Lanthanide (III) complexes that contain a self-immolative arm: Potential enzyme responsive contrast agents for magnetic resonance imaging

Article abstract of DOI:10.1002/chem.201101779

Enzyme-responsive MRIcontrast agents containing a "self- immolative" benzylcarbamate moiety that links the MRI-reporter lanthanide complex to a specific enzyme substrate have been developed. The enzymatic cleavage initiates an electronic cascade reaction that leads to a structural change in the Ln^III complex, with a concomitant response in its MRI-contrast- enhancing properties. We synthesized and investigated a series of Gd3+ and Yb³⁺ complexes, including those bearing a self-immolative arm and a sugar unit as selective substrates for bgalactosidase; we synthesized complex LnL¹, its NH₂ amine derivatives formed after enzymatic cleavage, LnL², and two model compounds, LnL³ and LnL⁴. All of the Gd³⁺ complexes synthesized have a single inner-sphere water molecule. The relaxivity change upon enzymatic cleavage is limited (3.68 vs. 3.15 mm^-1 s^-1 for complexes GdL¹ and GdL², respectively; 37 °C, 60 MHz), which prevents application of this system as an enzyme-responsive T₁ relaxation agent. Variable-temperature ¹⁷O NMR spectroscopy and ¹H NMRD (nuclear magnetic relaxation dispersion) analysis were used to assess the parameters that determine proton relaxivity for the Gd ³⁺ complexes, including the water-exchange rate (k_ex ²⁹⁸, varies in the range 1.5-3.9×10⁶ s ^-1). Following the enzymatic reaction, the chelates contain an exocyclic amine that is not protonated at physiological pH, as deduced from pH-potentiometric measurements (log K_H=5.12(±0.01) and 5.99(±0.01) for GdL² and GdL³, respectively). The Yb³⁺ analogues show a PARACEST effect after enzymatic cleavage that can be exploited for the specific detection of enzymatic activity. The proton-exchange rates were determined at various pH values for the amine derivatives by using the dependency of the CEST effect on concentration, saturation time, and saturation power. A concentration-independent analysis of the saturation-power-dependency data was also applied. All these different methods showed that the exchange rate of the amine protons of the Yb ^III complexes decreases with increasing pH value (for YbL ³, k_ex= 1300 s^-1 at pH 8.4 vs. 6000 s ^-1 at pH 6.4), thereby resulting in a diminution of the observed CEST effect.

Full text of DOI:10.1002/chem.201101779

DOI: 10.1002/chem.201101779
LanthanideACHTUNGTRENNUNG(III) Complexes That Contain a Self-Immolative Arm: Potential
Enzyme Responsive Contrast Agents for Magnetic Resonance Imaging
Thomas Chauvin,[a] Susana Torres,[b] Renato Rosseto,[b] Jan Kotek,[c] Bernard Badet,[b]
Philippe Durand,*[b] and ꢀva Tꢁth*[a]
Abstract: Enzyme-responsive MRI-
contrast agents containing a “self-im-
molative” benzylcarbamate moiety that
links the MRI-reporter lanthanide
complex to a specific enzyme substrate
have been developed. The enzymatic
cleavage initiates an electronic cascade
upon enzymatic cleavage is limited
(3.68 vs. 3.15 mmꢀ1 sꢀ1 for complexes
GdL1 and GdL2, respectively; 378C,
60 MHz), which prevents application of
this system as an enzyme-responsive T1
relaxation agent. Variable-temperature
ric measurements (log KH =5.12
and 5.99
G
AHCTUNGTRENNUNG
respectively). The Yb3+ analogues show
a PARACEST effect after enzymatic
cleavage that can be exploited for the
specific detection of enzymatic activity.
The proton-exchange rates were deter-
mined at various pH values for the
amine derivatives by using the depend-
ency of the CEST effect on concentra-
tion, saturation time, and saturation
1
17O NMR spectroscopy and H NMRD
reaction that leads to
a
structural
(nuclear magnetic relaxation disper-
sion) analysis were used to assess the
parameters that determine proton re-
change in the LnIII complex, with a
concomitant response in its MRI-con-
trast-enhancing properties. We synthe-
sized and investigated a series of Gd3+
and Yb3+ complexes, including those
bearing a self-immolative arm and a
sugar unit as selective substrates for b-
galactosidase; we synthesized complex
LnL1, its NH2 amine derivatives
formed after enzymatic cleavage, LnL2,
and two model compounds, LnL3 and
LnL4. All of the Gd3+ complexes syn-
laxivity for the Gd3+ complexes, includ-
298
ing the water-exchange rate (kex
,
power. A concentration-independent
varies in the range 1.5–3.9ꢃ106 sꢀ1).
Following the enzymatic reaction, the
chelates contain an exocyclic amine
that is not protonated at physiological
pH, as deduced from pH-potentiomet-
analysis of the saturation-power-de-
pendency data was also applied. All
these different methods showed that
the exchange rate of the amine protons
of the YbIII complexes decreases with
increasing pH value (for YbL3, kex =
1300 sꢀ1 at pH 8.4 vs. 6000 sꢀ1 at
pH 6.4), thereby resulting in a diminu-
tion of the observed CEST effect.
