Optimizing Water Exchange Rates and Rotational Mobility for High-Relaxivity of a Novel Gd-DO3A Derivative Complex Conjugated to Inulin as Macromolecular Contrast Agents for MRI

Article abstract of DOI:10.1002/cbdv.201700487

Thanks to the understanding of the relationships between the residence lifetime τ_M of the coordinated water molecules to macrocyclic Gd-complexes and the rotational mobility τ_R of these structures, and according to the theory for paramagnetic relaxation, it is now possible to design macromolecular contrast agents with enhanced relaxivities by optimizing these two parameters through ligand structural modification. We succeeded in accelerating the water exchange rate by inducing steric compression around the water binding site, and by removing the amide function from the DOTA-AA ligand [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono(p-aminoanilide)] (L) previously designed. This new ligand 10[2(1-oxo-1-p-propylthioureidophenylpropyl]-1,4,7,10-tetraazacyclodecane-1,4,7-tetraacetic acid (L₁) was then covalently conjugated to API [O-(aminopropyl)inulin] to get the complex API-(GdL₁)x with intent to slow down the rotational correlation time (τ_R) of the macromolecular complex. The evaluation of the longitudinal relaxivity at different magnetic fields and the study of the ¹⁷O-NMR at variable temperature of the low-molecular-weight compound (GdL₁) showed a slight decrease of the τ_M value (τ_M³¹⁰ =?331?ns vs. τ_M³¹⁰ =?450?ns for the GdL complex). Consequently to the increase of the size of the API-(GdL₁)x complex, the rotational correlation time becomes about 360 times longer compared to the monomeric GdL₁ complex (τ_R?=?33,700?ps), which results in an enhanced proton relaxivity.

Full text of DOI:10.1002/cbdv.201700487

Article Type: Full Paper
Optimizing Water Exchange Rates and Rotational Mobility for High-Relaxivity
of a Novel Gd-DO3A Derivative Complex Conjugated to Inulin as
Macromolecular Contrast Agents for MRI
Luigi Granato,1 Luce Vander Elst,1 Celine Henoumont,1 Robert N. Muller,1,2 Sophie
Laurent1,2
1
General, Organic and Biomedical Chemistry Unit, NMR and Molecular Imaging
Laboratory,
University of Mons, Avenue Maistriau, 19, B-7000 Mons, Belgium
2 Center for Microscopy and Molecular Imaging (CMMI), Rue A. Bolland, 8, 6041Gosselies,
Belgium
Corresponding author mail id: sophie.laurent@umons.ac.be
Abstract
Thanks to the understanding of the relationships between the residence lifetime τM of
the coordinated water molecules to macrocyclic Gd-complexes and the rotational
mobility τR of these structures, and according to the theory for paramagnetic
relaxation, it is now possible to design macromolecular contrast agents with
enhanced relaxivities by optimizing these two parameters through ligand structural
modification. We succeeded in accelerating the water exchange rate by inducing
steric compression around the water binding site, and by removing the amide
function
from
the
DOTA-AA
ligand
[1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid mono(p‐aminoanilide)] (L)
previously designed. This new ligand 10[2(1-oxo-1-p-propylthioureidophenyl-propyl]-
1,4,7,10‐tetraazacyclodecane‐1,4,7‐tetraacetic acid (L1) was then covalently
conjugated to API [O-(aminopropyl)inulin] to get the complex API-(GdL1)x with intent
to slow down the rotational correlation time (τR) of the macromolecular complex. The
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evaluation of the longitudinal relaxivity at different magnetic fields and the study of
17
the O NMR at variable temperature of the low-molecular-weight compound (GdL1)
showed a slight decrease of the τM value (M310=331ns vs M310=450ns for the GdL
complex). Consequently to the increase of the size of the API-(GdL1)x complex, the
rotational correlation time becomes about 360 times longer compared to the
monomeric GdL1 complex (R = 33700ps), which results in an enhanced proton
relaxivity.
Keywords
Contrast agents, Gd complexes, relaxivity, MRI, NMRD profiles
Introduction
Magnetic Resonance Imaging (MRI) has become an important technique in modern
diagnostic medicine, providing high-quality three-dimensional images of soft tissue
without the need for harmful ionizing radiation.[1] The difference in signal intensity
between tissues or anatomic spaces can be enhanced by manipulation of imaging
parameters and/or by the introduction of certain chemical (contrast) agents that
influence the relaxation of water, the most abundant substance in human tissues.
These contrast agents (CAs) are paramagnetic compounds that shorten the
relaxation times of local proton spins (T1 and T2) and result in an increase of the
signal intensity of T1-weighted images and decrease it in T2-weighted images.[ 2]
The majority of the approved CAs are gadolinium chelates that are based both on
macrocyclic tetraazapolyaminocarboxylate chelators (e.g., Dotarem®, Prohance®,
and Gadovist®) or open-chain polyaminocarboxylate chelators (e.g., Magnevist®,
Omniscan,® and Multihance®).
These low-molecular-weight chelates constitute the first generation of MRI contrast
agents and are extracellular fluid agents as they equilibrate rapidly between the
intravascular and interstitial space and do not interact specifically with any type of
cells.[3]
The efficiency of such contrast agents is evaluated on the basis of their relaxivity, or
how much the relaxation rates of water protons are increased in the presence of the
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agent at a given concentration. The observed relaxation rates of solvent protons
result from both paramagnetic (1/Ti,p) and diamagnetic (1/Ti,d) contributions. The
paramagnetic contribution is linearly related to the concentration of paramagnetic
species present, while the diamagnetic contribution corresponds to the relaxation rate
of the solvent (water) nuclei in the absence of a paramagnetic solute. The overall
relaxivity (r1) of the water nuclei observed in aqueous solution of paramagnetic Gd3+-
complexes is governed mainly by two mechanisms: an inner-sphere component from
the proton relaxation of a solvent molecule directly coordinated to the Gd3+ ion, and
an outer-sphere component from bulk solvent.[4-6] Current agent design focuses
is
mainly on attaining higher inner-sphere longitudinal relaxivity, r1 , from protons of
water molecules in the first coordination sphere of the metal. Equation 1 reveals that
if water exchange at the Gd3+ center is fast enough (small values of τM, the mean
water residence time), the paramagnetic relaxation enhancement experienced by the
bulk solvent will come from the relaxation rate (1/T1M) increase of the coordinated
solvent molecule (1/T1 is the longitudinal relaxation rate, q is the number of bound
solvent molecules, and PM = [Gd]/55.6, is the mole fraction of water coordinated to
the metal center). According to the Solomon-Bloembergen-Morgan (SBM) equations
of paramagnetic relaxation theory,[7-9] T1M for the dipole-dipole relaxation mechanism
is defined by Equation 2. This equation shows that modulation of the correlation time
τc defined in Equation 3 (where 1/Tie is the electronic longitudinal and transverse
relaxation rate) becomes critical in the obtainment of the high relaxivities predicted by
theory.[4,6]
Eq. 1
Eq. 2
Eq. 3
(i = 1, 2)
The relaxivities of current commercial agents based on polyaminocarboxylate
scaffolds are small compared to what is theoretically possible, with r1 values of only
4-5 mM-1 s-1 at 20 MHz. By virtue of these equations 1-3, scientists have focused on
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maximizing the hydration number q (q = 1 for all commercial agents), on optimizing
τM (150-1000 ns in commercial agents), and the rotational correlation time τR (in the
ps regime for small molecules) in order to increase the relaxivity.[10] It has indeed
been demonstrated that for small-molecular-weight Gd3+-complexes with optimized
τM, the overall relaxivity is mainly controlled by the rotational correlation time. Figure 1
depicts simulations of relaxivity profiles at several values of rotational correlation time
τR and water residence time τM with fixed values of electronic parameters.[11] It is
clear that every magnetic field requires different optimal parameters of τR and τM and
the maximum relaxivity can be achieved at fields corresponding to the proton Larmor
frequency 10-70 MHz (magnetic field 0.24-1.65 T) with τR as high as possible and τM
in the range 10-50 ns.
