinated
water, assuming q = 1. The temperature dependence of 1/T2r was fit to
a four-parameter model as previously described.25 The Gd−O
hyperfine coupling constant, A/ℏ, was assumed to be 3.8 × 106 rad
s−1.29
Determination of the Percent Binding to HSA. Ultrafiltration
measurements of protein binding solutions of a 0.1 mM gadolinium
compound and 4.5% HSA (200 μL total) were added to a 5000 MW
ultrafiltration unit (regenerated cellulose membrane, Millipore,
Bedford, MA). The samples were incubated at a set temperature for
30 min and subsequently centrifuged to filter off the nonprotein-
bound components. The filtrates were analyzed for the gadolinium
concentration by ICP-MS. The concentration of bound complex in the
protein samples was determined by subtracting the gadolinium
concentration (unbound complex) found in the filtrate from the
gadolinium concentration of the initial solution. Experiments were
performed in duplicate.
Measurement of Kinetic Inertness. Stock solutions of MS-325-L
(the ligand of the MS-325 complex),47 Gd(4b), and Gd(6) were
prepared. For experiment 1, MS-325-L was added to solutions of the
gadolinium complexes in pH 3 water and incubated at 37 °C. The final
concentrations of the gadolinium complexes and MS-325-L were 0.1
mM. A 10 μL aliquot was removed for HPLC analysis and analyzed at
time points 1, 20, 50, 72, 80, 96, 120,168, 192, 216, 240, 288, 312, 336,
360, and 480 h, while the remainder of the solution was incubated at
37 °C. For experiment 2, the gadolinium complexes were added to a
10 mM pH 3 citrate buffer, affording a 0.1 mM complex concentration,
and incubated at 37 °C. A 10 μL aliquot was removed and analyzed at
0, 0.75, 1.5, 2, 3, 4, 5, 6, 24, 72, 96, 120, 168, 192, 216, 240, 288 312,
336, and 408 h.
2,2′-[4-[2-Carboxy-2-(2-phenylpropanamido)ethyl]-10-
(phosphonomethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl]-
1
diacetic Acid (4a). H NMR (CD3OD, 500 MHz, ppm): 7.38−7.23
(m, 5H), 5.15 (s, br, 1H), 4.76 (m, 1H), 3.98−2.86 (m, 24H), 1.47 (t,
3H). 13C NMR (CD3OD, 125.7 MHz, ppm): 159.4, 140.9, 128.5−
126.8, 116.7, 114.4, 53.3, 49.5, 46.1, 17.1. 31P NMR (CD3OD, 202.4
MHz, ppm): 0.8. LC−ESI-MS. Calcd for C25H40N5O10P: m/z 601.2.
Found: m/z 602.4 ([M + H]+). Rt = 2.1 min (method B).
2,2′-[4-[2-Carboxy-2-[[1-(4-isobutylphenyl)ethyl]amino]-
ethyl]-10-(phosphonomethyl)-1,4,7,10-tetraazacyclodode-
1
cane-1,7-diyl]diacetic Acid (4b). H NMR (CD3OD, 500 MHz,
ppm): 7.28 (d, 2H), 7.20 (d, 2H), 4.73 (s, br, 1H), 3.91−2.87 (m,
24H), 2.45 (m, 2H), 1.84 (m, 1H), 1.48−1.29 (m, 5H), 0.89 (d, 6H).
13C NMR (CD3OD, 125.7 MHz, ppm): 172.8, 169.6, 140.5, 138.2,
129.5, 127.0, 54.7−48.2, 30.0, 21.3, 17.1. 31P NMR (CD3OD, 202.4
MHz, ppm): 3.5. LC−ESI-MS. Calcd for C28H48N5O9P: m/z 629.3.
Found: m/z 630.5 ([M + H]+). Rt = 5.6 min (method B).
2,2′-[4-[2-Carboxy-2-[2-(4-isobutylphenyl)propanamido]-
ethyl]-10-(phosphonomethyl)-1,4,7,10-tetraazacyclodode-
1
cane-1,7-diyl]diacetic Acid (5a). H NMR (CD3OD, 500 MHz,
ppm): 7.39−7.26 (m, 5H), 5.02 (s, br, 1H), 4.15−2.46 (m, 24H),
1.64−1.46 (m, 3H). 13C NMR (CD3OD, 125.7 MHz, ppm): 169.7,
166.3, 160.4, 142.7, 128.5−125.8, 119.6, 117.3, 114.9, 112.7, 53.3−
49.5, 27.2, 20.85. 31P NMR (CD3OD, 202.4 MHz, ppm): 6.1. LC−
ESI-MS. Calcd for C24H41N6O8P: m/z 573.3. Found: m/z 574.6 ([M +
H]+). Rt = 2.3 min (method B).