Keywords: enzymatic detection
·
lanthanides · MRI contrast agents ·
PARACEST · water exchange
thesized have
a single inner-sphere
water molecule. The relaxivity change
Introduction
ble, non-invasive assessment, characterization, and quantifi-
cation of gene and protein function, protein–protein interac-
tions, signal transduction, etc. makes molecular imaging par-
ticularly attractive in biomedical applications. Any molecu-
lar-imaging procedure requires an imaging probe that is
specific for a given molecular event.[1] Owing to its excep-
tional spatial and temporal resolution, magnetic resonance
imaging is one of the most-powerful state-of-the-art diagnos-
tic modalities. The contrast of MR images is often enhanced
by the use of paramagnetic agents, often GdIII complexes,
which allow a better delineation of morphology or func-
tion.[2] More recently, a fundamentally different contrast
mechanism based on chemical-exchange saturation transfer
has been proposed.[3] Paramagnetic chemical-exchange satu-
ration transfer (PARACEST) agents, typically lanthanide
complexes, contain paramagnetically shifted mobile protons
in slow exchange with bulk water.[4] The irradiation of these
protons affects the magnetic resonance signal of water pro-
tons through chemical exchange. PARACEST imaging
offers the advantage of 1) turning the contrast on and off at
will, 2) multiplex analysis by using more than one probe,
Molecular imaging aims at visualizing molecular events oc-
curring in vivo at a cellular level. The possibility of repeata-
[a] Dr. T. Chauvin, Dr. ꢀ. Tꢁth
Centre de Biophysique Molꢂculaire, CNRS
rue Charles Sadron
45071 Orlꢂans (France)
E-mail: eva.jakabtoth@cnrs-orleans.fr
[b] Dr. S. Torres, Dr. R. Rosseto, Dr. B. Badet, Dr. P. Durand
Centre de Recherches de Gif, ICSN-CNRS
Avenue de la terrasse
91198 Gif-surYvette Cedex (France)
E-mail: philippe.durand@icsn.cnrs-gif.fr
[c] Dr. J. Kotek
Department of Inorganic Chemistry
Universita Karlova v Praze
(Charles University in Prague)
Hlavova 2030, 12840 Prague 2 (Czech Republic)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201101779.
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Chem. Eur. J. 2012, 18, 1408 – 1418

FULL PAPER
and 3) a relatively straightforward design of responsive re-
porters because all factors that influence the exchange will
have an effect on the water signal. In recent years, much
effort has been devoted to the development of LnIII com-
plexes that are capable of providing an MRI-detectable re-
sponse to an in vivo stimulus. Among these, enzymatically
activated agents have specific advantages. Molecular mag-
netic resonance imaging is often seen as being limited by
the low (nanomolar) concentrations of the molecular tar-
gets. Enzymatic activity can be exploited to concentrate the
imaging probe at the target site by converting an MRI-silent
agent into an active one.
The detection of b-galactosidase (b-gal) is of particular in-
terest in biological applications. The lacZ gene encoding b-
galactosidase is a reporter routinely used to reveal genetic
manipulations in molecular biology.[5] b-gal is used to detect
gene expression on tissue sections, biopsies, and post-
mortem samples using colorimetric substrates that are not
suitable for long-term follow-up. NIR-fluorescent agents[6]
or MRI-contrast agents[7] that are activated upon cleavage
by b-gal and are applicable in non-invasive, live-animal
imaging could be valuable tools to visualize changes in lacZ
gene expression over time in deep tissues.[8] The first MRI
agent to detect b-gal was reported by Meade and co-work-
ers.[7] This galactopyranoside-capped GdIII complex showed
an increase in relaxivity upon exposure to b-gal, owing to in-
creased water access to the GdIII site. This probe has been
used for the detection of the lacZ gene in Xenopus laevis. In
another example, a GdIII chelate bearing a 2-difluoromethyl-
phenyl-b-galactopyranoside was used as a probe in which
the b-galactosidase-catalyzed cleavage of the glycosidic
bond induces formation of an electrophilic species that
reacts covalently with human serum albumin (HSA) or b-
galactosidase to yield macromolecular adducts and thus ach-
Scheme 1. Platform of self-immolative agents for the detection of enzyme
activities.
(GDEPT),[12] and are based on the intrinsic instability of
benzylcarbamates that possess an electron-donor substituent
at the ortho or para positions. Employing benzylcarbamates
as self-immolative units allows the scope of the substrate to
be any enzyme-recognized moiety that is capable of transi-
tionally reducing the electron-donating properties of the
substituent. This is a very general approach, which opens
the way to the detection of a large variety of enzyme activi-
ties. Therefore, one can create enzyme-activated molecular-
imaging probes that incorporate a MRI-reporting unit and a
self-immolative chain onto the appropriate substrate to
ensure enzyme specificity.
We have recently reported the first example of an activat-
able contrast agent based on this versatile platform (LnL1,
Scheme 1).[13] Destruction of the self-immolative linker in-
duced by enzyme cleavage was originally expected to afford
an unstable,[14] 2-glycylsubstituted GdDO3A complex that
would decompose into the bis-hydrated GdDO3A, thereby
increasing the hydration number and thus the relaxivity.
However, as established by HPLC/MS analysis of the enzy-
matic reaction mixture,[13] the self-immolative process led to
the cleavage of the carbamate to yield the stable complex
GdL2, which was also observed during the course of a study
by Yoo and Pagel (Scheme 1).[15] Indeed, literature examples
confirmed that metal-coordination can stabilize gem-dia-
mines.[16]
ieves
a relaxivity change upon enzymatic activation
(ca. 60% change in T1). This probe was used to visualize b-
gal expression in a mouse-tumor model.[9] Nagano and co-
workers reported a b-gal-activated contrast agent based on
the receptor-induced magnetization enhancement (RIME)
phenomenon. A galactoside unit is linked to the GdIII che-
late through an albumin binding moiety that only becomes
available for albumin interaction following enzymatic cleav-
age.[10] The compound exhibited an increase in relaxivity of
57% in phosphate-buffered saline (PBS, pH 7.4) with 4.5%
w/v HSA in the presence of b-galactosidase.
Our approach to enzyme-responsive MRI-contrast agents
is based on a “self-immolative” mechanism, where the
action of a specific enzyme on the substrate moiety of the
contrast agent initiates a self-immolation process. This “self-
destructive” cascade reaction is expected to result in the
transformation of the LnIII complex with a concomitant
change in the contrast-enhancement properties (Scheme 1).
In the context of MRI, Meade and co-workers have report-
ed a GdIII complex containing a self-immolative linker, de-
signed for detection of b-glucuronidase.[11] Self-immolative
units are applied in antibody-directed enzyme prodrug ther-
apy (ADEPT) or gene-directed enzyme prodrug therapy
Ytterbium-complex YbL2 demonstrated a PARACEST
effect. Exploiting this result,
a prototype compound,
Yb(dota-abz-bGal)ꢀ (YbL1, dota=1,4,7,10-tetraazacyclodo-
decane-N,N’,N’’,N’’’-tetraacetate) was designed to give a
PARACEST response to the activity of b-galactosidase
(Scheme 1).[13] In order to gain further insight into the pecu-
liar behavior of these aminal derivatives, herein, we report
the synthesis of various GdIII and YbIII complexes and their
physicochemical characterization with potential applications
as T1 or PARACEST MRI probes (Scheme 2). We also
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ꢀ. Tꢁth, P. Durand et al.