M = 10 ns ; R = 0.1 ns
M = 10 ns ; R = 1 ns
M = 10 ns ; R = 10 ns
M = 500 ns ; R = 10 ns
M = 500 ns ; R = 1 ns
M = 500 ns ; R = 0.1 ns
60
40
20
0
0.01
0.1
1
10
100
1000
Proton Larmor Frequency (MHz)
Figure 1 Simulations of relaxivity as a function of proton Larmor frequency (1H NMRD profile) with
298τM = 10 ns (black) and 500 ns (grey) and 298τR = 100 ps, 1 ns and 10 ns. T = 37 °C, 298τv = 40 ps,
RGdH = 3.1 Å. Adapted from [11]
A different situation takes place at higher frequencies 100-400 MHz (fields 2.35-9.4
T) where a shorter τM is more suitable and τR should have an intermediate value. In
the design of Gd3+-based CAs, this should be taken into account.In particular, optimal
values of about 1-30 ns for water residence times (τM; optimal value decreases with
increasing magnetic field strength) and ns values of the rotational correlation time (τR)
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are required to reach the peak in the relaxivity profile. It is therefore necessary to
increase water exchange rates and slow down molecular tumbling relative to
commercial agents, while also maintaining long electronic relaxation times with a high
number of inner-sphere water molecules to achieve the high relaxivities predicted by
theory. Although attaining a more favorable combination of these parameters relative
to current agents is desirable, it must come without sacrificing chelate stability, so
that toxicity due to free Gd3+ is avoided.
The aim of this article is to demonstrate how to enhance the relaxivity at 1.5 T (the
most common magnetic field used in MRI scanner) of the Gd3+-complexes by
modulating only the τM, and/or τR, while the number of coordinated water molecules
(q) stays constant. As previously demonstrated[6], the increase of q leads always to
complexes characterized by a lower stability due to the reduced coordination number
with gadolinium. As a consequence, only stable Gd3+-complexes of macrocyclic
DO3A-derivatives, that have one inner-sphere water molecule directly coordinated to
the paramagnetic centre, will be studied.[6]
The work is divided in two parts: a small monomeric Gd-complex (GdL1, scheme 1)
will first be synthesized by modifying a Gd-complex previously published (GdL)[12] in
order to optimize the τM value. It will secondly be covalently attached to inulin (API-
(GdL1)x, scheme 1) to achieve an optimal value of the τR at around 10 ns at 1.5 T
and again be compared to a previously synthesised macrocyclic Gd-complex (API-
(GdL)x).[12] Inulin is a polydisperse polysaccharide which consists of β(2→1) fructosyl
fructose units with one glucopyranose unit at the reducing end. Depending on the
source, the degree of polymerization (dp) of inulin varies from 10 to 30. This leads to
an average molecular weight distribution in a range between 1.5 and 5 kDa, which is
favourable for the preparation of macromolecular conjugates : it allows an increase of
the τR of the Gd-complex as well as an increase of the Gd3+ concentration per
molecule, which should be accompanied by an increase of the relaxivity of the
[13]
compound.
Furthermore, inulin and its derivatives possess a low viscosity in
aqueous solutions and do not metabolize in blood; both properties can be considered
as advantageous for the application of MRI CAs.
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O
O
O
O
HO
N
N
N
N
OH
HO
N
N
N
N
OH
S
HO
O
O
HO
NH
N
N
H
H
O
O
HN
S
L1
L
HN
O
OR'
OR'
O
O-
O
O
O
-O
R'O
R'O
R'O
N
N
O
R'O
N
O-
N
OR'
O
OR'
Gd3+
Gd3+
R'O
-O
-O
N
R'O
O
N
O
S
O
N
N
O
N
H
S
OR'
O
OR'
-O
N
N
H
N
O
H
O
N
H
O
O
H
R'O
O
R'O
O
R'O
dp = 25
ds = 1.4
O
O
R'O
dp = 32
ds = 0.9
OR'
API-(GdL)x
OR'
API-(GdL1)x
OR'
OR'
R' = H and/orCH2CH2CH2NH2
and/or L
R' = H and/orCH2CH2CH2NH2
and/or L
Scheme 1 : Representation of the Gd-complexes with the chemical structural difference between
ligand L1 and L previously published.[12]
Results and Discussion
1. Synthesis and relaxometric characterization of the GdL1 complex :
optimizing the water residence time τM.
The rate of exchange between the water molecule coordinated to a metal ion and
bulk water is quite important in the design of most efficient CA for MRI.[14] SBM theory
predicts that for a Gd3+-based T1 agent to achieve its highest T1 sensitivity (maximum
r1), the optimal bound water lifetime is about 10 ns at 1.5T.[6] Various strategies to
modify τM have been discussed in the literature[15] but for the purpose of this work, we
will briefly describe those factors that can be manipulated on a chemical basis by
ligand design. These effects can generally be attributed to stabilization or
destabilization of intermediates involved in the water exchange mechanism and/or
changes in the population of various CA isomers present in solution.
One of the strategies to increase the rate of water exchange is to modulate the steric
hindrance around the Gd3+ coordination sphere which forces the bound water
molecule to reside, on average, further away from the Gd3+-ion, thereby making it
easier for the water molecule to leave the coordination sphere and mix with the much
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larger pool of bulk water molecules. This has been demonstrated by a comparison of
the water exchange rate in the Gd3+-complexes of DOTA (macrocycle containing 12
atoms, τM = 122 ns) with TRITA (macrocycle containing 13 atoms, τM = 3.7 ns)[16] or
DO3A-N-prop (one acetate replaced by a propionate, τM = 16.4 ns)[17] (see Figure 2a
to inspect the ligand structures discussed in this section). While Gd-DO3A-N-prop
displays a near optimal τM value, the kinetic stability of the complex is somewhat
compromised compared with Gd-DOTA. This effect is even more pronounced in Gd-
TRITA, which has a relatively poor kinetic stability (k1 = 0.21 M-1s-1).[18] Adding a
charged group to the ligand also has an effect on water exchange. For example,
addition of an amino group to the propyl sidechain of DO3A-N-prop to yield DO3A-α-
amino-prop results in a dramatic shortening of the bound water lifetime (25 ns) in
comparison with the Gd-DOTA complex.[19] On the other hand, when a negatively
charged acetate group is added to DO3A-N-prop to form DOTA-SA, the water
exchange rate in the corresponding Gd3+-complex becomes slower (τM = 159 ns)[20]
compared to the DO3A-α-amino-prop derivative. An interesting feature of the Gd-
DOTA-SA complex is that its rotational correlation time is larger than for Gd-DOTA
(125 ps versus 53 ps, respectively) which must be attributed to the additional
negative charge of the extra carboxylate. It is believed that this carboxylate may
assemble extra water molecules in the second coordination sphere which makes the
effective molecular weight of the complex larger and hence molecular rotation
becomes slower. Gd-DOTA-SA also has the highest thermodynamic stability (log Kst
= 27.2) reported to date for DOTA-like complexes and this again can be attributed to
the excess negative charge.[20] Addition of a bulky group onto the α-position of an
acetate sidechain also has an impact on water exchange. For example, when four
methyl groups are introduced, one per acetate, as in Gd-DOTMA, the population of
coordination isomers changes from favouring the SAP isomer in Gd-DOTA to
preferring the TSAP isomer in Gd-DOTMA.[21] Given that water exchange has been
observed to be ~50-fold faster in TSAP isomers compared with SAP isomers (Fig. 2
b), it is not surprising to find that the measured water exchange lifetime is faster in
Gd-DOTMA (τM310 = 85 ns) than in Gd-DOTA (τM310 = 122 ns).