2,2′-[4-[2-Amino-3-[[2-[2-(4-isobutylphenyl)propanamido]-
ethyl]amino]-3-oxopropyl]-10-(phosphonomethyl)-1,4,7,10-
tetraazacyclododecane-1,7-diyl]diacetic Acid (5b). 1H NMR
(CD3OD, 500 MHz, ppm): 7.24 (d, 2H), 7.10 (d, 2H), 4.21−2.44 (m,
30H), 1.94−1.29 (m, 6H), 0.89 (d, 6H). 13C NMR (CD3OD, 125.7
MHz, ppm): 175.4, 169.8, 167.4, 140.2, 138.8, 128.9, 126.7, 54.6−48.1,
38.2, 30.1, 21.3, 17.1. 31P NMR (CD3OD, 202.4 MHz, ppm): 6.1.
LC−ESI-MS. Calcd for C31H54N7O9P: m/z 699.3. Found: m/z 700.6
([M + H]+). Rt = 6.8 min (method B).
Lanthanide Complexes. LC−ESI-MS. Calcd for
C16H28GdN5O9P [Gd(3)]: m/z 623.1. Found: m/z 625.1 ([M +
2H]+). Calcd for C16H28EuN5O9P [Eu(3)]: m/z 618.1. Found: m/z
620.1 ([M + 2H]+). Calcd for C24H36GdN5O9P [Gd(4a)]: m/z 755.1.
Found: m/z 757.2 ([M + 2H]+). Calcd for C24H36EuN5O9P [Eu(4a)]:
m/z 750.1. Found: m/z 752.1 ([M + 2H]+). Calcd for
C29H44GdN5O10P [Gd(4b)]: m/z 811.2. Found: m/z 813.4 ([M +
2H]+). Calcd for C29H44EuN5O10P [Eu(4b)]: m/z 806.2. Found: m/z
808.4 ([M + 2H]+). Calcd for C29H44TbN5O10P [Tb(4b)]: 812.2.
Found: m/z 814.4 ([M + 2H]+). Calcd for C24H38GdN6O8P
[Gd(5a)]: m/z 727.2. Found: m/z 728.2 ([M + H]+). Calcd for
C24H38EuN6O8P [Eu(5a)]: m/z 722.2. Found: m/z 723.3 ([M + H]+).
Calcd for C31H51GdN7O9P [Gd(5b)]: m/z 852.3. Found: m/z 853.5
Imaging Protocol for MS-325/Gd(4b). Nu/nu mice were
anesthetized with isoflurane (1−2%) and placed in a specially
designed cradle with the body temperature maintained at 37 °C.
The tail vein was cannulated for intravenous delivery of the contrast
agent while the animal was positioned in the scanner. Imaging was
performed at 4.7 T, using a custom-built volume coil to acquire
coronal T1-weighted images with a field of view that covered the major
organs of the thorax and abdomen (heart, stomach, liver, intestines,
and kidney). The contrast agent dose was 100 μmol kg−1. T1-weighted
images were acquired using a 3D Fast Low Angle Shot (FLASH)
sequence: TR/TE/flip angle = 15.3 ms/1.54 ms/40°, FOV = 4.8 × 2.4
× 2.4 cm, matrix = 192 × 96 × 96, 1 average.
6993
dx.doi.org/10.1021/ic5008928 | Inorg. Chem. 2014, 53, 6985−6994

Inorganic Chemistry
Article
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ASSOCIATED CONTENT
■
S
* Supporting Information
Obtained relaxivity values, kinetic inertness challenge data,
temperature-dependent fraction bound of Gd(4b) and Gd(5b)
to HSA, correlation plot of europium luminescence lifetime
derived q values versus relaxivity, and LC−MS traces. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: caravan@nmr.mgh.harvard.edu.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
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Notes
The authors declare no competing financial interest.
́
̌
(30) Rudovsky, J.; Cígler, P.; Kotek, J.; Hermann, P.; Vojtísek, P.;
ACKNOWLEDGMENTS
Lukes,
̌
I.; Peters, J. A.; Vander Elst, L.; Muller, R. N. Chem.Eur. J.
■
2005, 11, 2373−2384.
P.C. acknowledges the National Institute for Biomedical
Imaging and Bioengineering for funding for this project
(NIBIB Award R01EB009062) and the National Center for
Research Resources for instrumentation grants (Grants
S10OD010650, S10RR023385, and P41RR14075). E.B. thanks
the Swiss National Science Foundation (advanced.postdoc
mobility fellowship) for support.
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