Results and Discussion
Synthesis: We designed a synthetic pathway applicable to all
of the targeted ligands (L1–L4) including the O-glycosylated
derivative, L1 (Scheme 3). N-benzyloxycarbonyl a-amino-
glycine intermediates (3a–3d) were obtained by amine addi-
tion to N-benzyloxycarbonyl imine (or iminium) acetates
generated in-situ from activated a-heteroglycine precursors,
such as a-acetoxy (2a and 2b) or a-chloroglycines (2c and
2d).[17] In this condensation reaction, we used the less-reac-
tive ester derivative of N1,N4,N7-cyclentriacetic acid
(DO3A)[18] rather than cyclen to avoid handling the poorly
stable aminal intermediate. The commercially available
triethyl ester of DO3A was preferred to the tri-tert-butyl
ester because of the known instability of the glycosidic bond
of L1 under acidic conditions, which are necessary for the
ester deprotection. During the course of this work, the syn-
thesis of complex LnL2 and its peptide derivatives was re-
ported from the condensation of DO3A-ter-butyl ester with
an a-bromoglycine derivative.[15] In our synthesis, each of
the fully protected intermediates 3a–3d could be isolated,
but they all exhibited limited stability. Therefore, we pre-
ferred to synthesize complexes LnL1 and LnL4–LnL6 from
a-heteroglycines 2a–2d without purification of the inter-
Scheme 2. Complexes studied in this work. (Ln=Gd, Yb).
extend the study to the N-methylated analogues as model
compounds.
The starting GdIII complex GdL1, enzymatic-conversion
product GdL2, as well as two model compounds, GdL3 and
GdL4, were characterized with regard to their contrast-agent
efficacy (Scheme 2). Variable-temperature 17O NMR and
1H NMRD measurements were performed to assess the pa-
rameters that influence their proton relaxivity. Lumines-
cence lifetime and UV/Vis absorption measurements were
performed on some EuIII derivatives to quantify the bound
water molecules and assess the hydration equilibria. The
protonation states of the complexes bearing an exocyclic
amine were determined by pH-potentiometric titrations. In
addition, and to complete the work reported in our first
communication,[13] we report the determination of proton-
exchange rates of these Yb-derivatives as PARACEST
probes.
mediates. Polymer-supported 1,5,7-triazabicycloACTHNUTRGNE[UNG 4.4.0]dec-5-
ene (TBD resin) was used as a base for the condensation re-
actions and the more-lipophilic 1-naphthylmethylcarbamate
was substituted for the benzyl one in the case of complex
LnL6 in order to facilitate the final purification of the lan-
thanide complexes.
The more-activated a-chloroglycines 2c and 2d were
more suitable for the condensation of the N-methylated de-
rivatives. In this case, triethylamine was used as the base but
the intrinsic basicity of the DO3A ester was also sufficient
to catalyze the reaction. The saponification and the subse-
quent complexation to the lanthanide were done in a classi-
cal way. Finally, the carbamate moiety of complexes LnL4–
LnL6 was removed by catalyzed hydrogenolysis using Pd/C
Scheme 3. Synthesis of LnL1–L6: a) see Ref. [13]; b) Ac2O, Py, CH2Cl2, 08C; c) SOCl2, CH2Cl2, reflux; d) 2b, TBD resin, CH2Cl2, RT; e) 2c, Et3N,
CH2Cl2, RT; f) 2d, CH2Cl2, RT; g) i) NaOH, EtOH/H2O 1:3 to 1:5, RT; ii) LnCl3·xH2O, pH 6.5–7, RT; h) H2O, Pd/C, H2, RT. Py=pyridine.
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LanthanideACHTUNGTRENNUNG(III) Complexes That Contain a Self-Immolative Arm
FULL PAPER
under an atmosphere or a slight pressure of hydrogen to
“normal” one.[25] Whilst the anomeric effect is negligible for
+
give the a-amino-glycine/dota complexes, LnL2 and LnL3.
neutral aminals, it is of higher magnitude for R2NCHRNR3
ions[23] and we suspect that it could be partly responsible for
the low basicity of the exocyclic amine nitrogen atom of
complexes GdL2 and GdL3.
Characterization of the GdIII complexes
Protonation constants of the GdL2 and GdL3 complexes:
The protonation constant of the non-coordinating exocyclic
amine nitrogen atom has been determined by pH-potentio-
metric titrations for complexes GdL2 and GdL3 ([Eq. (1)],
using equilibrium concentrations):
Moreover, according to the established mechanism for the
hydrolysis of gem-diamine, the low basicity of the exocyclic
amine atom could be also responsible for the unexpected
stability of complex LnL2 and LnL3, especially under acidic
conditions.[20]
Determination of the number of coordinated water mole-
cules: For all four GdIII complexes, the coordination of a
single inner-sphere water molecule is expected base on pre-
½GdLHꢂ
ð1Þ
KH
¼
½GdLꢂ½Hþꢂ
The values obtained are log KH =5.12
A
N
vious results for analogous lanthanideACTHNGUTERNNU(G III) complexes. This
coordination was confirmed by luminescence-decay meas-
urements on the EuIII complexes of ligands L1 and L2. The
luminescence lifetimes are tH O =0.900 ms, tD O =2.740 ms
protonation constants are about four orders of magnitude
lower than those typically reported for primary or secondary
amines. This result indicates that the exocyclic amino groups
in both complexes are unprotonated at physiological pH,
thus conferring a global negative charge on the com-
plexes.[19] The surprisingly low basicity of the exocyclic
amine nitrogen atoms in complexes GdL2 and GdL3 cannot
only be rationalized by the presence of the gem-diamino
motif or of the carboxylic-acid substituent because protona-
tion constants of 7.8 and 8.9 have been reported for a
mono-acetylated 1,1-diamine compound[20] and an a-amino-
glycine derivative,[21] respectively. The coordination of the
endocyclic nitrogen atom to the positively charged metal
cation obviously contributes to the high acidity, as previous-
ly suggested for other gem-diamine-containing metal com-
plexes.[16h] Indeed, a spectacular decrease of the protonation
constant has been reported for the primary amine in Co2+
complexes formed with the a-diaminomalonate moiety(log
KH=1.6)[16i] or for the tertiary amine in the Ni2+ complex
formed with azacyclam (log KH <2).[16h] In fact, coordination
of the metal at such a short distance from the uncoordinated
amine atom affords a strong electrostatic repulsion towards
positively charged species, which makes the approach of the
hydrogen ion difficult.[16h] Long-range interactions between
the nitrogen atom and the electrons of the metal were also
suggested to explain this effect.[16a] The crystal structures re-
ported for a-diamine-containing metal complexes also re-
2
2
for complex EuL1 and tH O =0.882 ms, tD O =2.565 ms for
2
2
complex EuL2. From these values and using well-established
relationships,[26] we obtain q=0.8
(ꢁ0.3) for both complexes.