Another way to modulate the τM is to replace the negatively carboxylate groups (-
COO-) with neutral amide groups (-CONHR) which is one effective way to slow the
water exchange rate in DOTA-based complexes. Given that the oxygen atom of an
amide group is less basic than an oxygen atom of carboxylate, a single amide
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substitution can have a dramatic effect on the rate of water exchange. For example,
water exchange in the mono-amide complex, Gd-DOTA-1-Bz-NO2, is about 5-fold
slower (τM = 625 ns) than that in Gd-DOTA. Typical Gd-DOTA-bis-amide complexes
display even slower water exchange as evidenced by the τM value reported for Gd-
DOTA-2DMA (811 ns)[22] while Gd-DOTA-tetra-amide complexes such as Gd-
DOTAM have bound water lifetimes extending into the several µs range (19 µs).[23] It
has also been shown that the bound water lifetime decreases with an increase in the
number of methyl substituents on the amide nitrogen (compare Gd-DTMA [τM = 17
µs] versus Gd-DOTTA [τM = 7.8 µs]).[23]
This indicates that the increased steric bulkiness provided by the two methyl
substituents can be used to fine-tune water exchange rates in such complexes.
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A
B
Figure 2 (a) Structures of some DOTA-based ligands discussed in this section; (b) two
diastereoisomeric forms, M (SA) and m (TSA), of Ln (III) complexes with DOTA-type ligands. Adapted
from [6]
On the basis of these observations, we set off with the idea of inducing steric
compression around the water binding site in dissociatively exchanging Gd3+-
complex by adding a methyl group (-CH3), and by removing the neutral amide groups
(-CONHR) from the DOTA-derivative ligand (L), previously synthesised and
described elsewhere (Scheme 1),[12] in order to accelerate the water exchange
without decreasing its well-known complex stability.
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The new complex GdL1 was obtained by following the reactions outlined in Scheme
2. This route required the synthesis of a particular linker (2), which is later added to
the 1,4,7,10-tetraazacyclodecane-1,4,7-tris-(acetic acid t-butyl ester) (3 DO3A-tBu),
by using the same alkylation condition employed in the previously published
synthesis.[12]
O
i
ii
NH2
NH
O
Br
O
NH
O
O
O
O
1
2
But O
N
N
N
N
O tBu
HO
N
N
N
N
OH
iv
v
But O
O
HO
O
O
O
O
O
NH HN
But O
But O
N
N
O tBu
iii
4
5
NH
NH2
NH HN
N
HN
HBr
*
O
O
3
vi
O
O
-O
-O
N
N
N
O-
O
O
OH
Gd3+
HO
N
N
N
N
O
vii; viii
xi; viii
O
HO
N
O
O
HN
S
HN
GdL1
N
6
C
S
OH
OR
OR
O
O
O
HO
HO
RO
RO
RO
RO
O
OH
OR
OR
-O
HO
RO
RO
O
O
O
O
O
O
N
-O
OH
OH
OR
S
OR
OR
ix; x
Gd3+
N
O
N
N
O
N
HO
RO
H
H
OH
O
OR
O
N
RO
O
n= 32
ds= 0.9
O
O
n= 32
ds= 0.9
O
n=32
O
O-
OR
O
OH
OR
OR
OH
OR
OR
API-(GdL1)x
7
API
R = H and/or CH2CH2CH2NH2
R' = L1 and/or H
Scheme 2 Synthesis of API-(GdL1)x and GdL1: (i) acetyl chloride, K2CO3, CH2Cl2, 0 °C; (ii)
Bromopropionyl bromide, 1,2,4-trichlorobenzene, AlCl3, 70 °C; (iii) tert-butyl bromoacetate, NaHCO2,
CH3CN; K2CO3, CH3CN; (iv) K2CO3, CH3CN, room temperature (rt); (v) 0.1 M HCl, CH3CN, 100 °C, 4h;
(vi) CSCl2, CCl4/water, rt; (vii) water, 1‐propylamine, pH 9-10; (viii) GdCl3·6H2O, pH 6.5-7.0; (ix)
acrylonitrile, aqueous NaOH, 45 °C; (x) Li, liquid NH3; EtOH, 50 °C; (xi) water, pH 9-10, room
temperature.
The preparation of the 4-acetamido-α-bromopropiophenone (2) was attempted by
using of conventional Friedel-Crafts conditions, where bromopropionyl bromide was
added at 70 °C in a pre-incubated mixture of acetanilide and aluminium trichloride, in
1,2,4-trichlorobenzene as a solvent.
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The final GdL1 complex was obtained via the same complexation method previously
described for synthesising the agent GdL.[12]
Variable-temperature 17O NMR measurements
The determination of the water exchange rate kex (or the residence lifetime of water
molecule in the first coordination sphere τM = 1/kex) is of utmost importance. This
parameter contributes directly to the modulation of the electron-nuclear dipolar
interaction as it controls the efficiency of the transfer of the paramagnetic effect to the
bulk water. The water exchange rate of GdL1 was determined from a variable
17
temperature O NMR study, more specifically from the temperature dependence of
the transverse relaxation rate 1/T2 of the water in an aqueous solution of the
compound of interest (figure 3a).
The experimental data were fitted with a theoretical model as described previously,[24]
310
which gives a τM value of 331 ± 44 ns. This is lower than that calculated for GdL
(450 ± 20 ns), but higher than that for Gd-DOTA (122 ± 10 ns) complex. A detailed
discussion of these calculated results is presented in the next paragraph, where the
Nuclear Magnetic Relaxation Dispersion (NMRD) data are discussed.
Figure 3 (a) Temperature dependence of the reduced 17O transverse relaxation rate at 11.75 T; (b)
comparison between 1H NMRD profiles of GdL1 1.78 mM (spheres), GdL 2.60 mM (triangles) and Gd-
DOTA (stars). The lines represent simulation using the best-fit parameters (Table 1).
Simultaneous Fitting of NMRD and 17O NMR Data
In the NMRD studies, the longitudinal proton relaxation rate enhancements are
generally expressed in terms of the relaxivity r1 (mM-1 s-1). The observed
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paramagnetic relaxation results from a sum of contribution originating from short-
distance interactions (inner-sphere mechanism) and from longer-distance interactions
(outer-sphere mechanism). The influence of τM on proton relaxivity is well
understood. When this exchange time is short relative to the relaxation time of the
protons of the coordinated water molecules, the overall proton relaxivity is enhanced
by a temperature decrease. On the contrary, when the exchange time is long, the
global relaxivity is levelled off or even decreased by a reduction of the temperature.
The fittings of the proton NMRD profiles were performed as described in the
experimental section according to the classical paramagnetic relaxation
formalisms.[7,25,26] The following strategy was adopted: after subtraction of the
diamagnetic relaxation rate of the solvent, the outer-sphere and the inner-sphere
contributions were simultaneously fitted after attribution of fixed or calculated values
to the various parameters. The relative diffusion constant D was fixed to the diffusion
constant of water (D = 3 x 10-9 m2 s-1), d, the distance of closest approach for the
17
outer sphere contribution was set at 0.36 nm, τM values were those obtained by O
NMR and q, the number of water molecules in the first coordination sphere of Gd3+,
was given a value of one. The parameters describing the electronic relaxation times,
τv and τs0 (the electronic relaxation time at zero field), and the rotational correlation
time (τR) were optimized for the outer-sphere and the inner-sphere contributions
simultaneously. The structure of the complex GdL1 as well as the shape of its NMRD
profile do not justify in this case the introduction of a second sphere contribution.
