Gd(DOTAla): A single amino acid Gd-complex as a modular tool for high relaxivity MR contrast agent development

Article abstract of DOI:10.1021/ja309187m

MR imaging at high magnetic fields benefits from an increased signal-to-noise ratio; however T₁-based MR contrast agents show decreasing relaxivity (r₁) at higher fields. High field, high relaxivity contrast agents can be designed by carefully controlling the rotational dynamics of the molecule. To this end, we investigated applications of the alanine analogue of Gd(DOTA), Gd(DOTAla). Fmoc-protected DOTAla suitable for solid phase peptide synthesis was synthesized and integrated into polypeptide structures. Gd(III) coordination results in very rigid attachment of the metal chelate to the peptide backbone through both the amino acid side chain and coordination of the amide carbonyl. Linear and cyclic monomers (GdL1, GdC1), dimers (Gd₂L2, Gd₂C2), and trimers (Gd ₃L3, Gd₃C3) were prepared and relaxivities were determined at different field strengths ranging from 0.47 to 11.7 T. Amide carbonyl coordination was indirectly confirmed by determination of the hydration number q for the EuL1 integrated into a peptide backbone, q = 0.96 ± 0.09. The water residency time of GdL1 at 37 °C was optimal for relaxivity, τ_M = 17 ± 2 ns. Increased molecular size leads to increased per Gd relaxivity (from r₁ = 7.5 for GdL1 to 12.9 mM ^-1-s^-1 for Gd₃L3 at 1.4 T, 37 °C). The cyclic, multimeric derivatives exhibited slightly higher relaxivities than the corresponding linearized multimers (Gd₂C2: r₁ = 10.5 mM^-1-s^-1 versus Gd₂C2-red r₁ = 9 mM^-1-s^-1 at 1.4 T, 37 °C). Overall, all six synthesized Gd complexes had higher relaxivities at low, intermediate, and high fields than the clinically used small molecule contrast agent [Gd(HP-DO3A)(H₂O)].

Full text of DOI:10.1021/ja309187m

Article
pubs.acs.org/JACS
Gd(DOTAla): A Single Amino Acid Gd-complex as a Modular Tool for
High Relaxivity MR Contrast Agent Development
Eszter Boros, Miloslav Polasek, Zhaoda Zhang, 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
ABSTRACT: MR imaging at high magnetic fields benefits
from an increased signal-to-noise ratio; however T1-based MR
contrast agents show decreasing relaxivity (r1) at higher fields.
High field, high relaxivity contrast agents can be designed by
carefully controlling the rotational dynamics of the molecule.
To this end, we investigated applications of the alanine
analogue of Gd(DOTA), Gd(DOTAla). Fmoc-protected
DOTAla suitable for solid phase peptide synthesis was synthesized and integrated into polypeptide structures. Gd(III)
coordination results in very rigid attachment of the metal chelate to the peptide backbone through both the amino acid side chain
and coordination of the amide carbonyl. Linear and cyclic monomers (GdL1, GdC1), dimers (Gd2L2, Gd2C2), and trimers
(Gd3L3, Gd3C3) were prepared and relaxivities were determined at different field strengths ranging from 0.47 to 11.7 T. Amide
carbonyl coordination was indirectly confirmed by determination of the hydration number q for the EuL1 integrated into a
peptide backbone, q = 0.96 0.09. The water residency time of GdL1 at 37 °C was optimal for relaxivity, τM = 17 2 ns.
Increased molecular size leads to increased per Gd relaxivity (from r1 = 7.5 for GdL1 to 12.9 mM−1 s−1 for Gd3L3 at 1.4 T, 37
°C). The cyclic, multimeric derivatives exhibited slightly higher relaxivities than the corresponding linearized multimers (Gd2C2:
r1 = 10.5 mM−1 s−1 versus Gd2C2-red r1 = 9 mM−1 s−1 at 1.4 T, 37 °C). Overall, all six synthesized Gd complexes had higher
relaxivities at low, intermediate, and high fields than the clinically used small molecule contrast agent [Gd(HP-DO3A)(H2O)].
fold increase in relaxivity at low fields once it is associated with
human serum albumin (HSA).15
INTRODUCTION
■
Magnetic resonance imaging (MRI) is one of the most
important modalities used for noninvasive investigation of
disease in the clinic. MRI is the imaging technology of choice
whenever high-resolution tissue contrast is required. Another
advantage is the use of harmless magnetic fields for MRI as
opposed to ionizing radiation in the case of CT.1,2
While 1.5 T remains the dominant field strength for clinical
MRI, there is now a large installed base of 3 T scanners and the
major equipment vendors also offer 7 T whole body human
scanners. Small animal scanners operate almost exclusively at
field strengths of 4.7 T and higher. The primary benefit of high
field is the increased signal-to-noise ratio, which enables greater
spatial resolution and reduced acquisition time. In addition, the
inherent T1 of tissue increases with increasing magnetic field.16
Thus, a contrast agent with equivalent relaxivity at a high and
low field would provide much greater contrast at the high field.
However, the relaxivity of many T1-contrast agents decreases
more rapidly with applied field than the inherent tissue T1
increases.
Relaxivity above 0.1 T depends on a variety of parameters,
some of which are depicted in Figure 1.19 As the magnetic field
increases, the optimal correlation time, τc, for maximum
possible relaxivity decreases, as it is inversely dependent on
the proton Larmor frequency ωH. While the contribution from
the electronic relaxation time (T1e) is negligible at fields above
1.5 T, contributions from the mean water residency time (τM)
and the rotational correlation time (τR) become the levers for
generating high relaxivity Gd-based agents.20
A large fraction of scans performed in the clinical setting are
further enhanced by the use of contrast agents.3 Contrast
agents shorten the relaxation times of water molecules in their
proximity and increase tissue contrast on relaxation weighted
imaging sequences. Currently, most clinically employed
contrast agents are nonspecific, small molecule gadolinium
complexes which are able to increase the longitudinal relaxation
rate 1/T1 of water protons in the extracellular space.4 The
extent to which a contrast agent can enhance relaxation
depends on its concentration and its relaxivity (r1), an inherent
property of the molecule. Most approved contrast agents have
low relaxivities (r1) which makes them effective only at
relatively high concentrations (≥0.1 mM).4 There has been a
considerable research effort to increase the relaxivity of contrast
agents5−9 Compounds with high relaxivity can be detected at
lower doses,10 or provide greater contrast at equivalent dose to
compounds with lower relaxivity. Additionally, an attachment
of a targeting moiety allows for target specific delivery of the
contrast.10−12 The clinically approved blood pool agent MS-
325 (gadofosveset, Ablavar) is an example of a contrast agent
with a high relaxivity;13,14 this small molecular compound
carries an albumin-targeting moiety and will display an over 8
For the design of high field, high relaxivity contrast agents it
is instructive to consider the equation for two site exchange
Received: September 16, 2012
Published: November 16, 2012
© 2012 American Chemical Society
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> 1 will also occur at lower fields and relaxivity will be low at
high fields.
q/[H2O]
r1IS
=
T
+ τM
(1)
1M
⎡
⎤
⎥
⎦
3τ
c
⎢
⎣
1/T = C
1M
1 + ω 2τ2
(2)
H c
Figure 1. Molecular parameters that influence relaxivity: rotation (τR),
water exchange (τM), hydration number (q) and electronic relaxation
(T1e).