The absence of hydration equilibria was confirmed by UV/
Vis absorption spectra of aqueous solutions of complex
EuL1 and EuL2. Both complexes exhibited one temperature-
invariant absorption band in the 578–582 nm region, which
!
5
corresponded to a D0 7F0 transition (see the Supporting
Information). This transition is very sensitive to the coordi-
nation environment and the observation of a single band ex-
cludes the co-existence of differently hydrated species.[27]
1
17O NMR and H NMRD measurements on the GdIII com-
plexes: The water-exchange rate was determined for the
GdIII complexes of ligands L1, L2, L3, and L4 from a varia-
ble-temperature 17O NMR study. In order to assess the
effect of the protonation of the amine on the water-ex-
change parameter of complex GdL2, measurements at two
different pH values (pH 4.6 and 6.6) were performed, which
correspond to conditions in which the amine is mainly in its
protonated and unprotonated forms, respectively (log KH =
5.12). Owing to the instability of the complex below pH 4, a
further decrease in pH value to attain a fully protonated
amine form was not possible. In addition, variable-tempera-
ture proton-relaxation rates (25 and 378C) were measured
for complexes GdL1, GdL2, and GdL4 as a function of the
proton Larmor frequency (NMRD profiles), with the objec-
tive of determining parameters that describe rotation. The
17O NMR chemical shifts (Dwr), transverse relaxation rates
(1/T2r), and the proton relaxivities (r1) were analyzed simul-
taneously using the Solomon–Bloembergen–Morgan (SBM)
theory of paramagnetic relaxation (see the Supporting Infor-
mation). If we are not interested in detailed information
about the electron-spin relaxation and if we restrict the
analysis of the NMRD data to medium- and high magnetic
fields, this SBM approach gives reliable information on dy-
namic processes like water-exchange and rotational correla-
tion times for small complexes.[28] Therefore, we included
only relaxivity values above 6 MHz in the simultaneous fit
vealed a very peculiar structural behavior: the C N bond
length for the coordinated nitrogen atom is longer than typi-
ꢀ
cal C N single bonds, whilst that of the uncoordinated nitro-
gen atom is shorter than the typical length. In addition, the
N-C-N bond angle is flatter than that of an sp3 nitrogen
atom. All of these results indicate a near-sp2 hybridization
of the uncoordinated amine nitrogen atom and a behavior
that is more characteristic of aniline than of an aliphatic
amine.[16a,b,d,f–l,22] One can also evoke the anomeric effect ob-
served in systems containing X-C-Y motifs (X, Y=OR, NR2,
halogen),[23] the exact origin of which is still under debate[24]
and which affects both structural and electronic properties.
It has been established that an atom involved in an anome-
ric effect has a lower proton or cation affinity than a
Chem. Eur. J. 2012, 18, 1408 – 1418
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1411

ꢀ. Tꢁth, P. Durand et al.
and the following parameters have been adjusted: the
water-exchange rate (kex298), the activation enthalpy for
¼
water exchange (DH ), the scalar coupling constant (A/ꢀh),
the rotational correlation time (tR298) and its activation
energy (ER), and the parameters describing electron-spin re-
laxation, the mean square of the zero-field splitting (D2), the
correlation time for the modulation of the zero-field split-
ting (tV298), whilst its activation energy (EV) has been fixed
to 1 kJmolꢀ1. The scalar coupling constants obtained in this
way are in the usual range for GdIII complexes; this observa-
tion justifies the assumption of q=1. The experimental
17O NMR and NMRD data and the fitted curves for GdL1
are presented in Figure 1, the data and curves for the other
complexes are shown in the Supporting Information.
The shape of the NMRD curves and their temperature
dependence (r1 decreases with increasing temperature) fol-
lows the general trend observed for typical small-molecular-
weight complexes. At low temperatures, and for all GdIII
complexes studied, the transverse 17O relaxation rates (1/
T2r) increase with increasing temperature, thereby indicating
that these systems are in slow exchange, as expected for
dota-analogue complexes. Here, 1/T2r is directly determined
by the water-residence time (tm =1/kex). At higher tempera-
tures, the systems turn to a fast-exchange regime, where the
transverse 17O-relaxation rates are also influenced by the
longitudinal electronic-relaxation rate (1/T1e) and the nucle-
ar hyperfine coupling constant (A/ꢀh). All parameters ob-
tained in the fit are shown in Table 1.
Water exchange and rotation: The exchange rates for all
four systems are slightly lower than that of [GdACTHNUGRTNEUNG(dota)]
(Table 1), with small differences from one complex to the
other. The mechanism remains dissociatively activated, as
shown by the positive activation entropies. In general terms,
the rate and mechanism of the water-exchange rates are
closely related to the inner-sphere solution structures of the
complexes. The water-exchange rate is little affected by sub-
stituents that do not directly interfere in the inner coordina-
tion sphere. The destruction of the self-immolative arm does
not lead to a significant change in the water-exchange pro-
cess: complexes GdL1 and GdL2 exhibit similar water-ex-
change rates despite the removal of one pending arm. It is
also known that in dissociative-
Figure 1. Temperature dependence of a) the reduced transverse 17O relax-
ation rates (1/T2r), and b) 17O chemical shifts (Dwr) for GdL1. c) Proton
relaxivities (r1) as a function of the Larmor frequency at 258C (n) and
378C (~). The lines represent the best fits to the experimental points.