The parameters obtained from the fitting are listed in Table 1 with the curve fits
shown in Figure 3b. For comparison, previously published data for GdL and Gd-
DOTA have been included in the Table 1.[12,27] It can be noted that the relaxivities
measured for the three complexes are all in the same range. In particular, the r1 of
GdL1, measured at 20 MHz and 310 K, is 4.7 mM-1 s-1. This value is slightly higher
than that of the GdL complex previously calculated (r1 = 4.2 mM-1 s-1),[12] and also
higher compared to the relaxivity of the Gd-DOTA complex taken as reference (3.5
mM-1 s-1).[27]
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1
Table 1 Parameters of GdL1, and GdL obtained from the analysis of H NMRD data compared with
Gd-DOTA as a reference.
Parameters
τV 310(ps)
GdL [11]
10.5 ± 1.0 (18)a 7 ± 1.0
Gd-DOTA
GdL1
19.2±1.1 (23.8)a
450 ± 20b
331 ± 44b
93.1 ± 2.2
74.8 ± 1.2
τM 310(ns)
τR310(ps)
τS0310(ps)
122 ± 10b
53 ± 1.3
404 ± 24
84 ± 3.4
92.5 ± 3.2
(a) The first values were obtained from the fitting of the proton NMRD profile; the values in parentheses
were obtained from the fitting of the 17O NMR data.
(b) The τM values are those obtained from the fitting of the 17O NMR data.
As mentioned above there is a small difference between the values of τM obtained by
17O NMR measurement for the two Gd3+ complexes. Both the rate and the
mechanism of the water exchange are intimately related to the structure of the
complexes in solution. The steric constrain of the methyl group present on the linkage
arm of the GdL1 complex explains a steric compression of these atoms around the
site occupied by the water molecule in the first coordination sphere. This destabilizes
the bound water molecule and accelerates the water exchange. The increase of the
water exchange rate observed for GdL1 complex can also be explained through the
replacement of the amide moiety by an oxo-group. The explanation of this structural
behaviour is reported in the literature for macrocyclic complexes when one
carboxylate of DOTA is substituted by an amide or OH coordination group. An amide
group is less strongly coordinated towards the lanthanide ion than a carboxylate,
which is exhibited by smaller stability constants of the amide complexes compared to
carboxylates in solution,[28-30] and by longer Gd-amide oxygen distances in the solid
state, when compared to Gd-carboxylate oxygen distances.[31] As a consequence,
the inner-sphere is less crowded in amide than in carboxylate complexes. In
dissociatively activated water exchange processes the steric crowding is of primary
importance, that is a tightly coordinating ligand pushes the water molecule to leave,
and thus favours the dissociative activation step.[6]
The value of the rotational correlation time (τR310) follows the general trend of the
relative molecular mass of the typical DOTA derivative complexes.[6] The higher τR
value for the new complex may be explained through an increased rigidity of the
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molecular structure acquired after the modification of the ligand L1 via the
replacement of the amide bound.
As expected, the τs0 value of GdL1 complex is lower than that of Gd-DOTA. It is
usually assumed that the large value of τs0 of Gd-DOTA is related to the rigidity and
/or the symmetry of the complex. Since GdL1 complex is expected to be less rigid
and non-symmetric, its τs0 value is lower and comparable to that of GdL.
Kinetic stability of the new monomeric GdL1 complex
It has been demonstrated that gadolinium chelates could be sensitive to
transmetallation by endogenous ions such as Cu2+, Ca2+, and Zn2+.[48-49] Only zinc
can displace significant amount of Gd3+ because its concentration in the blood is
relatively high (55-125 mol/L), whereas copper is present in very small amount (1-10
mol/L) and calcium ions have low affinity to organic ligands. Transmetallation
between Gd3+ and zinc will result in the formation of zinc chelate which is excreted in
urine, while the released Gd3+ becomes attached to endogenous anions such as
phosphate, citrate, hydroxide or carbonate and deposit in tissues as insoluble toxic
compounds. For these reasons we investigated the kinetic stability of this new
monomeric GdL1 complex by a relaxometric study of its transmetallation with ZnCl2.
The technique is performed by measuring the evolution of the water proton
p
paramagnetic longitudinal relaxation rate (R1 ) of a phosphate buffer solution (pH = 7)
p
containing GdL1 complex and ZnCl2 at equal concentration (2.5 mmol/L). The R1
relaxation rate is obtained after subtraction of the diamagnetic contribution of the
proton water relaxation (0.2826 s-1) from the observed relaxation rate R1 = (1/T1).
Upon transmetallation of a paramagnetic Gd3+ ion by a diamagnetic Zn2+ ion in
phosphate buffer, the released Gd3+ precipitates as GdPO4, which does not
contribute to the relaxivity. Therefore, the overall relaxivity of the solution decreases
over time with a rate depending on the rate of transmetallation. This decrease in
relaxivity is a good estimation of the kinetic stability of the Gd3+ complexes. The result
of this experiment (Fig. 4) shows a very good kinetic stability of Gd3+ complex GdL1,
similar to the previous GdL, as compared with Magnevist, which shows significant
decomplexation during the same time period (5 days).
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P
P
Figure 4. Evolution of the water proton paramagnetic longitudinal relaxation rate R1 (t)/R1 (t0) vs time
of 2.5mM Zn2+ aqueous solution for GdL1 (2.5mM), GdL (2.5mM) and Magnevist (2.5mM).
2. Conjugation of the Ligand L1 to Inulin: From Small Molecule to
Macromolecular Contrast Agents
Small molecular Gd3+-complexes have a relatively low relaxivity and extravasate non-
selectively from blood into the interstitium of both normal tissue and tumor, which has
been a major limitation for their clinical applications. As mentioned before, at the
magnetic field strengths currently employed in MRI (0.5-1.5 T), the ability of Gd3+-
chelates to enhance the longitudinal water proton relaxation rate is mainly
determined by the value of their molecular reorientational time, τR. Therefore, the
achievement of higher water proton relaxation rates may be pursued through an
increase of this parameter.
In order to slow down the reorientational motion of Gd3+-complexes, several
techniques have been used. Conceptually, they represent different strategies to
increase the effective molecular weight (more precisely, molecular volume)
accompanied by an enhancement of τR. It should be emphasized that the water
residence time τM should be optimized for a particular magnetic field to take full
advantage of the increased τR. Usually, the lengthening of the rotational motion is
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pursued through a covalent or noncovalent binding of the paramagnetic complex to
macro-/supramolecular carrier. In order to attach paramagnetic ions to larger
structures, a class of chelates known as bifunctional chelates have been developed
based on DTPA and DOTA. These chelating agents are typically modified to have an
electrophilic group that is available for conjugation to nucleophilic groups on
biomolecules. For example, these functional groups include anhydride, bromo- or
iodo-acetamide, isothiocyanate, N-hydroxysuccinimide (NHS) ester and maleimide.
In cases where the biomolecules contain only electrophilic functionality, such as a
carboxylic acid group, the common strategy is to use cross-linking agents that
provide a link between the two moieties or introduce functionality that makes
conjugation more amenable.
A vast number of MR macromolecular contrast agents have been reported over the
last 30 years, ranging from protein-, to albumin-, polymer-, dendrimer-, carbohydrate-
, liposomes-, micelles-based molecules.[6,12,32-37] These macromolecular carriers can
be linear or of a spherical shape.