For instance, MS-325 was designed for high relaxivity at low
fields (≤1.5 T). Serum albumin binding of MS-325 results in a
very long τR resulting in high relaxivity at 1.5 T, but a
precipitous decline in relaxivity with increasing field
strength.21,22 Small molecule agents with very short correlation
times such as [Gd(DTPA)(H2O)]2‑ (Magnevist) display a
modest relaxivity decrease with increasing field strength but
exhibit relatively low relaxivity due to their rapid tumbling.23
We have previously investigated the interplay of water exchange
and rotational correlation time for Gd-based T1 agents at fields
ranging from 0.47 to 9.4 T,21 and showed that the optimal
ranges are 5 < τM < 25 ns and 0.5 < τR < 2 ns to yield high
relaxivity over a range of fields. A number of compounds with a
corresponding, intermediate τR value between 0.35 − 1 ns has
been reported,18,24,25 however none of these structures allow
for the simple adjustment of τR without sacrificing rigidity of
the Gd-complex or complete redesign of the entire scaffold.
written in terms of inner-sphere water relaxivity, eq 1, and the
Solomon equation, eq 2, which describes the field dependence
of T1 relaxation of the coordinated inner-sphere water
hydrogen atoms.3 Eq 1 teaches that the inner-sphere water
relaxation time T1M and the water residency time, τM, should be
as short as possible. With regards to T1M, eq 2 indicates that the
correlation time should be as large as possible, but while still
meeting the requirement of ωHτc < 1, where ωH is the proton
Larmor frequency and C is a constant. For a given Larmor
frequency, there is an optimal correlation time. Unless water
exchange is exceedingly fast (>109 s−1), the correlation time at
1.5 T and higher will essentially be the rotational correlation
time, τR. If τR is very long (nanoseconds and longer), then
relaxivity will be very high at low fields, but the condition ωHτc
Figure 2. (Top row) Schematic depiction of previously explored Gd complexes with τR between 0.35 and 1 ns (A, B, C), as well as the novel
approach described here (D). (Bottom row) Examples for molecules reported using approaches A,17 B5 and C.18
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Figure 3. (Top) Structures of approved Gd-based agents [Gd(DOTA)(H2O)]− and [Gd(HP-DO3A)(H2O)]. (Bottom) Various approaches to
conjugated DOTA derivates. E and F show previously investigated Gd(DOTA) type complexes with optimal water exchange properties. G
represents a previously explored lysine derivative of DOTA without the dual attachment strategy. Compound H represents our approach using dual
attachment to the peptide to limit internal motion of the complex.
This paper describes synthesis and investigation of a unique,
modular system, capable of the construction of a new
generation of high relaxivity T1 contrast agents for high
magnetic fields. Peptide structure and Gd complex incorpo-
ration can be easily modified using solid phase peptide
synthesis, without change of the local complex environment.
attachment of the metal complex using the dual anchor
strategy, (2) multimerization and (3) easy adjustment of
molecular size would provide a construct highly suitable for
high field applications.
The immediate coordination environment around the Gd ion
influences important parameters such as kinetic inertness and
water exchange kinetics. While q ≥ 2 complexes can provide
great relaxivity enhancement due to two or more possible sites
of interaction for water molecules with the paramagnetic
metal,30−34 only few have the kinetic inertness with respect to
Gd dissociation/transchelation required for in vivo applications.
For q = 1, a myriad of kinetically inert Gd(DOTA) type
complexes have been well characterized and provided us with
the information necessary for choosing a suitable system.35,36
DOTA monoproponiamide derivatives, where the amide
oxygen atom forms a 6-membered chelate ring upon
coordination of Gd(III), were found to have a mean water
residency time of 10−20 ns (at 37 °C), which is within the ideal
range required for our purposes (Figure 3, compound E).37
Geraldes and co-workers have reported the synthesis and
investigation of Gd(DO3A-N-α-aminopropionate) (Figure 3,
Compound F).38 This system provided the basis for our
investigations. We reasoned that derivatization and multi-
merization of DO3A-N-α-amino-propionate could be achieved
by using an Fmoc-analogue of this system and standard peptide
synthesis, similar to an approach previously explored by Sherry
and co-workers (Figure 3, compound G),39 as well as by
Stephenson et al.40 In our system, the complex is linked to the
peptide backbone via the short methylene linkage of the alanine
side chain. Gadolinium coordination of the amide carbonyl,
which is also used for coupling to the polypeptide, provides the
second point of attachment and results in a rigid incorporation
STRUCTURAL DESIGN
■
Control and optimization of τR requires rigid attachment of the
corresponding Gd complex to a molecular construct of
appropriate size. Conjugating the Gd complex to a targeting
vector or molecular scaffold is typically done through a single
linkage, and this results in fast internal motion about that
linkage and concomitant lower relaxivity. Tweedle and co-
workers pioneered the dual anchor strategy,26 which was also
employed by Desreux and colleagues to rigidify attachment of
the metal complex for the construction of fatty acid derivatized
Gd(DOTA).27 A similar, multisite attachment strategy was
employed for the design of metallostars, where a metallic
barycenter is used as a point of attachment for multiple
Gd(DTTA) type complexes (Figure 2, approach A).24 As
attachment of multiple copies of the Gd complex increases size,
the enhancement of τR combined with increase of the Gd-
complex payload will further expedite molecular relaxiv-
ity.25,28,29 Meade and colleagues employed click chemistry to
attach multiple Gd complexes to rigid, all-organic barycenters
(Figure 2, approach B).5,25 Alternatively, Parker and co-workers
showed that the rigid Gd complex can itself be placed at the
barycenter of a molecule of variable size (Figure 2, approach
C).18 Most of these constructs display enhanced relaxivity at
high fields compared to systems with either very short or very
long τR. We concluded that a combination of (1) rigid
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Scheme 1. Synthesis of Compound 6
a
Scheme 2. Synthesis of New Contrast Agents Described in This Paper
a
(i) (1) 4 equiv Fmoc-X (X = Gly, Phe, Cys(Acm)), 4 equiv HATU, 4.5 equiv NMM, DMF, 12 h, (2) 20% piperidine in DMF, 2 h; (ii) (1) 1.5
equiv 6, 2 equiv HATU, 3 equiv NMM, (2) 20% piperidine in DMF; (iii) 10 equiv I2, DMF, 6 h; (iv) TFA/DDT/TIPS/Water (9.5:0.25:0.25:0.25),
6 h; (v) DMSO (2% v/v), H2O (pH 8), 12 h.
of the complex into the polypeptide that should restrict internal
motion and enable control over τR. This design would be
capable of satisfying all our criteria: fast water exchange, tunable
rotational dynamics; limited internal motion, and ease of
derivatization using solid phase synthesis. Moreover, by using
the Gd(DOTAla) moiety itself to increase molecular size, the
overall molecular relaxivity is increased via multimerization
(Figure 3, compound H).