For GdL2–GdL4, see the Supporting Information.
Table 1. Kinetic and structural parameters obtained for complexes [GdL1–4
17O NMR and NMRD data. Values in italic type were fixed in the fitting procedure.
ACHTUNGTREN(NUGN H2O)] from the fitting of
ly activated water-exchange
processes, the charge of the
complex is important: a higher
overall negative charge favors
the release of the water mole-
cule, thus accelerating the ex-
change. We observe the same
Ligand
kex298 ꢃ10ꢀ6 [sꢀ1
L1[a]
L2[a,c]
L3[b]
L4[a]
DOTA4ꢀ[a,d]
]
2.5
U
3.9
1.5
46.0
39.7
N
1.9
G
2.2
N
4.1
49.8
48.5
ACHTUNGTRENNUNG
¼
DH [kJmolꢀ1
]
59.0
55.2
36.3
51.6
16.6
ACHTUNGTRENNUNG
¼
DS [Jmolꢀ1 K]
92(ꢁ5)
25(ꢁ4)
7(ꢁ6)
ACHTUNGTRENNUNG
298
trend in the kex value deter-
tRH298 [ps]
130
23.0
85(ꢁ7)
–
–
110
21.4
ꢀ3.6
77(ꢁ4)
mined for complex GdL2 at the
two different pH values: at
pH 6.6, the complex is not pro-
tonated at the exocyclic amine
atom, and therefore its overall
ER [kJmolꢀ1
]
25.0
ꢀ3.8
ꢀ3.8
16.1ACHTUNGTRENNUNG
A/ꢀhꢃ10ꢀ6 [Hz]
ꢀ3.6
A
ꢀ3.8
ꢀ3.7
ACHTUNGTRENNUNG
[a] determined by 17O NMR and NMRD spectroscopy. [b] From 17O NMR spectroscopic relaxation rates and
chemical shifts. [c] Values determined at pH 6.6 (top line) and 4.5 (bottom line), respectively; at pH 4.5 only
charge is ꢀ1 whilst at pH 4.6 it from 17O NMR data. [d] From Ref. [29]
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LanthanideACHTUNGTRENNUNG(III) Complexes That Contain a Self-Immolative Arm
FULL PAPER
is protonated and becomes neutral. Consequently, the
water-exchange rate of complex GdL2 is reduced by a factor
of approximately 2 between pH 6.6 and 4.6.
The rotational correlation times calculated for these GdIII
complexes are reasonable for the size of the molecule and
their trend reflects their increasing size from GdL2 to GdL4
and GdL1.
Variation in relaxivity upon enzymatic cleavage: Contrary to
our original expectations, the cleavage of GdL1 and the sub-
sequent self-immolative reaction does not proceed until the
formation of the GdDO3A complex, which would result in
an increase in the hydration number from q=1 to 2. There-
fore, the variation in relaxivity upon enzymatic cleavage is
only caused by a decrease in the complex size, that is, the
rotational correlation time, and it amounts to between 10–
20%, depending on the temperature and the proton Larmor
frequency (Table 2). Such a small change in relaxivity is
Figure 2. CEST spectra recorded for YbL3 (378C, [YbL3]=20 mm) at var-
ious pH values.
showed a broad peak corresponding to the CEST effect of
the amine proton (d=ꢀ26 ppm; Figure 3). This signal
broadens with decreasing pH value and disappears below
Table 2. Relaxivity of the GdIII complexes before (GdL1) and after enzy-
matic cleavage (GdL2).
r1 [mmꢀ1 sꢀ1
]
20 MHz
60 MHz
258C
378C
258C
378C
GdL1
GdL2
5.57
3.72
3.38
4.83
4.11
3.68
3.15
4.38[a]
[a] r1 =4.83 mmꢀ1 sꢀ1 was reported for GdL2 at 600 MHz.[15]
likely insufficient for the in vivo detection of enzymatic ac-
tivities using these complexes. However, one can imagine
that the cleavage of larger macromolecular probes of com-
plexes GdL2 or GdL3 attached to a dendrimeric structure
through self-immolative linkers would result in considerable
diminution of the rotational correlation time, and thus of
the relaxivity.
Characterization of the YbIII complexes: In a previous com-
munication on the YbIII complexes of L1 and L2,[13] we re-
ported that no PARACEST effect is detectable for complex
YbL1, despite the presence of a carbamate proton, in con-
trast to the PARACEST effect found for the TmIII complex
of related derivatives.[30] After incubation with b-galactosi-
dase, a PARACEST effect is observed at d=ꢀ16.7 and d=
ꢀ20.5 ppm, owing to the two slowly exchanging, magnetical-
ly non-equivalent amine protons. A PARACEST effect was
also reported for the TmL2 complex at d=+8 ppm, but this
effect was difficult to exploit.[30] Here, our objective was to
complete the preliminary study of the substrate and the
product of the enzymatic reaction with respect to their
PARACEST behavior, in particular to determine their
proton-exchange rate. In order to assess the effect of amine
substitution on the PARACEST properties, we also investi-
gated the methyl-amine derivative YbL3. As expected, a
single CEST peak was detected for the secondary amine of
YbL3 at dꢃꢀ25 ppm, in the same region as for complex
YbL2. The CEST effect exhibits a strong pH-dependency
(Figure 2). High-resolution NMR spectroscopic analysis
1
Figure 3. 500 MHz H NMR spectra of a 20 mm aqueous solution of YbL3
at various pH values (378C, dbulkwater =0 ppm).
pH 8, thereby indicating an increasingly fast proton ex-
change when the pH value is decreased. The fact that the
secondary amine group is also capable of providing a CEST
effect opens up the possibility of functionalizing this amine
group to link the PARACEST probe to biomacromolecules,
or to form macromolecular structures.