The second part of this work will describe the enhancement of the relaxivity r1, when
the monomer GdL1 complex is conjugated to the inulin as linear carrier. The reasons
of choosing the inulin as a biomolecule to synthesize a macromolecular contrast
agent were defined in the introduction.
Synthesis and relaxometric characterization of the API-(GdL1)x complex
The new chelate L1 (Scheme 1) was conjugated to inulin following the synthetic route
outlined in Scheme 2. As we know inulin is a polydisperse polysaccharide consisting
of β(2→1) fructosyl fructose units with one glucopyranose unit at the reducing end.
Depending on the source, the degree of polymerization (dp) of inulin varies from
about 10 to 30. In the present study the dp of the polysaccharide used was estimated
at 32.
The O-(aminopropyl)inulin (API) was obtained functionalising the inulin through a
cyanoethylation by using acrylonitrile and base, followed by reduction of the cyano
groups with metallic Li in liquid ammonia-ethanol at low temperature. The theoretical
maximum degree of substitution (ds) can be three, corresponding to the three
available hydroxyl groups per sugar unit in the starting material. However previous
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studies have indicated that the cyanoethyl groups in O-cyanoethylinulin are
distributed uniformly along the inulin chain and that, within each fructose unit, the
4‐position is the most reactive towards cyanoethylation.[38]
The final macromolecular ligand [API-(L1)x] was synthesised by reacting the amino
group of the API and the isothiocyanate moiety of the intermediate structure DO3A-
NCSPP[6] (Scheme 2) of ligand L1. By the study of the 13C and 1H NMR spectroscopy
of the product API-(L1)x, an average degree of substitution of 0.9 was obtained.
Variable-temperature 17O NMR measurements and NMRD study
The API-(GdL1)x complex was synthesized in the same way as for the complex
GdL1 already mentioned and described previously. The value of τM was estimated
from the analysis of the temperature dependence of the reduced transverse 17O
relaxation rates, which is almost 2 fold higher than the analog macromolecular
complex previously synthesized [API-(GdL)x][12] (Scheme 1 and Table 2). For a
comparison, Figure 5a shows the 17O NMR measurements of the two
macromolecular complexes, with the theoretical fitting performed as previously
described.[24]
Figure 5 (a) Temperature dependence of the reduced 17O transverse relaxation rate at 11.75 T; (b)
comparison between 1H NMRD profiles of API-(GdL1)x 1.13 mM (spheres), and API-(GdL)x 1.18 mM
(rectangles). The lines represent simulation using the best-fit parameters (Table 2).
To assess the efficacy of the novel macromolecular contrast agent, API-(GdL1)x, we
measured the longitudinal relaxation rate enhancements of water protons as a
function of the Larmor frequency induced by this complex. In Figure 5b, the resulting
NMRD profile at 310 K is compared with that of API-(GdL)x complex of the
1
corresponding conjugated chelate L (Scheme 1). The H NMRD profiles of these
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macromolecular complexes were fitted using a model that takes into account
inner‐sphere, outer-sphere and second‐sphere contributions to the paramagnetic
relaxation rate. Some parameters were fixed during fitting procedure: d, the distance
of closest approach (d = 0.4 nm when a second sphere contribution is present and d
= 0.36 nm in the absence of second sphere contribution); D, the relative diffusion
constant (D = 3×10-9m2 s-1); and r, the distance between the Gd3+ ion and the proton
nuclei of water (r = 0.31 nm). The residence lifetime of the inner sphere, τM, was set
17
to the value determined by O NMR, the distance between Gd3+ and the protons of
the second sphere water molecules, rss was fixed at 0.36 nm. The rotational
correlation time (τR), the electronic relaxation time at zero field (τso) and its correlation
time (τv), as well as the number of second‐sphere water molecules (qss) and the
correlation time modulating the second sphere interaction (τss), were treated as
adjustable parameters. The conjugation of the paramagnetic complex to the inulin
carrier resulted in an increase in τR (33.7 ns) and consequently in a higher relaxivity
comparing to the monomer GdL1 (Table 2). The obtained τR value is higher than that
of the previously synthesized API-(GdL)x complex, probably due to the higher degree
of polymerization (dp = 32 vs dp = 25) and the increased rigidity of the complex GdL1
as previously discussed.
Table 2 Parameters of API-(GdL1)x, GdL1, API-(GdL)x and GdL obtained from the analysis of 17O
NMR and 1H NMRD data compared with Gd-DOTA as a reference.
API-(GdL)x
API-(GdL1)X
dp=32; ds=0.9
Parameters
dp=25;
GdL[11]
Gd-DOTA GdL1
ds=1.4[11]
r1b [mM−1 s−1]
r1c [mM−1 s−1]
21.7
4.1
3.5
3.5
4.7
4.7
14.5
759.5
4.1
417.6
10.5
(18)a
±
1.0
τV 310(ps)
45±1.0 (6.2)a
7 ± 1.0
19.2±1.1 (23.8)a 26.5± 0.6 (15.4)a
τM 310(ns)
τR310(ps)
τS0310(ps)
qSS
625 ± 34
1460 ± 70.7
153 ± 7.0
5 ± 0.6
450 ± 20
122 ± 10
53 ± 1.3
404 ± 24
331 ± 44
1096 ± 52
33700 ± 12.5
105 ± 6.1
84 ± 3.4
93.1 ± 2.2
92.5 ± 3.2
74.8 ± 1.2
c
c
c
c
2.10 ± 0.27
23 ± 2.5
τSS(ps)
23.4 ± 1.6
(a) The first values were obtained from the fitting of the proton NMRD profile; the values in parentheses were
obtained from the fitting of the 17O NMR data; (b) r1 corresponds to the relaxivity per Gd-complex, (c) per
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molecule, both calculated at 20 MHz and 37 °C; (c) The structure of the complexes GdL and GdL1 as well as the
shape of their NMRD profiles do not justify the introduction of a second sphere contribution.
The shape of the NMRD curve of the API-(GdL1)x complex, particularly the
maximum around 20-30 MHz, is characteristic for high-molecular-weight complexes
and reflects the effect of the relatively low tumbling time of the complex.
Although the increase of the value of τR (by increasing the molecular weight of the
paramagnetic API-(GdL1)x complex) causes the increase of total relaxivity, it turns
out to be lower than that one measured for the API-(GdL)x complex at the same
magnetic field strengths (Fig. 5b). One of the reasons to describe this outcome is
because the residence lifetime (τM) of the inner-sphere water becomes a limiting
factor for the relaxivity.
In our particular case, although the water exchange rate measured for the monomer
GdL1 complex (Scheme 1) is higher than that of the GdL (Scheme 1) due to the
reasons above described, the residence lifetime became larger when the GdL1 is
conjugate with API. API-(GdL1)x has indeed a higher degree of polymerisation (dp =
32 vs dp = 25), and especially a lower degree of substitution (averaged ds = 0.9 vs
ds = 1.4) than the macromolecular complex previously reported [API-(GdL)x].[12] As a
consequence, a higher number of -OH groups around the second coordination
sphere of the Gd3+ complex limits the access to the water bulk to be coordinated to
the paramagnetic centre.
The number of second-sphere water molecules, qss obtained from the fitting is lower
as compared to the value calculated for API-(GdL)x (qss = 2 vs qss = 5). In summary,
the lower relaxivity of API-(GdL1)x compared to API-(GdL)x results from the slower
water exchange rate and from a lower number of second sphere water molecules.