RESULTS AND DISCUSSION
■
Synthesis of Fmoc-DOTAla. For the use of DO3A-N-α-
aminopropionate in solid phase peptide synthesis, construction
of the corresponding Fmoc-derivative (“Fmoc-DOTAla-tBu3”,
compound 6) was required. Fmoc is easily deprotected under
mildly basic conditions while the ligand-carboxylates remain
protected41 hence it is more suitable for our purposes than a
potential Boc-derivative.5 As cyclen has high inherent basicity,
the Fmoc protective group can only be introduced after
alkylation of all secondary amines on cyclen.
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We employed a synthetic strategy similar to the one used by
Sherry and co-workers39 in order to afford 6 in 15% over all
yield after 5 synthetic steps (Scheme 1). Commercially available
serine derivative 1 was converted into the mesylate 2. Our
initial attempted introduction of this sterically crowded synthon
onto commercially available t-butyl protected DO3A failed.
Instead, 2 was used for the mono-N-alkylation of cyclen.
Compound 3 was further alkylated after removal of excess
cyclen mesylate salt using tert-butyl bromoacetate in order to
afford compound 4, which was isolated using preparative
HPLC. Simultaneous removal of the benzyl and carboxybenzyl
protective groups using H2 and Pd/C yielded compound 5.
Using Fmoc-Cl in a mixuture of H2O and dioxane under basic
conditions for 5 results in the introduction of the desired Fmoc
protective group. Compound 6 is purified using preparative
HPLC. It was found that using extended reaction times over 12
h leads to a considerable amount of side products of which
some are more difficult to separate from the final product.
Synthesis and Evaluation of Monomeric Metallopeptide.
As a next step, we aimed to incorporate 6 into the linear model
sequence H-Cys(Acm)-Gly-DOTAla-Gly-Phe-Cys(Acm)-NH2
(H3L1, Scheme 2). The corresponding Gd(III) complex
would allow us to study water exchange kinetics, while the
analogous Eu(III) complex provides information on the
hydration number (q) at the metal center. Rather forcing
conditions encompassing HATU in the presence of NMM in
DMF were required for significant product formation. We
synthesized H3L1 using PEGA-Rink resin and standard manual
solid phase synthesis (Scheme 2). Incorporation of a Phe
residue was employed to provide a UV handle for detection and
purification. Two Cys residues were used as the terminal amino
acids, serving as potential sites of secondary structure
modification through intramolecular disulfide bond formation.
The tert-butyl esters were removed simultaneously with
cleavage from the resin using a typical acidic cleavage cocktail
(TFA/DDT/TIPS/H2O (9.25:0.25:0.25:0.25)). The crude
peptide H3L1 was isolated using ether precipitation. Complex-
ation with either GdCl3·6H2O or EuCl3·6H2O by mixing of
aqueous solutions of the metal salt and the crude peptide at pH
3, followed by slow adjustment of the pH to 6.5 using an
aqueous 0.1 M NaOH solution yielded the corresponding
crude metal complex. The metallopeptide was purified using
preparative HPLC.
ence of the transverse relaxation time T2 of H217O in the
presence and absence of GdL1. The data in Figure 4 were fit to
Figure 4. Temperature dependence of the 17O NMR (11.7 T) reduced
transverse relaxation rates of GdL1 (6.8 mM). Solid line represents fit
to the data to determine the water exchange rate.
Table 1. Water Exchange Kinetic Parameters for GdL1 and
Comparison with the Gd(III) Complexes of Propionamide
a
and Propionate Derivatives E and F (Figure 3)
Gd complex
E37
F38
GdL1
310
k
ex
× 106 (s−1
)
84
42
60
6
ΔH⧧ (kJ mol−1
)
34.0
12
19.1
24
41.7 3.2
17
310τM (ns)
2
a
310
T
= 160 124 ns and ΔE⧧ = 21 18 kJ mol−1 for GdL1.
1e
a 4 parameter model as described previously.22 The water
exchange rate at 310 K, 310kex, its activation enthalpy, ΔH⧧, the
310
electronic relaxation time at 310 K, T1e, and its activation
energy, ΔE⧧ were iteratively varied to fit the observed reduced
relaxation rate data R2r. The hyperfine coupling constant was
fixed at 3.8 × 106 rad/s.45 At the high field used, τM dominates
the scalar correlation time and results in an accurate estimate of
water exchange, while the relative contribution of T1e to 17O
nuclear relaxation is much lower and this parameter is less well-
defined, Table 1. The water residency time (τM = 1/kex) was
determined to be 17 2 ns at 310 K, which is similar to the
Gd-DOTA-monopropionamide derivative reported by Geraldes
and co-workers (Table 1, Figure 4).38 This similar water
exchange rate is also consistent with the amide carbonyl as a
donor. We further note that this water residency time is in the
optimal range for high relaxivity at all field strengths.
This class of Ln-DOTA derivatives is typically 9-coordinate.
If the amide carbonyl from the peptide backbone was
coordinated to the lanthanide, then we would expect a single
aqua coligand, that is, q = 1. The luminescence lifetime of the
Eu(III) complex was measured in H2O and D2O. A custom-
designed multimodal confocal imaging system built by Yaseen
et al.42 was used to measure the luminescence lifetime of the
Multimeric Metallopeptides. GdL1 demonstrated that
the GdDOTAla moiety could be incorporated into a peptide,
and the resultant complex had the expected single inner-sphere
water coligand with an optimal water exchange rate. However
GdL1 is still a rather small molecule with a relatively short τR.