Determination of the proton-exchange rates: To explain the
evolution of the CEST effect for complexes YbL2 and YbL3,
we determined the exchange rate of the mobile protons on
both complexes at various pH values. Several methods are
available to estimate the proton-exchange rate from the
CEST effect. One of them uses the concentration depend-
ence of the saturation transfer, as shown in Equation (2):[31]
ꢀ
ꢁ
Ms
M0
1
¼
ð2Þ
kex½CꢂnT1
1 þ
111
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1413

ꢀ. Tꢁth, P. Durand et al.
where Ms is the signal intensity of the bulk water protons
taken immediately after the pulse is applied to the ex-
changeable protons of the CEST agent, M0 is the initial in-
tensity of bulk water protons in the absence of saturation,
kex is the exchange-rate constant, n is the number of saturat-
ed protons per CEST molecule, [C] is the concentration of
the CEST agent, and T1 is the longitudinal relaxation time
of bulk water protons.
One can also determine the proton-exchange rate from
the saturation time and saturation-power dependency of the
intensity of the water proton (QUEST and QUESP experi-
ments refer to quantification of the exchange rate as a func-
tion of saturation time or saturation power, respectively).[32]
Assuming that the steady state is reached upon saturation of
the solute, Equation (3) applies,[32] where xCA is the fraction-
al concentration of exchangeable protons of the contrast
agent, tsat is the saturation time, a is the saturation efficiency,
and kex is the rate of proton exchange on the amine:
plot) should be linear with an x-axis intercept
(M /
G
ꢀ1/kex2 [Eq. (9)]:
1
k2ex
1
w12
ꢀ
¼
ð9Þ
The derivation of this linear relationship assumes that kex is
much higher than the longitudinal- and transverse relaxation
rates.
We have used these various methods to assess the ex-
change rate on the complexes YbL2 and YbL3. Variation of
the concentration and QUEST measurements were per-
formed on complex YbL2, whilst the exchange rate of YbL3
was determined from: 1) a simultaneous analysis of QUEST
and QUESP data, 2) a fit of the saturation-time-dependent
data to Equation (7), and 3) a fit of the QUESP data to
Equation (8). First, we will discuss the data for complex
YbL3, because, for this system in particular, we assessed the
value of QUESP measurements in calculating proton-ex-
change rates. Proton-exchange rates reported in the litera-
ture are typically determined from saturation-time-depen-
dent experiments, and we wanted to assess whether the satu-
ration-power-dependency experiments make the determina-
tion more accurate in combination with QUEST data, as
previously suggested for diamagnetic systems,[32] and wheth-
er the concentration-independent approach of the QUESP
analysis is applicable.
Â
Ã
Ms
kexaxCA
1W þkexxCAÞtsat
ð3Þ
1 ꢀ
¼
ꢄ 1 ꢀ eꢀðR
M0 R1W þ kexxCA
The saturation efficiency depends on the pulse power,
[Eq. (4)], where R1,2S and R1,2W are the longitudinal- and
transverse relaxation rates of the solute and the bulk water
in the saturated state, respectively:
w12
w21 þ pq
ð4Þ
ð5Þ
ð6Þ
a ¼
Figure 4 shows the dependence of the CEST effect of
complex YbL3 on saturation time and saturation power at
k2exxCA
R2W þ kexxCA
k2exxCA
R1W þ kexxCA
p ¼ R2S þ kex
q ¼ R1S þ kex
ꢀ
ꢀ
The values of 1ꢀMs/M0 obtained as a function of saturation
time are often analyzed according to Equation (7), as re-
ported by Zhang et al,[33] where both kex and the longitudinal
relaxation time, T1, are used to make the fit:
ꢀ
ꢁ
ð7Þ
MS
M0
1
kexxCAT1
exxCAT1Þtsat
1 ꢀ
¼ 1 ꢀ
þ
eꢀð1þk
1 þ kexxCAT1 1 þ kexxCAT1
Each of these techniques requires that the agent concentra-
tion is known. Recently, Dixon et al. have reported a modi-
fied analysis of the QUESP experiment as a concentration-
independent method to assess the proton-exchange rate in
PARACEST agents.[34] They derived Equation (8), where w1
is the amplitude of the radiofrequency applied for the satu-
ration pulse given in radsꢀ1
:
ꢀ
ꢁ
MS
M0 ꢀ MS
55:5
c
1
k2ex
1
w12
¼
kexR1w
þ
ð8Þ
Figure 4. QUEST (top) and QUESP (bottom) data for YbL3. The curves
represent a simultaneous fit to Equations (3)–(6).
2
A plot of M /
(M0-MS) versus 1/w1 (referred to as an omega
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LanthanideACHTUNGTRENNUNG(III) Complexes That Contain a Self-Immolative Arm
FULL PAPER
different pH values, measured at d=ꢀ26 ppm. All of the
QUEST and QUESP data have been fitted simultaneously
to Equations (3)–(6) to obtain the rate of proton exchange.
The fitted values of R1W and R2W were in the range 0.4–0.5-
considerably higher than the relaxation rates. This simple
analysis of the QUESP data, when applicable, provides a
very convenient way of determining the kex value, because
this method avoids the concentration effect and also does
not include the fitting of the longitudinal- and transverse re-
laxation rates, which are not directly accessible by independ-
ent measurements.
A
these results led to no significant alteration of the calculated
proton-exchange rate. R1S is negligible compared to kex and
has no influence on the fit. The transverse relaxation rate of
the solute R2S has been calculated in the fit to give 130-
For complex YbL2, the rate constants of the exchangeable
NH2 protons (d=ꢀ23 ppm and d=ꢀ16 ppm) were either
estimated from: 1) a concentration-dependent study of the
saturation transfer in the range of 1–75 mm (pH 7.4;
Figure 6) by fitting the results to Equation (2) (Table 4), or
A
values remained unaffected even by a relatively large (sever-
al fold) variation of R2S. The QUEST experiments were also
fitted to Equation (7) (see the Supporting Information). The
QUESP data were also analyzed according to Equation (8)
(for pH 8.4, see Figure 5; data for the other pH values are
Figure 6. Concentration dependence of the CEST effect for YbL2 at d=
ꢀ16 ppm (n) and d=ꢀ23 ppm (p). The curve represents the fit to Equa-
tion (2).
Figure 5. Omega plot for YbL3 at pH 8.4. The curve represents the fit to
Table 4. kex values obtained from the best fit of QUEST data of YbL2 to
Equation (8).