Conclusions
We have reported a similar synthetic pathway for the complexes described
previously, with the intent to improve the relaxivity properties of these new complexes
by modifying important parameters as τM and τR. On the basis of structural
considerations in the inner sphere of the monomeric GdL1 complex, we succeeded in
accelerating water exchange by inducing steric compression and by removing the
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amide group around the water bonding site. The increased steric crowding was
achieved by adding a methyl group, whereas the amide group was replaced by an
oxo-group from the linker of the DO3A-derivative chelate. Despite these
modifications, the kinetic stability of the new complex toward transmetalation with
Zn2+ is not reduced, which is comparable with that of GdL, considering also that both
macrocyclic structures are very similar.
Analysis of the variable temperature 17O NMR and NMRD data suggests that the
conjugated inulin-based complexes [API-(GdL1)x and API-(GdL)x], having different
degree of polymerization (dp = 32 vs dp = 25) and different degree of substitution
(averaged ds = 0.9 vs ds = 1.4), show a dramatic increase in relaxivity for both
macromolecular complexes. The substantial increase of the rotational correlation
time (τR) of the API-(GdL1)x complex doesn’t reflect in the enhancement of its overall
relaxivity, because its water exchange rate (τM) remains still a limiting factor. As
mentioned before, even if the τM of GdL1 is shorter than that of GdL, when the
monomeric complex is conjugated to inulin, the efficacy of the macromolecular
complex is reduced because of a slowing down of the residence lifetime and a
decrease of number of second sphere water molecules.
Experimental Section
General methods and materials
All reagents and chemicals were obtained from commercial sources and used without
further purification. Solvents were distilled and/or dried prior to use by standard
methodology except for those, which were reagent grades. Inulin (dp 32) was
donated by Sensus Cooperatie Cosun U.A. (Roosendaal, the Netherlands). The new
O-(Aminopropyl)inulin (API) was synthesized from inulin by following a procedure
described previously.[38] Water was demineralized prior to use. Unless stated
otherwise, all the reactions were carried out under a nitrogen atmosphere and the
reaction flasks were pre-dried by heat gun under vacuum. The pH values of the
solution were adjusted using aqueous solution 1M NaOH, 1N pyridine or 1N HCl.
Most of the reactions were monitored by TLC on silica plates detecting the
components by UV light or by applying the Dragendorff reagent, while other product
formations were monitored via analytical HPLC (Waters W600, with a quaternary
pump gradient module) with an UV detector coupled with a Waters micromass ZQ
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system (Waters, Belgium). For low molecular weight characterization, ESI-QTOF-MS
were obtained on a Q‐TOF Ultima mass spectrometer (Micromass, Manchester, UK),
whereas for high molecular weight products, MALDI spectra were obtained on
MALDI-TOF spectrometer (Micromass, Manchester, UK). The purification of the
compounds was performed on KP-silica and KP-C18 Biotage cartridges over a
Biotage flash chromatography instrument, Uppsala, Sweden. A final purification step
of high molecular weight compounds was performed either with a Millipore stirred cell
ultra-filtration system using membranes with 0.5, 1, 3 kDa MW cut-off, or via dialysis
procedure by using membranes with 150-300 Da MW cut-off. 1H-NMR, 13C-NMR
spectra were recorded on either with a Bruker Avance‐300 MHz or a Bruker Avance
II‐500 MHz spectrometer, at 25°C. Chemical shifts are given in ppm (δ) values. Data
are reported as follows: chemical shift (multiplicity: s = singlet, d = doublet, t = triplet,
q = quadruplet, m = multiplet, br = broad resonance) J = coupling constant (Hz),
integration, peak assignment in italic form.
Synthesis of Acetanilide (1)
To a stirred solution of aniline (10.2 g, 110 mmol) and K2CO3 (12.9 g, 165 mmol) in
CH2Cl2 at 0 °C, acetyl chloride (7.6 g, 55 mmol) in CH2Cl2 (volume) was added
dropwise after 10 min.
The resulting reaction mixture was stirred at this temperature for a further 4 h, and
monitored via TLC with Hexane/EtOAc (7:3) as a mobile phase mixture.
After 24h, the reaction was stopped and poured into water. The aqueous layer was
extracted twice with methylene chloride. The organic phase was combined, washed
three times with a dilute hydrochloric acid/water solution (pH=2), and dried over
Na2SO4, and evaporated to dryness to give a pure compound (12.1 g, 81.1% yield)
1
as a pale white solid. ESI MS: m/z 136 [M + H]+; H NMR (500 MHz, CDCl3, 25 °C,
TMS): δ 2.10 (3H, s, CH3), 7.02 (1H, t, ArH, J = 7Hz), 7.28 (2H, t, ArH, J = 7Hz),
13
7.55 (2H, d, ArH, J = 7Hz), 9.75 (1H, s, NHCO). C NMR (500 MHz, CDCl3, 25 °C,
TMS): δ 24.05, 119.97, 123.78, 128.50, 140.32, 169.21.
Synthesis of 4-Acetamido-α-bromopropiophenone (2)
Bromopropionyl bromide (8.8 g, 41 mmol) was added at 70 °C to a stirred mixture of
acetanilide (5.0 g, 37 mmol) and aluminium trichloride (15.0 g, 112 mmol) in 1,2,4-
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trichlorobenzene (25 ml) so that the temperature did not exceed 80 °C . The mixture
was stirred for a further 30 minutes at 80 °C, cooled to 60 °C and methanol (5 ml)
added slowly with cooling to keep the temperature below 70 °C, followed by cautious
addition of water (50 ml). On cooling, the product crystallized out of the organic layer
and dichloromethane was added to redissolve it. The organic layer was separated,
washed with water (twice), aqueous sodium bicarbonate, dried (Na2SO4), filtered and
concentrated at 60 °C. Petroleum ether (60 °C - 80 °C) was then added to crystallise
a solid which was collected and dried to give 4-acetamido-α-bromopropiophenone
(9.18 g; 91% yield), m.p. 107°C - 112°C. ESI MS: m/z 271-273 [M + H]+;1H NMR (500
MHz, CDCl3, 25 °C, TMS): δ 1.89-1.90 (3H, d, BrCHCH3, J = 7Hz), 2.09 (3H, s,
COCH3), 5.66 (1H, q, BrCH, J = 10 Hz), 7.68 - 797 (5H, 2 d + 1 br s, ArH (J = 8 Hz),
NHCO). 13C NMR (500 MHz, CDCl3, 25 °C, TMS): δ 20.58, 24.18, 42.85, 120.12,
130.56, 131.40, 145.08, 172.00, 194.23.
Synthesis of 1,4,7,10-tetraazacyclodecane-1,4,7-tris(acetic acid t-butyl
ester) hydrobromide salt (3)
1,4,7,10-Tetraazacyclododecane (10.0 g, 58 mmol) and sodium hydrogen carbonate
(16.1 g, 192 mmol) were stirred in freshly distilled acetonitrile (200 ml) cooled to 0 °C
under N2. Tert-butyl bromoacetate (37.4 g, 192 mmol) was added dropwise over 30
min. After addition, the reaction mixture was allowed to reach ambient temperature
and stirred for a further 48 h. The inorganic solids were removed by filtration and the
filtrate evaporated under reduced pressure to leave a pale-white solid. The crude
solid product was stirred in toluene for 24 h, offering the title ester (3) as a white solid
(24.36 g, 81.6%). ESI MS: m/z 515 [M + H]+, m/z 537 [M + Na]+; 1H NMR (500 MHz,
CDCl3, 25°C, TMS): δ 1.47 [27H, s, C(CH3)3], 2.88-2.94 (12H, br m, CH2CH2), 3.11
(4H, br s, CH2CH2), 3.30 (2H, s, NCH2COO), 3.38 (4H, s, NCH2COO), 10.03 (2H, br
s, NH2). 13C NMR (500 MHz, CDCl3, 25 °C, TMS): δ 28.21, 28.25, 47.54, 49.22,
51.38, 58.25, 81.71, 81.87, 169.65, 170.54.