In order to increase τR and enhance the molecular relaxivity, we
also synthesized dimeric and trimeric structures. The cysteines
were either left protected (“linear” structures) or were
deprotected and used to induce intramolecular cyclization
(“cyclic” structures) in order to highlight the possibility of
secondary structure modification with our approach (Scheme
2). Multimers H6L2 and H9L3 were furnished using the same
synthesis methodology as for the linear, model monomer
peptide H3L1. On-bead deprotection of the Acm protective
group on the Cys amino acids using I2 was done in order to
43
5
Eu(III) excited state D1 as previously reported. Lumines-
cence lifetimes were measured and averaged and used for the
modified Horrocks equation44 (eq 3), which accounts for the
amide donor as one of the ligands.
⎡
⎢
⎤
⎥
⎛
⎝
⎞
⎠
1
1
⎜
⎜
⎟
⎟
qH O = 1.2
−
− 0.325
2
⎢
⎣
⎥
⎦
τ
τ
H2O
D2O
(3)
The value obtained for the hydration number q was 0.96
0.09, which suggests that the carbonyl of the peptide backbone
is indeed coordinated to the metal ion.
Water exchange kinetics for the inner-sphere water ligand
were determined by measurement of the temperature depend-
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afford the cyclized analogues H6C1, H6C2 and H9C3.46 As
cyclization was only 60% complete for compounds H6C2 and
H9C3, cyclization was driven to completion using 2% DMSO in
H2O at pH 8 (Scheme 2).47 The Gd complexes GdL1, Gd2L2
Gd3L3, GdC1, Gd2C2 and Gd3C3 were formed and purified
using the same methodology as described for the monomer.
Isolated yields for the cyclic products were considerably lower
due to intermolecular disulfide bond formation resulting in
polymeric side products, which are separated by HPLC
purification. All Gd complexes were characterized using LC−
ESI-MS.
Table 2. Half-times for Gd Transchelation to MS-325 at pH
3, 37 °C, with a Gd/MS-325-L ratio of 1:1 at 0.1 mM Gd
Complex Concentration
Gd complex
GdL1
t1/2 (h)
39
3
3
4
6
Gd2L2
52
61
91
Gd3L3
Gd(DO3A-HP)
Gd(DTPA)
0.42 0.0.18
Kinetic Inertness. The development of new contrast agents
requires compounds with high thermodynamic stability and
kinetic inertness with respect to Gd dechelation. Tei et al.
showed that a GdDOTA-monoproponiamide deriviative had a
very high stability constant, log KML = 20.2,37 and we expected
that our system with the same donor set would exhibit similar
thermodynamic stability. To address kinetic inertness, we
measured the full transchelation of Gd(III) from the complexes
GdL1, Gd2L2 Gd3L3 to a DTPA derivative with higher
thermodynamic stability. Each of these complexes was
challenged with one equivalent of the ligand of MS-325 (MS-
325-L) on a per gadolinium basis (e.g., Gd3L3 was challenged
with 3 equivalents of MS-325-L). MS-325-L is a DTPA
derivative with a biphenyl moiety that enables easy separation
and monitoring of the free ligand from the MS-325 gadolinium
complex by HPLC. Figure 5 shows the conversion of MS-325-L
to MS-325 as a function of time for the three metallopeptides at
pH 3 (10 mM citrate buffer) and 37 °C.48
the trimer, followed by the dimer. The approved macrocyclic
agent [Gd(HP-DO3A)(H2O)] showed even slower trans-
chelation kinetics. These results allowed us to conclude that
multimers based on Gd(DOTAla) are also suitable for in vivo
applications due to satisfactory kinetic inertness in comparison
with clinically utilized Gd based agents. We were also able to
confirm that multimerization has no detrimental effect on
decomplexation of the metal complex, rather it appears to have
a stabilizing effect.
Relaxivity. Per Gd relaxivities were determined by
measuring T1 at 37 °C using 20, 60, 200, 400, and 500 MHz
spectrometers. Relaxivities for MS-325 (with and without the
presence of HSA) as a reference compound with a long τR and
[Gd(HP-DO3A)(H2O)] as a reference for very short τR were
also measured, and all the relaxivity data is tabulated in Table 3,
together with results obtained from literature for the
compounds with similar estimated τR.
At low fields such as 0.47 and 1.4T, the compounds with the
highest rotational correlation times (MS-325/HSA and the
trimers) exhibit the highest relaxivity. Additional rigidity
through cyclization seems to provide only minor relaxivity
increase for the dimeric and trimeric systems (Gd2C2 and
Gd3C3). At intermediate field (4.7 T), only a moderate
decrease in relaxivity is observed for the metallopeptides. In
comparison, HSA-associated MS-325 exhibits a peak molecular
relaxivity of above 40 mM−1 s−1,49 followed by rapid decrease in
relaxivity upon increase of the magnetic field. Because we use
the rigid GdDOTAla amino acid for multimerization, both the
per Gd relaxivity and per molecule relaxivity increase with
increased molecular size. Figure 6A illustrates this effect where
we plot the field dependent molecular relaxivity of GdL3 along
with that of the approved contrast agents [Gd(HP-DO3A)-
(H2O)] in PBS and MS-325 in the presence of excess HSA. At
0.47 T the molecular relaxivity of Gd3L3 is similar to MS-325 in
HSA solution. As the field is increased the molecular relaxivity
of Gd3L3, with its intermediate rotational correlation time,
becomes higher than that of MS-325/HSA: 50% higher at 1.4 T
and 350−450% higher at fields from 4.7 to 11.7 T. The
molecular relaxivity of Gd3L3 is 5- to 11-fold higher than that of
[Gd(HP-DO3A)(H2O)] at all fields measured. On a per Gd
basis, the relaxivity of Gd3L3 is 50−220% higher than either
HSA-bound MS-325 or [Gd(HP-DO3A)(H2O)] at high fields
(4.7−11.7 T), Figure 6B.
Figure 5. Kinetic inertness of Gd(DOTAla) derivatives. Trans-
■
●
chelation of Gd from linear complexes GdL1( ), Gd2L2( ), Gd3L3
(
◆
▲
) and [Gd(HP-DO3A)(H2O)] ( ) to MS-325 at pH 3, 37 °C.
Data shown for the first 168 h of the reaction.