Equation (7) at 310 K.
pH
kex [sꢀ1
d=ꢀ16 [ppm] d=ꢀ23 [ppm] d=ꢀ16 [ppm] d=ꢀ23 [ppm]
]
T1sat [s]
6.4
6.9
7.4
–
3500
2800
2100
N
–
1.4
1.1
1.3
ACHTUNGTRENNUNG
shown in the Supporting Information). They indeed fall on a
2000
1600
N
1.1
1.2E
(ꢁ0.1)
ACHTUNGTRENNUNG
(glyOEt)4]3+
straight line, as shown for the [EuACHTUNTRGENNUG(DOTA)ACHTUNGTRENNUGN
ACHTUNGTRENNUNG
system.[34] The best-fit values are listed in Table 3.
[a]
1700
A
2200
ACHTUNGTRENNUNG
8.0
1000
G
1700
R
1.1G(ꢁ0.1)
1.1
N
Table 3. kex [sꢀ1] values obtained for YbL3 at 310 K.
[a] From concentration-dependent measurements.
pH value
6.4
QUEST+ QUESP 5500
(ꢁ500) 3800
2900
(ꢁ800) 3200
6000
(ꢁ800) 4300
0.288ꢃ10ꢀ3 0.285ꢃ10ꢀ3 0.286ꢃ10ꢀ3 0.287ꢃ10ꢀ3
6.9
(ꢁ500) 1500
(ꢁ800) 1800
(ꢁ600) 1600
7.4
(ꢁ500) 1200
(ꢁ500) 1500
(ꢁ500) 1300
8.4
(ꢁ500)
(ꢁ500)
(ꢁ500)
from 2) the dependence of the CEST effect on the satura-
tion time (see the Supporting Information). These results in-
dicate that the CEST effect does not increase when the satu-
ration time is increased beyond 2 s. From the measurements,
we were able to distinguish the two amine protons. We
fitted the QUEST experiments performed at different pH
values to Equation (7) and the kex values obtained with this
method are reported in Table 4.
The exchange rates of the amine protons obtained for
complexes YbL2 and YbL3 are of the order 103 sꢀ1, similar
to those reported in the literature for amide protons of
Yb3+-DOTAM complexes (DOTAM=1,4,7,10-tetrakis(car-
bamoylmethyl)-1,4,7,10-tetrazacyclododecane), which are
well-known to give rise to an observable CEST effect. For
complex YbL2, the proton at d=ꢀ16 ppm exchanges slightly
G
G
G
ACHTUNGTRENNUNG
QUEST [Eq. (7)]
“omega plot”
xCA
E
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
[a]
[a] Fixed in the fit.
Very similar proton exchange rates were obtained from
the different approaches (Table 3) for a given pH value. The
method based on the omega plot was previously validated
for a Eu3+ complex in which the CEST effect originated
from the slow exchange of the coordinated water mole-
cules.[34] For our YbL3 complex, the proton exchange is
around ten times faster than in [Eu
which is important with respect to the condition of kex being
(glyOEt)4]3+,
ACHUTGTNRNEUG(N DOTA)ACHTUTGNRENNUGN
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1415

ꢀ. Tꢁth, P. Durand et al.
more slowly than the proton at d=ꢀ23 ppm. A similar dif-
ference in the exchange rate has been previously reported
for the two magnetically non-equivalent NH2 protons of the
Yb3+-DOTAM complex.[35] To the best of our knowledge,
no proton-exchange rates have been reported for amine
groups in metal complexes. The exchange rate of the amine
function of acetyl-lysine-NH2 was 4000 sꢀ1, whilst exchange
rates for arginine of 700 sꢀ1 and 1200 sꢀ1 correspond to the
eNH and hNH2 groups, respectively.[36,37] For the amine pro-
tons of both complexes YbL2 and YbL3, we observe a sys-
tematic decrease of the exchange rate with increasing pH
value, which is also translated by a diminution of the ob-
served CEST effect. This trend is similar to that observed
for aniline protons[37] and for the amine of the TmL2 com-
plex.[38]
However this trend is opposite to the typical behavior of
the amide protons in Ln3+-DOTAM complexes, for which
the proton exchange is a base-catalyzed process, with a con-
comitant increase of the CEST effect with increasing basici-
ty.[39] For instance, the CEST effect measured in a 30 mm so-
lution of the tetraglycinate-derivative Yb-DOTAM-Gly
changes from 0 to about 65% between pH 5.5 and 8.0,[39]
whilst for complex YbL3, it decreases from 65% at pH 6.3
to 15% at pH 9.0. The aniline-like behavior of the amine
groups of complexes YbL2 and YbL3 could be explained by
their near-sp2 hybridization caused by lanthanide coordina-
tion of the gem-diamine, moiety as previously discussed. For
the proton exchange of amines, both acid- and base-catalysis
has been reported.[36] It is interesting to note that a plot of
log kex values versus the pH value for both complexes (and
for both protons of YbL2) gives a straight line, in accordance
with the general formula of acid catalysis (kex =k0+kH[H+];
also see the Supporting Information).
various pH values by using the dependency of the CEST
effect on concentration, saturation time, and saturation
power. We also applied a concentration-independent analy-
sis of the saturation power dependency data, as recently
proposed by Sherry and co-workers.[33] These different meth-
ods gave similar results. In contrast to the typically base-cat-
alyzed amide-proton exchange on [LnACTHNUTRGNEUNG(dota)]/tetraamide
complexes, the exchange of the amine protons becomes
faster upon decreasing the pH value, which then leads to an
increasing CEST effect at lower pH values.