Synthesis of 10[2(1-oxo-1-p-acetamidophenyl propyl)]-1,4,7,10 tetraaza
cyclodecane-1,4,7-tris(acetic acid t-butyl ester) (4)
Into a two-necked round bottom flask, under dry condition (N2), a suspension of
1,4,7,10-tetraazacyclodecane-1,4,7-tris(acetic acid t‐butyl ester) hydrobromide salt
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(3) (4.64 g, 9.02 mmol) and K2CO3 (1.87 g, 13.5 mmol) in HPLC‐grade CH3CN (80
ml) was stirred for 30min at RT. 4-acetamido-α-bromopropiophenone (2.43 g, 9.02
mmol) was first dissolved in 40 ml of CH3CN, and immediately added dropwise to the
reaction mixture over 30 min, [reaction monitored by TLC: CH2Cl2/CH3OH, 9:1]. After
24h, the reaction was stopped and poured into ethyl acetate (200 ml). The organic
layer was washed three times with a dilute hydrochloric acid water solution (pH=2),
dried over Na2SO4, and evaporated to dryness to give a pale-yellow solid compound
(6.0 g). A 370.8 mg aliquot of residue product was purified by flash chromatography
on silica gel using CH2Cl2/CH3OH (9:1) as mobile phase. After evaporation of the
solvent, 241 mg (2.4 g, 65.0 % of yield) of 4 was obtained as a pale-yellow powder.
1
ESI MS: m/z 726 [M + Na]+; H NMR (500 MHz, CDCl3, 25 °C, TMS): δ 1.04 [9H, s,
C(CH3)3], 1.35 [9H, s, C(CH3)3], 1.26-1.27 (3H, d, NCHCH3, J = 7 Hz), 1.48 [9H, s,
C(CH3)3], 2.06 (3H, s, COCH3), 1.90-3.76 (22H, br m, 3xNCH2COOH, 4xCH2NCH2),
4.54-4.55 (1H, q, NCHCH3, J = 10 Hz), 7.57-7.80 (4H, 2 d, ArH, J = 10 Hz), 7.84 (1H,
13
s, NHCO). C NMR (500 MHz, CDCl3, 25 °C, TMS): δ 11.26, 24.13, 28.35, 28.44,
28.47, 46.67, 53.93, 54.26, 54.66, 56.58, 56.66, 57.12, 60.11, 82.90, 83.00, 83.04,
120.03, 131.10, 132.47, 144.47, 171.96, 174.41, 174.50, 175.21, 204.61.
Synthesis of 10[2(1-oxo-1-p-aminophenyl propyl)]-1,4,7,10-tetraaza
cyclodecane-1,4,7-tetraacetic acid (DO3A-APP) (5)
Two routes of synthesis were used to prepare compound 10[2(1-oxo-1-p-
aminophenyl propyl)]-1,4,7,10-tetraazacyclodecane-1,4,7-tetraacetic acid (DO3A-
APP) (5).
Route1: Into a microwave reaction vials, 10[2(1-oxo-1-p-acetamidophenyl propyl)]-
1,4,7,10-tetraazacyclodecane-1,4,7-tris(acetic acid t-butyl ester) (4) (1.0 g, 1.42
mmol) was dissolved in a mixture of 0.1M HCl (7 ml) standard aqueous solution, and
3 ml of HPLC‐grade CH3CN in order to have a better homogeneous reaction mixture.
The reactants were subjected to microwave radiation for 30 min. at 100 °C. The final
product was first neutralised at pH=6.8 by using a standard aqueous NaOH (0.1 M)
solution, then washed two times with ethyl ether, and dried under rotary evaporator.
In order to remove the NaCl salt from the final product, the water solution was
dialysed for 7 days by using a membrane with a molecular weight cut-off of 150-300
Da, and freeze-dried to give a pure pale-yellow solid compound 5 (0.582 g, 83% of
yield).
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Route 2: Into a round bottom flask, 10[2(1-oxo-1-p-acetamidophenyl propyl)]-
1,4,7,10‐tetraazacyclodecane-1,4,7-tris(acetic acid t-butyl ester) (4) (3.922 g, 5.57
mmol) was dissolved in a mixture of 0.1M HCl (25 ml) standard aqueous solution,
and 10 ml of HPLC‐grade CH3CN in order to have a better homogeneous reaction
mix. Reaction was stirred under reflux for 4h. The final product was first neutralised at
pH=6.8 by using a standard aqueous NaOH (0.1 M) solution, then washed two times
with ethyl ether, dried under rotary evaporator first, and then freeze-dried to give a
pale-yellow solid compound (3.229 g). In order to remove the NaCl salt from the final
product, a 100 mg aliquot of residue product was dialysed for 7 days by using a
membrane with a molecular weight cut-off of 150-300 Da, and freeze-dried to give a
pure pale-yellow solid compound 5 (0.064 g, 71% of yield). ESI MS: m/z 494 [M +
1
H]+; H NMR (500 MHz, DMSO‐d6, 25 °C, TMS): δ 1.22-1.23 (3H, d, NCHCH3, J = 7
Hz), 2.90–3.83 (22H, br m, 3xNCH2COOH, 4xCH2NCH2), 4.82 (1H, q, NCHCH3, J =
13
10 Hz), 6.66-7.66 (4H, 2 d, ArH, J = 10 Hz). C NMR (500 MHz, DMSO‐d6, 25 °C,
TMS): δ 14.70, 44.20, 45.49, 48.67, 50.52, 52.17, 55.93, 57.26, 58.66, 59.58, 60.66,
62.12, 64.11, 114.49, 125.19, 131.10, 153.47, 169.41, 170.50, 177.21, 202.61.
Synthesis of 10[2(1-oxo-1-p-isothiocyanophenyl propyl)]-1,4,7,10-tetraaza
cyclodecane-1,4,7- tetraacetic acid (DO3A-NCSPP) (6)
DO3A-APP (5) (0.582 g, 1.18 mmol) was dissolved in water (10 ml), and the pH was
adjusted to 2 by several drops of concentrated HCl aqueous solution. A mixture of
thiophosgene (0.170 g, 1.47 mmol, 1.2 equiv.) and 5ml of CCl4 was added in one
portion, and the reaction was vigorously stirred at room temperature for 48 h. The
excess of thiophosgene was removed by washing the aqueous mixture, using first
CH2Cl2 (3 times) and later ethyl ether (3 times). The water fraction was separated and
freeze-dried to get a pale-yellow solid compound (6). (0.560 g, 89% of yield). ESI MS:
1
m/z 536 [M + H]+; H NMR (500 MHz, DMSO‐d6, 25 °C, TMS): δ 1.37-1.38 (3H, d,
NCHCH3, J = 7 Hz), 3.06–3.91 (22H, br m, 3xNCH2COOH, 4xCH2NCH2), 4.88-4.93
13
(1H, q, NCHCH3, J = 10 Hz), 6.83-7.80 (4H, 2 d, ArH, J = 10 Hz). C NMR (500
MHz, DMSO‐d6, 25 °C, TMS): δ 14.40, 48.67, 55.93, 57.26, 58.66, 59.58, 60.66,
62.12, 64.11, 120.03, 131.10, 132.47, 136.52, 144.47, 175.41, 175.50, 176.21,
205.61.