Transchelation was monitored using LC-MS, via formation
of the MS-325 complex. For comparison, we also measured
transchelation from the approved contrast agents [Gd(HP-
DO3A)(H2O)] (ProHance, gadoteridol) and [Gd(DTPA)-
(H2O)]2‑ (Magnevist, gadopentetate). Although thermody-
namically favored, it is apparent from Figure 5 that trans-
chelation takes place over days even at pH 3. We estimated
half-times for these transchelations (time to 50% of the
equilibrium value). For the approved contrast agent [Gd-
(DTPA)(H2O)]2‑, the half-time was 25 min. On the other
hand, the metallopeptides were much more inert with half-time
in the 2−3 day range (Table 2). Transchelation was slowest for
In order to further illustrate this, we imaged a series of
phantoms at 4.7 T (Figure 7). Water is used as a reference for
background, [Gd(HP-DO3A)(H2O)] as an example of a
compound with a short τR, and MS-325 bound to HSA as an
example of a complex with a long τR. Gd3L3 is shown at two
different concentrations: either equimolar on a per Gd basis, or
on a per molecule basis. It is evident that Gd3L3 provides better
contrast at this field strength then either FDA approved
compound, highlighting superiority in performance of our
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Table 3. Measured per Gd Relaxivities as a Function of Proton Larmor Frequency at 37 °C for the Linear and Cyclic Systems
Described in This Work along with [Gd(HP-DO3A)(H2O)] and MS-325 Measured in the Presence and Absence of 4.5%
a
Human Serum Albumin (HSA)
relaxivity [mM−1 s−1] per Gd
20 MHz
60 MHz
200 MHz
400 MHz
500 MHz
GdL1
8.1
10.8
13.2
8.3
7.4
9.9
7
5.8
6.1
6.1
5.1
5.7
6.6
3.0
4.8
4.1
4.9
4.9
4.7
4.5
4.5
5.5
2.9
4.7
3.7
n.a.
n.a.
Gd2L2
8.3
9
Gd3L3
12.2
7.1
GdC1
7.3
7.5
9.2
3.6
5.7
5.0
Gd2C2
11.4
12.7
4.3
10.6
12.3
3.2
Gd3C3
[Gd(HP-DO3A)]
MS-325
e
e
6.8
5.4
e
e
MS-325 w HSA
42
23.8
b
b
b
b
{Fe[Gd2bpy(DTTA)2(H2O)4]3}4−
[Bnt(Gd(HPN3DO3A)(H2O))3 ]
[Gd(gDOTA-Glu12)(H2O)]−
20.1
26.8
15.9
8.3
c
c
c
∼15
15.4
n.a.
4.8
d
d
23.5
∼25
n.a.
n.a.
n.a.
c
For comparison, literature data with examples of compounds A (q = 2),17 B,5 C (data obtained at 25 °C)18 are included. Reference 17. Reference
5. Reference 18. Data at 20 and 60 MHz from ref 22; n.a., data not available.
a
b
d
e
◆
●
Figure 6. Relaxivities of Gd3L3( ), MS-325 with excess HSA (∇) and [Gd(HP-DO3A)(H2O)] ( ) as a function of magnetic field at 37 °C. (A)
Relaxivity plotted per molecule showing that Gd3L3 with its intermediate correlation time is a much more potent relaxation agent than slow or fast
tumbling compounds at 60 MHz and higher frequencies. (B) Relaxivity plotted per Gd shows that the intermediate correlation time of Gd3L3 results
in higher relaxivities at high fields.
compound with intermediate τR at fields above 1.5 T. Under
these conditions, the signal intensity of Gd3L3 at equimolar
Gd(III) ion concentrations was 65% greater than [Gd(HP-
DO3A)(H2O)] and 55% greater than MS-325/HSA. On a per
molecule basis, the Gd3L3 phantom was 190% and 170%
brighter than Gd(HP-DO3A) and MS-325/HSA, respectively.
At 9.4 T, the per Gd relaxivities were 6.1 and 6.6 mM−1 s−1
for the trimeric metallopeptides. These values are also found to
be higher than the relaxivities measured at 9.4 T for previously
reported trimeric compounds of similar composition and
hydration number.5,30 For compounds based on q = 2
complexes, higher relaxivities can be obtained.17
Investigation of the effect of tertiary structure on relaxivity,
was done by examination of the effect of disulfide bond
reduction on T1 at 0.47 and 1.41 T. T1 was measured for each
sample at 37 °C and then the samples were incubated with 20
eq. TCEP for 30 min at room temperature to reduce the
intramolecular disulfide bond and give the linear peptide.
Subsequently, the T1 values were remeasured and concen-
trations redetermined in order to calculate relaxivities. A slight
decrease in relaxivity (7−14%) was observed for Gd2C2-red (9
mM−1 s−1) and Gd3C3-red (11.5 mM−1 s−1). Over all,
reduction of the disulfide bond has only a slight effect on
relaxivity. We hypothesize that the large Gd-chelate side chain
and the Gd(III) coordination by the amide carbonyl imposes
defined structure to the peptide that dominates the overall
molecule structures for both the linear and the cyclic multimers.
Introduction of a secondary structure modification such as the
cyclization has only a marginal influence on the relaxivity.
Nevertheless, facile introduction of the disulfide bridge by use
of standard peptide synthesis methodology demonstrates the
modularity of our system.
The high field relaxivities that we have obtained are
consistent with an intermediate rotational correlation time.
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Fmoc-DOTAla was performed using method A: Injection of crude
mixture onto preparative HPLC on a Rainin, Dynamax (column: 250
mm Polaris C18) by using A, 0.1% TFA in water; B, 0.1% TFA in
MeCN, flow-rate 15 mL/min, from 5% B to 95% B over 20 min.
Purification of Gd complexes was performed using method B:
Injection of crude mixture onto analytical column (Phenomenex
Luna, C18(2) 100/2 mm) using A, water; B, MeCN, flow-rate 0.8
mL/min, 15 min gradient from 2% B to 60% B over 15 min.
Monitoring of UV absorption was done at 220 nm. HPLC purity
analysis (both UV and MS detection) was carried out on an Agilent
1100 system (column: Phenomenex Luna, C18(2) 100/2 mm) with
UV detection at 220, 254, and 280 nm by using a method C: A
gradient of 95% A (0.1% formic acid in water) to 95% B (0.1% formic
acid in MeCN), flow-rate 0.8 mL/min, over 15 min. Kinetic inertness
measurements were also carried out using the LCMS agilent system,
using method D: A gradient of 95% A (ammonium formate, 20 mM,
pH 6.8) with 5% (9:1 MeCN/20 mm ammonium formate) to 95% B
(9:1 MeCN/20 mM ammonium formate), flow-rate 0.8 mL/min, over
15 min.
Figure 7. Gradient echo MR image acquired at 4.7 T (TE = 6 ms and
TR = 30 ms, flip angle = 90°) of equimolar solutions of [Gd(HP-
DO3A)(H2O)], MS-325 in HSA (0.66 mM), Gd3L3 in H2O at both
equal Gd(III) ionic concentration and equal molecular concentration.
The synthesis of ligands was carried out as shown in Schemes 1 and
2. 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.