Experimental Section
NMR measurements: 1H and 13C NMR spectra were recorded at
300 MHz and 75 MHz, respectively, and calibrated using tetramethylsi-
lane as an internal reference. Variable-temperature 17O NMR measure-
ments of aqueous solutions of the GdIII complexes were obtained on a
Bruker Avance 500 spectrometer (11.75 T, 67.8 MHz) and referenced to
an acidified water solution (aqueous HClO4, pH 4). Longitudinal
17O NMR relaxation times (T1) were measured by the inversion-recovery
pulse sequence,[40] and the transverse relaxation times (T2) were obtained
by the Carr–Purcell–Meiboom–Gill spin-echo technique.[41] To eliminate
susceptibility corrections to the chemical shifts, the samples were sealed
in glass spheres that fitted into 10 mm NMR tubes.[42] To improve sensi-
tivity in the 17O NMR spectra, 17O-enriched water (10% H217O, Cortec-
net) was added to the solutions to yield 1% 17O enrichment. The temper-
ature was calculated according to a previous calibration with ethylene
glycol and methanol.[43] The concentrations and pH values of the samples
were:
[GdL1]=14.2 mmolkgꢀ1
,
pH 7.10;
[GdL2]=13.7 mmolkgꢀ1
,
,
pH 4.50; [GdL2]=10.3 mmolkgꢀ1
,
pH 6.60; [GdL3]=24.7 mmolkgꢀ1
pH 6.90; [GdL4]=12.6 mmolkgꢀ1, pH 7.20. The pH values of the solu-
tions were adjusted by using diluted solutions of NaOH and HCl. The
saturation-transfer experiments were performed on a Bruker Avance 500
spectrometer by irradiating the sample at 0.1 ppm increments. CEST
spectra were recorded using pre-saturation pulses of 3 s duration at
25 mT, unless otherwise stated. Spectra were measured by recording the
signal intensity of the bulk water as a function of the presaturation fre-
quency. For QUEST experiments (quantification of the exchange rate as
a function of saturation time), data were collected by varying the satura-
tion time (0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5 s) at constant power (25 mT). The
QUESP data (quantification of the exchange rate as a function of satura-
tion power) were collected by varying the saturation power whilst the
saturation time remained constant (3 s). The QUEST and QUESP data
were fitted with Scientist (MicroMath, Inc.).
Conclusions
We have designed and synthesized DOTA derivatives of a-
aminoglycine and their corresponding lanthanide complexes
as building blocks for self-immolative imaging probes. A
synthetic pathway giving access to the platform with or with-
out the self-immolative linker and without the difficulties as-
sociated with the intrinsic instability of these ligands was
elaborated. The physicochemical properties of several Gd3+
complexes were investigated: a chelate bearing a self-immo-
lative arm and a sugar unit as a selective substrate for b-gal-
actosidase, its enzymatic product, and two model com-
pounds. They all have one inner-sphere water molecule and
consequently only slightly different proton relaxivities. This
similarity precludes the application of the GdL1 complex as
enzyme-specific T1 relaxation agents. The water exchange
for all four systems is slightly slower than for GdDOTA.
NMRD measurements: The 1/T1 nuclear magnetic relaxation dispersion
(NMRD) profiles of the GdIII complexes were recorded on a Stelar
SMARtracer FFC fast-field-cycling relaxometer covering magnetic fields
from 2.35ꢃ10ꢀ4 T to 0.25 T, which corresponded to a proton Larmor fre-
quency range of 0.01–10 MHz. The relaxivity at higher fields was record-
ed using a Bruker WP80 adapted to variable field measurements and
controlled by the SMARtracer PC NMR console. The temperature was
controlled by a VTC90 temperature-control unit and fixed by a gas flow.
The temperature was determined according to a previous calibration
with a platinum resistance temperature probe. The relaxivity at 500 MHz
was measured on a Bruker Avance 500 (11.75 T) spectrometer. The si-
multaneous-least-squares fit of the 17O NMR and 1H NMRD data were
performed by using Micromath Scientist version 2.0 (Salt Lake City, UT,
USA). The reported errors correspond to one standard deviation ob-
tained by statistical analysis.
The complexes containing exocyclic NH2 or NHACTHNURTGNENG(U CH3)
UV/Vis spectrophotometry: Absorbance spectra were recorded on a
Perkin–Elmer Lambda 19 spectrometer in thermostated cells between 25
and 508C for EuL1 (cEu ꢃ2.5 mm, pHꢃ6.80) and EuL2 (cEu ꢃ2.4 mm, pH
ꢃ6.90). The measurements were carried out in a cylindrical cuvette with
10 cm optical path-length between l=577–581.0 nm.
amine groups are not protonated at physiological pH, as evi-
denced by pH-potentiometric measurements. Their Yb3+ an-
alogues show a pH-dependent PARACEST effect. The
proton-exchange rate was determined for both complexes at
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LanthanideACHTUNGTRENNUNG(III) Complexes That Contain a Self-Immolative Arm
FULL PAPER
Luminescence: The luminescence measurements of EuIII complexes were
performed on Luminescence Thermo Spectronic spectrometer
Chenu, R. Lougerstay-Madec, C. Monneret, J.-C. Florent, D. Carrez,
A. Croisy, NMR Biomed. 2000, 13, 306–310; c) R. Madec-Louger-
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a
AMINCO Bowman Series 2. The luminescence spectra were obtained
!
after excitation at the EuIII 5L6 7F0 band (l=396 nm). All measurement
were performed at room temperature.
pH potentiometry: Protonation constants of the GdL2 and GdL3 com-
plexes were determined by pH-potentiometric titration at 258C in 0.1m
KCl. The samples (3 mL) were stirred whilst a constant flow of N2 was
bubbled through the solutions. Titrations of about 3 mm GdL2 and GdL3
solutions (starting pHꢃ4.5) were carried out by adding standardized
KOH solution with a Metrohm 702 SM Titrino automatic burette. A
Metrohm 692 pH/ion-meter was used to measure the pH value. The con-
centration of H+ ions was determined from the measured pH values
using the correction method proposed by Irving et al.[44] The protonation
and stability constants were calculated from parallel titrations with the
PSEQUAD program.[45] The errors given correspond to a single standard
deviation.
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Acknowledgements
This work was financially supported by the Centre National de la Re-
cherche Scientifique, the Interdisciplinary program “Imagerie du Petit
Animal” (CNRS), the European network (EMIL) No. 50356, and the
ANR (ENZYMRI and ENZYMAGE). This work was performed within
the European COST Action D38 “Metal-Based Systems for Molecular
Imaging Applications”. Financial support from the Grant Agency of the
Czech Republic for J.K. (No. P207/11/1437), from the Institute of
Chemistry of Natural Susbtances (ICSN) for S.T., and from the CNRS
for R.R. is greatly acknowledged.
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