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Synthesis of 10[2(1-oxo-1-p-propylthioureidophenyl propyl)]-1,4,7,10-
tetraazacyclodecane-1,4,7- tetraacetic acid (L1)
Propylamine (0.012 g, 0.2 mmol) was dissolved in water (5ml), and the pH was
adjusted to 9-10 with diluted NaOH aqueous solution. DO3A-NCSPP (6) freshly
prepared (0.08 g, 0.15 mmol), was added, and the reaction mixture was stirred at
room temperature for 24 h. The crude product (0.16 g) was purified by flash-
chromatography (RP-C18 column) with AcN/H2O (8:2) as mobile phase, yielding 89%
1
of pure pale-yellow L1 solid (0.08 g). ESI MS: m/z 595 [M+ H]+; H NMR (500 MHz,
D2O, 25 °C): δ 0.73-0.81 (3H, br m, CH3CH2CH2), 1.18-1.19 (3H, d, NCHCH3, J = 7
Hz), 1.39-1.49 (2H, br m, CH3CH2CH2), 2.71-3.90 (24H, br m, 3xNCH2COOH,
4xCH2NCH2, CSNHCH2CH2CH3), 4.73-4.81 (1H, q, NCHCH3, J = 10 Hz), 7.23-7.76
(4H, 2 d, ArH, J = 10 Hz).
Synthesis of conjugated API-(L1)x ligand
The O-(Aminopropyl)inulin (API) sample with degree of substitution (ds) of 0.9 was
synthesized from inulin (dp = 32) by following a procedure described previously[12].
API (0.245 g, 1.12 mmol amino groups), was dissolved in water (25 ml), and the pH
was adjusted to 9-10 with diluted NaOH. DO3A-NCSPP (6) freshly prepared (0.757
g, 1.41 mmol), was added to the reaction mixture. The reaction was stopped after 4
days, and purified by ultrafiltration using a membrane with a molecular weight cut-off
of 3 kDa. The product was freeze‐dried, resulting in 0.6 g of API-(L1)x as a yellow
1
powder. H NMR (500 MHz, D2O, 25 °C): δ 1.20 (br s, NCHCH3), 1.80 (br s,
CH2CH2NH2), 2.8-3.0 (br m, CH2CH2NH2), 3.1-3.3 (br m, NCH2CH2N), 3.3-3.6 (br m,
NCH2COOH), 4.14 (1H, br m, NCHCH3), 4.77 (inulin H, br m), 7.35 and 7.74 (4H, 2
br d, ArH). Comparison between integrals of the aromatic protons and those of the
sugar protons indicated an averaged ds of 0.9.
Preparation of (GdL1) and API-(GdL1)x complexes
GdL1 and API-(GdL1)x complexes were prepared by a stepwise addition of
GdCl3·6H2O to a solution of 80 mg of L1 (0.13 mmol) and 200 mg of API-(L1)x
respectively, in 8 ml of H2O, maintaining the pH between 6.5 and 7.0 by addition of 1
M NaOH aqueous solution. After 4 days the reaction mixture was stopped, and the
excess of free lanthanide for the GdL1 complex was removed by centrifugation as
Gd(OH)3 precipitate, which appeared at pH 10 after addition of 1 M NaOH, whereas
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for API-(GdL1)x complex the excess of free gadolinium was removed by ultra-
filtration using a membrane with a cut‐off of 3 kDa. The absence of free Gd3+ ions
was confirmed by a xylenol orange test, with a final pH of both solutions around 6.5-
7.00. The solutions were freeze-dried to give a pale-yellow solid for both final
complexes: GdL1 72 mg, ESI MS: m/z 771 [M + Na]+; and 0.23 g API-(GdL1)x.
NMRD profiles
1
1/T1 H NMRD profiles were measured on a Stelar Spinmaster FFC fast field cycling
NMR relaxometer (Stelar, Mede, Pavia, Italy) over a range of magnetic fields
1
extending from 0.24 mT to 0.7 T and corresponding to H Larmor frequencies from
0.01 to 30 MHz using 0.6 ml samples in 10 mm o.d. tubes. The samples were
prepared by dissolution of appropriate amounts of freeze-dried complexes of GdL1
and API-(GdL1)x in Milli-Q water with the pH in the range 6.5-7.0. The exact Gd3+
concentration was determined by proton relaxivity measurements at 20 MHz and 37
°C after complete hydrolysis in concentrated HNO3. Additional relaxation rates at 20
and 60 MHz were obtained with Bruker Minispec mq-20 and mq-60 spectrometers
(Bruker, Karlsruhe, Germany), respectively. The 300 MHz data were obtained on a
Bruker Avance-300 high resolution spectrometer.
1
Fitting of the H NMRD was performed with data processing software that uses
different theoretical models describing observed nuclear relaxation phenomena
(Minuit, CERN Library).[39,40] The theoretical model takes into account the inner-
sphere interaction, the outer‐sphere interaction and when necessary the second
sphere contribution.[6] It has to be noted that, as the macromolecular complex API-
(GdL1)x is a mixture of different compounds characterized by an averaged ds of 0.9,
the different parameters extracted from the fitting procedure for this complex are
obtained as an average for the mixture of compounds.
17O NMR studies
17O NMR relaxation rate measurements were performed on a Bruker Avance II-500
spectrometer using 5 mm diameter sample tubes containing 325 μl (pH 6.5-7.00) of
GdL1 (6.81 mM) and API-(GdL1)x (14.24 mM) solutions. The temperature was
17
regulated by air or N2 flow controlled by a BVT-3200 unit. O transverse relaxation
times of pure water (diamagnetic contribution) were measured using a CPMG
sequence and a subsequent two-parameter fit of the data points. The 90 and 180°
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pulse lengths were 27.5 and 55 μs, respectively. Broadband proton decoupling was
applied during the acquisition of all 17O NMR spectra. 17O T2 of water in complex
solution was obtained from line width measurement. The data are presented as the
R
P
reduced transverse relaxation rate 1/T2 = 55:55/([Gd complex]⋅q⋅T2 , where [Gd
complex] is the molar concentration of the complex, q is the number of coordinated
P
water molecules and T2 is the paramagnetic transverse relaxation rate obtained
after subtraction of the diamagnetic contribution from the observed relaxation rate.
The treatment of the experimental data was performed as already described.[24,26,50] It
has to be noted that, as the macromolecular complex API-(GdL1)x is a mixture of
different compounds characterized by an averaged ds of 0.9, the τM value for this
complex is obtained as an average for the mixture of compounds.
Transmetallation
The stability of the monomeric GdL1 complex was determined by a transmetallation
1
method monitoring the H longitudinal relaxation rates of water during 5 days at
37°C.[42] The measurements were performed on a Bruker Minispec mq-20 spin
analyzer at 20 MHz using 7 mm sample tubes containing 2.5 mM of Gd3+-complex
and 2.5 mM of ZnCl2 in 300 μl of phosphate buffer solution (26 mM KH2PO4, 41 mM
Na2HPO4, pH = 7).
Acknowledgements
This work was supported by the Walloon Region (program First spin-off), the FNRS
(Fond National de la Recherche Scientifique), the UIAP VII, ARC Programs of the
French Community of Belgium, the FEDER, the Walloon Region (Holocancer and
Gadolymph programs), Interreg and the COST Actions (TD 1004, TD1402 and
CA15209). The authors thank the Center for Microscopy and Molecular Imaging
(CMMI, supported by the European Regional Development Fund and the Walloon
Region).
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Author Contribution
The contributions of the respective authors are as follows: Luigi Granato performed
the syntheses and the physico-chemical characterization of the different compounds;
Luce Vander Elst executed the 17-oxygen NMR analysis and the NMRD profiles;
Celine Henoumont interpreted data and revised the manuscript; Robert N. Muller and
Sophie Laurent revised the manuscript and supervised the work. All authors read and
approved the manuscript.
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