Luminescence. Measurements were collected by using the
confocal portion of a custom-designed multimodal microscope.42,43
Briefly, a continuous-wave diode laser (l = 532 nm, B&W Tek)
provided excitation light that was temporally gated by an electro-
optical modulator (ConOptics, Danbury, CT) with extinction ratio of
approximately 200 at 532 nm. The excitation beam passed through
several conditioning optics, including a beam expander with pinhole
spatial filter, polarizer, shutter, dichroic mirror, scan lens, and tube lens
and a 20× magnification objective lens (XLumPlan FL, Olympus, NA
= 0.95). With the use of a customized control software and
galvanometric scanners (Cambridge Technology, Inc. Lexington,
MA) the excitation beam was guided to selected locations in the
approximately 600 μm field of view. The emitted luminescence was
descanned and collected by using an avalanche photodiode photon
counting module (APD, SPCM-AQRH-10, Perkin−Elmer, Waltham,
MA) sampled at 50 MHz with a high-speed DIO card (National
Instruments, Austin, TX). Data were processed by using custom-
written software in C and MATLAB (Mathworks, Natick, MA).
Detected luminescent photons were binned into 50 ms long bins, to
yield time-dependent phosphorescence decay profiles. With the use of
a nonlinear least-squares fitting routine, the resulting time-courses
were fit with a single-exponential function. A sample’s luminescence
lifetime is equal to its fitted profile’s calculated time constant.
(R)-tritert-Butyl 2,2′,2″-(10-(3-(B)-2-(((benzyloxy)carbonyl)-
amino)-3-oxopropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)-
triacetate (4). Cyclen (1.52 g, 8.8 mmol) was dissolved in MeCN (50
mL). K2CO3 (1 equiv, 0.61 g) was added and the reaction mixture was
preheated to 50 °C. (R)-Benzyl 2-(((benzyloxy)carbonyl)amino)-3-
((methylsulfonyl)oxy) propanoate (2 equiv, 1.8 g, 4.4 mmol) was
dissolved in MeCN (20 mL) and added dropwise to the preheated
solution. After 16 h, the precipitate was removed by filtration and the
solvent evaporated. The residue was taken up in EtOAc and extracted
twice with H2O (80 mL) and once with brine (80 mL). The organic
fraction was dried with Na2SO4 and the solvent was evaporated in
vacuo to afford the crude monocyclen derivative (1.48 g, 3 mmol),
which was resuspended in dry MeCN (50 mL) together with K2CO3
(10 equiv, 4.24 g). tert-Butyl bromoacetate (3.2 equiv, 1.45 mL, 1.91
g) was added dropwise and the mixture was stirred for 16 h at room
temperature. The solvent was then removed and the residue was
resuspended in EtOAc and extracted with H2O and brine. The organic
fraction was collected, dried with Na2SO4 and the solvent was
evaporated in vacuo to afford the crude product which was purified
using preparative HPLC, method A. Yield: 1.03 g (1.24 mmol, 28%).
1H NMR (CDCl3, 500 MHz, 298 K): δ = 7.31−7.30 (m, Bn−H,
By assuming that the contributions of second-sphere and outer-
sphere water can be estimated from a related q = 0 complex,50
we estimate τR of these metallopeptides to be in 150−600 ps
range, based on the magnitude and field dependence of their
relaxivities. More precise estimates of τR could be obtained by
additional relaxation measurements using high resolution NMR
with other Ln surrogates of Gd.51 Compared to the other
multimers reported in Table 3, our relaxivities are similar. For a
specific field strength the rotational dynamics will dictate the
optimal relaxivity. The modular amino acid approach presented
here offers the possibility to tune such a high field relaxivity by
systematically controlling the size and nuclearity of the
complex.
CONCLUSIONS
■
In conclusion, we were able to synthesize a single amino acid
Gd chelate, Gd(DOTAla), suitable for solid phase peptide
synthesis. The chelate is unique as it provides rigid and stable
attachment of the metal complex to the rest of the molecule by
using the amido-carbonyl of the corresponding peptide
backbone as a point of attachment. Gd(DOTAla) when
incorporated into a peptide exhibits one inner-sphere water
ligand that has an optimal rate of water exchange for
relaxometric purposes. The macrocyclic structure of the chelate
provides high thermodynamic stability and kinetic inertness
with respect to transchelation or Gd dissociation. The rigid
incorporation of Gd(DOTAla) into a peptide scaffold allows
design of contrast agents with defined rotational dynamics.
Here, we described 6 new compounds containing 1−3
Gd(DOTAla) per peptide in a linear or cyclic peptide
framework. By careful control of the rotational dynamics, it is
possible to design contrast agents with high relaxivities at both
low and high magnetic fields. These new contrast agents were
superior to commercial contrast agents [Gd(HP-DO3A)-
(H2O)] and MS-325/HSA at high fields. The modularity of
design, the ease of solid phase synthesis, high kinetic inertness,
and optimal water exchange rate renders the Gd(DOTAla)
scaffold a suitable platform for the development of high field T1
agents based on Gd.
EXPERIMENTAL SECTION
■
General Methods and Materials. 1H and 13C NMR spectra were
recorded on a Varian 11.7 T NMR system equipped with a 5 mm
broadband probe. Purification via HPLC of intermediates toward
10H), 5.14−5.04 (m, CH2−Bn, 4H), 4.75 (brs, α-CH, 1H), 3.75−3.05
(m, cyclen-H/N−CH2−COOtBu, 24H), 1.47−1.42 (m, CH3, 27H);
13C NMR (CDCl3, 125 MHz, 303 K): δ = 167.8, 167.7, 160.9, 160.7,
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Article
136.2, 134.8, 128.6, 128.5, 128.2, 127.9, 119.5, 117.2, 114.9, 112.6,
83.3, 68.0, 67.2, 55.0, 54.7, 50.9, 50.15, 27.9; LC/MS (ESI+):
C44H67N5O10 m/z: calcd. 826.5 [M + H]+; found: 826.4 [M + H]+.
(R)-2-Amino-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-
tetraazacyclododecan-1-yl)propanoic Acid (5). Compound 4 (5.5 g,
6.7 mmol) was dissolved in EtOH (600 mL). Pd/C (2.9 g, 10% w/w)
was added to afford a slurry which was subjected to H2 (35 psi) for 3
h. The Pd/C was filtered off and the filtrate was reduced in vacuo to
afford the product (3.85 g, 6.4 mmol) as a colorless oil which was used
H2N−C(Acm)PG-DOTAla-GC(Acm)CONH2 (H3L1), HPLC: Rt
= 2.4/3.1 min, MS-ESI: m/z: 1043.4 (calcd. 1043.3) [M + H]+.
H2N−C(Acm)PG-DOTAla-G-DOTAla-GC(Acm)CONH2
(H6L2), HPLC: Rt = 1.3/1.48 min, MS-ESI: m/z: 1514.6 (calcd.
1514.5) [M + H]+.
H2N−C(Acm)PG-DOTAla-G-DOTAla-G-DOTAla-GC(Acm)-
CONH2 (H9L3), HPLC: Rt = 1.21/1.35 min, MS-ESI: m/z: 994.0
(calcd. 994.2) [M + 2H]2+.
H2N−C(Scycl)PG-DOTAla-GC(Scycl)CONH2 (H3C1), HPLC: Rt
= 1.35/1.6 min, MS-ESI: m/z: 898.4 (calcd. 898.3) [M + H]+.
H2N−C(Scycl)PG-DOTAla-G-DOTAla-GC(Scycl)CONH2
(H6C2), HPLC: Rt = 1.1/1.2 min, MS-ESI: m/z: 1372.5 (calcd.
1372.3) [M + H]+.
1
without further purification in the subsequent reaction step. H NMR
(CD3OD, 500 MHz, 298 K): δ = 4.18 (brs, α-CH, 1H), 4.85 − 3.15
(m, cyclen-H/N−CH2−COOtBu, 24H), 1.53−1.50 (m, CH3, 27H);
13C NMR (CD3OD, 125 MHz, 303 K): δ = 170.4, 161.4, 161.2, 83.4,
H2N−C(Scycl)PG-DOTAla-G-DOTAla-G-DOTAla-GC(Scycl)-
CONH2 (H9C3), HPLC: Rt = 1.24 min, MS-ESI: m/z: 922.95 (calcd.
922.8) [M + 2H]2+.
54.3, 50.1, 49.1, 26.9; LC/MS (ESI+): C29H55N5O8 m/z: calcd. 602.4
[M + H]+; found: 602.5 [M + H]+.
(R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(4,7,10-
tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-
yl)propanoic Acid (6). Compound 5 (2.395 g, 3.98 mmol) was
dissolved in dioxane (60 mL). Na2CO3 (1.27 g, 11.9 mmol, 3 equiv)
was dissolved in H2O. The two solutions were mixed and cooled to 0
°C. Fmoc-Cl (1.125 g, 4.3 mmol) was dissolved in dioxane (5 mL) and
added to the reaction mixture. The solution was allowed to warm to
room temperature and stirred for 4 h. The solvent was then removed
and the solid residue was dissolved in MeCN. The residual solid was
filtered off and the filtrate was purified using preparative HPLC,
method A. The product fractions were pooled and the solvent was
removed in vacuo to afford the clean product as a white solid (1.81 g,
2.2 mmol, 55%). 1H NMR (CDCl3, 500 MHz, 298 K): δ = 7.30−7.26
(m, FmocAr−H, 10H), 4.76 (brs, α-CH, 1H), 4.30 (m, CH2−Fmoc,
2H), 4.15 (q, CH-Fmoc, 1H), 3.75−3.05 (m, cyclen-H/N−CH2−
COOtBu, 24H), 1.45−1.36 (m, CH3, 27H); 13C NMR (CDCl3, 125
MHz, 303 K): δ = 171.1, 169.7, 143.6, 141.2, 127.8, 127.2, 125.2,
119.9, 84.8, 83.3, 67.5, 54.0, 50.7, 48.3, 46.8, 27.8; LC/MS (ESI+):
C44H65N5O10 m/z: calcd. 824.5 [M + H]+; found: 824.4 [M + H]+.
Solid-phase Peptide Synthesis. Solid-phase peptide synthesis was
carried out manually following standard Fmoc protocols using PEGA
Rink amide resin. All peptide sequences were derived from one solid
support 0.33 mmol scale using single step couplings of four equivalents
of Fmoc-amino acids, two equiv of coupling agent (HATU) and 3
equiv of N-methylmorpholine (NMM) in DMF at RT (refer to
Scheme 2). Coupling with commercial amino acids was completed
within 12 h (Step i), while Fmoc-DOTAla was only used in a 1.5 equiv
excess and allowed to react with the free N terminus of the peptide for
48 h (Step ii). The coupling step was followed by rinsing with DMF
and deprotection with 20% piperidine in DMF for 2 h. After
subsequent thorough rinsing with DMF and dichloromethane, a small
aliquot of solid support was removed from the batch and deprotected
u s i n g c l e a v a g e c o c k t a i l ( T F A / D D T / T I P S / W a t e r
(9.25:0.25:0.25:0.25)) room temperature for 2 h. The resin was
filtered off and the filtrate concentrated with a gentle nitrogen flow.
The intermediate was precipitated with cold diethyl ether, collected
and characterized by ESI-MS. If coupling was found to be complete,
the next coupling step was initiated on the main peptide batch. Once a
sequence was complete, the corresponding aliquot was removed from
the main resin batch and completed by addition of the terminal Fmoc-
Cysteine-S-Acm. For cyclic sequences, treating the resin-bound
peptide with 10 equiv of I2 in DMF for 6 h completed side chain
deprotection with simultaneous cyclization of the Cys residues (Step
iii). After thorough rinsing of the resin with DMF and dichloro-
methane following the final processing step on-bead (Fmoc
deprotection for linear systems, I2 cyclization for cyclic sequences),
the crude peptide was afforded by cleaving from the resin using the
acidic cleavage cocktail (see above) and isolated by cold ether
precipitation, redissolution in water and lyophilization (Step iv).
Because the on-bead cyclization proceeds to only approximately 60%
completion, the crude peptide is further cyclized using 2% DMSO in
basic H2O (pH ∼7.5). As epimerization occurs on the stereocenter of
DOTAla, multiple peaks are detected for the corresponding
diastereomers.
Gadolinium Complex Formation. Complexes were prepared by
adding GdCl3•6H2O stock solution to a solution of ligand at pH 3
while stirring. The pH was gradually adjusted to pH 6.5 using 0.1 M
NaOH solution. Complete complex formation was checked by LCMS
(no residual ligand detectable). The solution was filtered and purified
using preparative HPLC, method B. The Eu(III) complex is formed in
analogous fashion.
H2N−C(Acm)PG-DOTAla(Gd)-GC(Acm)CONH2 (GdL1),
HPLC: Rt = 2.9/3.3 min, MS-ESI: m/z: 1197.3 (calcd. 1197.2) [M
+ H]+.
H2N−C(Acm)PG-DOTAla(Gd)-G-DOTAla(Gd)-GC(Acm)-
CONH2 (Gd2L2), HPLC: Rt = 2.6/3.0 min, MS-ESI: m/z 912.5
(calcd. 912.5) [M + 2H]2+.
H2N−C(Acm)PG-DOTAla(Gd)-G-DOTAla(Gd)-G-DOTAla-
(Gd)-GC(Acm)CONH2 (Gd3L3), HPLC: Rt = 3.2 min, MS-ESI: m/
z: 1225.95 (calcd. 1225.8) [M + 2H]2+.
H2N−C(Scycl)PG-DOTAla(Gd)-GC(Scycl)CONH2 (GdC1),
HPLC: Rt = 1.13 min, MS-ESI: m/z: 1053.2 (calcd. 1053.4) [M + H]+.
H2N−C(Scycl)PG-DOTAla(Gd)-G-DOTAla(Gd)-GC(Scycl)-
CONH2 (Gd2C2), HPLC: Rt = 1.25 min, MS-ESI: m/z 840.7 (calcd.
841.5) [M + 2H]2+.
H2N−C(Scycl)PG-DOTAla(Gd)-G-DOTAla(Gd)-G-DOTAla-
(Gd)-GC(Scycl)CONH2 (Gd3C3), HPLC: Rt = 1.35/1.9 min, MS-ESI:
m/z: 1153.8 (calcd. 1154.6) [M + 2H]2+.
Reduction of Disulfide Bond for Relaxivity Measurements.
Complex solutions of purified, cyclic Gd complexes (concentrations
of 0.1−0.025 mM, 110 μL) in HEPES buffer (50 mM, pH 7.4) were
mixed with TCEP solution (20 mM in HEPES, 10 μL) and incubated
room temperature. Reduction was checked by LCMS analysis and
found to be complete after 30 min.
H2N−C(SH)PG-DOTAla(Gd)-GC(SH)CONH2 (GdC1-red),
HPLC: Rt = 2.35 min, MS-ESI: m/z: 1055.2 (calcd. 1055.2) [M + H]+.
H2N−C(SH)PG-DOTAla(Gd)-G-DOTAla(Gd)-GC(SH)-
CONH2 (Gd2C2-red), HPLC: Rt = 2.8−3.1 min, MS-ESI: m/z 841.7
(calcd. 842.0) [M + 2H]2+.
H2N−C(SH)PG-DOTAla(Gd)-G-DOTAla(Gd)-G-DOTAla-
(Gd)-GC(SH)CONH2 (Gd3C3-red), HPLC: Rt = 3.1−3.3 min, MS-
ESI: m/z: 1155 (calcd. 1155) [M + 2H]2+.
Measurement of Kinetic Inertness. Stock solutions of MS-325-L
and GdL1, Gd2L2 and Gd3L3 were prepared in 50 mM citrate buffer
at pH 3.0. MS-325-L was added to solutions of the Gd complexes and
incubated at 37 °C. The final concentrations of the metal complexes
were 0.1 mM, while the concentration of MS-325-L was adjusted
according to the amounts of Gd complexes per metallopeptide
present. A 10 μL aliquot was removed for HPLC analysis and analyzed
while the remainder of the solution was incubated at 37 °C. A 10 μL
aliquot was removed and analyzed at 5, 10, 25, 46, 78, 96, 122, 141 and
244 h. As a reference, Gd(HP-DO3A) was subjected to MS-325-L
under same conditions and measured at time points 0.3, 1.5, 4, 6, 8, 24,
168 and 336 h.
Measurement of Relaxivity. Longitudinal relaxation times T1, were
measured on Bruker Minispecs mq20 (0.47 T) and mq60 (1.41 T), a
Bruker Bioscan horizontal bore 4.7, 9.4 and 11.7 T Varian NMR
spectrometers. T1 was measured by using an inversion recovery
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Journal of the American Chemical Society
Article
method with 10 inversion time values ranging from 0.05 × T1 to 5 ×
T1. Relaxivity was calculated from a linear plot of 3 or 4 different
concentrations (ranging from 0.01 to 0.5 mM, depending on amount
of compound isolated) versus the corresponding inverse relaxation
times. All samples were measured at 37 °C using either the internal
temperature control of the instrument (0.47, 1.41, 9.4, and 11.7 T) or
a warm air blower (4.7 T). MS-325/HSA was prepared in a 4.5% w/v
solution of HSA (0.66 mM) in PBS. The MS-325 concentration (in
presence of HSA) ranged from 0.05 to 0.15 mM.
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17O NMR of GdL1 Solution for Determination of τM. 17O NMR
measurements of solutions were performed at 11.7 T on 150 μL
samples contained in 2-mm-shigemi tubes inside a 5 mm standard
NMR tube on a Varian spectrometer. Temperature was regulated by
air flow controlled by a Varian VT unit. 17O transverse relaxation times
of distilled water (pH 3) containing 5% enriched 17OH2 or a 6.88 mM
solution of GdL1 (pH 7.4, 50 mM HEPES buffer) were measured
using a CPMG sequence. The concentration of the sample was
determined by ICP-MS. Reduced relaxation rates, 1/T2r were
calculated from the difference of 1/T2 between the GdL1 sample
and the water blank, and then divided by the mole fraction of
coordinated water. The temperature dependence of 1/T2r was fit to a
4-parameter model as previously described.22 The Gd−O hyperfine
coupling constant, A/ℏ, was assumed to be 3.8 × 106 rad/s,45 the Gd−
O distance was assumed to be 3.1 Å.52
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AUTHOR INFORMATION
■
Corresponding Author
caravan@nmr.mgh.harvard.edu
(26) Ranganathan, R. S.; Fernandez, M. E.; Kang, S. I.; Nunn, A. D.;
Ratsep, P. C.; Pillai, K. M. R.; Zhang, X.; Tweedle, M. F. Invest. Radiol.
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Notes
(27) Vanasschen, C.; Bouslimani, N.; Thonon, D.; Desreux, J. F.
Inorg. Chem. 2011, 50, 8946−8958.
The authors declare no competing financial interest.
(28) Sieving, P. F.; Watson, A. D.; Rocklage, S. M. Bioconjugate Chem.
1990, 1, 65−71.
ACKNOWLEDGMENTS
■
Dr. Mohammad A. Yaseen is warmly acknowledged for
measurement of the luminescence lifetimes of Eu(L1). Dr.
(29) Aime, S.; Botta, M.; Crich, S. G.; Giovenzana, G.; Palmisano, G.;
Sisti, M. Bioconjugate Chem. 1999, 10, 192−199.
(30) Livramento, J. B.; Helm, L.; Sour, A.; O’Neil, C.; Merbach, A. E.;
Daniel Schuhle is acknowledged for helpful discussions. This
̈
́
́
Toth, E. Dalton Trans. 2008, 1195−1202.
work was supported in part by awards R01EB009062 from the
National Institute of Biomedical Imaging and Bioengineering
and P41RR14075 from the National Center for Research
Resources. E.B. acknowledges the Swiss National Science
Foundation for a fellowship for prospective researchers.
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