Toward MRI and Optical Detection of Zwitterionic Neurotransmitters: Near-Infrared Luminescent and Magnetic Properties of Macrocyclic Lanthanide(III) Complexes Appended with a Crown Ether and a Benzophenone Chromophore

TLDR: The creation of an imaging agent suitable for NIR detection of NTs for an enhanced sensitivity in biological systems using the benzophenone (BP) moiety as the sensitizer of lanthanide luminescence was attempted.

Abstract: Thanks to their versatile magnetic and luminescence features, lanthanide complexes have gained a central position in biomedical imaging as magnetic resonance imaging (MRI) contrast agents and optic...

Figures

Figure 3. Plot of the longitudinal water proton relaxation rate of a 0.1 mM GdL3 solution in HEPES (25mM, pH 7.4) as a function of HSA concentration at 20 MHz and 37°C.

Figure 8. (Left) Emission spectra of YbL3 upon continuous illumination at 300 nm during 40 min and (right) corresponding absorption spectra (100 µM in 25 mM HEPES buffer at pH 7.4, room temperature). Arrows indicate the direction of changes.

Figure 5. Relaxivity profile of 1 mM GdL3 in response to addition of glutamate in the presence of 0.6 mM HSA in HEPES (25 mM, pH 7.4) at 37°C and 20 MHz.

Figure 9. Conversion ratios of L3 and LnL3 (Ln = YbIII, NdIII, EuIII) upon continuous illumination at 300 nm (100 µM in 25 mM HEPES buffer at pH 7.4, room temperature).

Figure 2. Proton relaxivities, r1, of 1 mM aqueous solution of GdL3 in HEPES (25 mM, pH7.4), as a function of the Larmor frequency in the absence of the human serum albumin (HSA) at 25°C (▲) and 37°C (■) and in presence of 0.6 mM HSA at 37°C (◊), after subtraction of the diamagnetic contribution of HSA.

Figure 6. Absorption spectra of LnL3 (Ln = YbIII, GdIII and NdIII; 100 µM in 25 mM HEPES buffer, pH = 7.4, room temperature).

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Toward MRI and Optical Detection of Zwitterionic
Neurotransmitters: Near-Infrared Luminescent and
Magnetic Properties of Macrocyclic Lanthanide(III)
Complexes Appended with a Crown Ether and a
Benzophenone Chromophore
Fatima Oukhatar, Svetlana Eliseeva, Celia Bonnet, Matteo Placidi, Nikos
Logothetis, Stéphane Petoud, Goran Angelovski, Éva Tóth
To cite this version:
Fatima Oukhatar, Svetlana Eliseeva, Celia Bonnet, Matteo Placidi, Nikos Logothetis, et al.. To-
ward MRI and Optical Detection of Zwitterionic Neurotransmitters: Near-Infrared Luminescent and
Magnetic Properties of Macrocyclic Lanthanide(III) Complexes Appended with a Crown Ether and
a Benzophenone Chromophore. Inorganic Chemistry, American Chemical Society, 2019, 58 (20),
pp.13619-13630. �10.1021/acs.inorgchem.9b00887�. �hal-02355920�

1
Towards MRI and Optical Detection of Zwitterionic Neurotransmitters:
Near-Infrared Luminescent and Magnetic Properties of Macrocyclic
Lanthanide(III) Complexes Appended with a Crown Ether and a
Benzophenone Chromophore
Fatima Oukhatar,
†§
Svetlana V. Eliseeva,
†
Célia S. Bonnet,
†
Matteo Placidi,
§
Nikos K.
Logothetis,
§₸
Stéphane Petoud,
*†
Goran Angelovski
*§
and Éva Tóth
*†
†
Centre de Biophysique Moléculaire, CNRS UPR 4301, Université d’Orléans, rue Charles Sadron,
45071 Orléans, Cedex 2, France.
§
Department of Physiology of Cognitive Processes and MR Neuroimaging Agents, Max Planck
Institute for Biological Cybernetics, Max-Planck-Ring 8, 72076 Tubingen, Germany.
₸
Department of Imaging Science and Biomedical Engineering, University of Manchester,
Manchester, UK.
* E-mail: eva.jakabtoth@cnrs-orleans.fr
* E-Mail: goran.angelovski@tuebingen.mpg.de
* E-mail: stephane.petoud@inserm.fr
KEYWORDS. Magnetic resonance imaging, luminescence, near-infrared, bimodal agents,
lanthanide complexes, neurotransmitters, benzophenone, human serum albumin.

2
ABSTRACT
Thanks to their versatile magnetic and luminescence features, lanthanide complexes have gained
a central position in biomedical imaging as MRI contrast agents and optical imaging probes. In
addition, appropriate chemical design allows the modification of the magnetic relaxation
properties of Gd
3+
complexes and the optical properties of visible or near-infrared emitting
lanthanide chelates upon interaction with various biomarkers, which makes them ideal candidates
for the creation of responsive agents. In this Forum article, we demonstrate such design principles
as well as the difficulties encountered in the context of neurotransmitter detection. Lanthanide(III)
complexes of a macrocyclic ligand incorporating a benzophenone chromophore and a monoaza-
crown ether (LnL
3
) have been synthesized as responsive probes to monitor amino acid
neurotransmitters either in magnetic resonance imaging (Ln=Gd) or in near-infrared optical
detection (Ln=Nd or Yb). The parameters characterizing the water exchange and the rotational
dynamics of the Gd
III
complex were assessed by
17
O NMR and
1
H NMRD. In the presence of
zwitterionic neurotransmitters, the inner sphere water molecule is replaced by the carboxylate
function of the neurotransmitters in the Gd
III
complex, leading to a decrease of the longitudinal
relaxivity from 6.7 mM
-1
s
-1
to 2-2.5 mM
-1
s
-1
(300 MHz and 37 °C). Apparent affinity constants
range from K
a
= 35 for -aminobutyric acid (GABA) to 80 M
-1
for glycine and glutamate, and there
is no selectivity with respect to hydrogen-carbonate (K
a
= 232; pH 7.4). The Gd
III
complex interacts
with human serum albumin (HSA) resulting in a 60% increase in relaxivity (20 MHz, 37 °C) in
the absence of neurotransmitters. The HSA-bound complex, however, revealed to be less
responsive to neurotransmitters due to the displacement of the Gd
III
-bound water by HSA, which
was confirmed by the hydration number calculated from luminescence lifetimes of the HSA-bound
Eu
III
complex.
The creation of an imaging agent suitable for near-infrared (NIR) detection of neurotransmitters
for an enhanced sensitivity in biological systems using the benzophenone moiety as sensitizer of
lanthanide luminescence was also attempted. Upon excitation at 300 nm of the benzophenone
chromophore in aqueous solutions of NdL
3
and YbL
3
, characteristic near-infrared emissions of
Nd
III
and Yb
III
were observed due to
4
F
3/2
→
4
I
J
(J = 9/2 – 13/2) and
2
F
5/2
→
2
F
7/2
transitions,
respectively, indicating that this chromophore is a suitable antenna. Despite these promising
results, luminescence titrations of Nd
3+
and Yb
3+
complexes with neurotransmitters were not
conclusive due to a chemical conversion of the ligand triggered by light, preventing quantitative

3
analysis. The observed photochemical reaction of the ligand is strongly dependent on the nature
of the lanthanide chelated; it is considerably slowed down in the presence of Nd
III
and Eu
III
.
INTRODUCTION
Responsive magnetic resonance imaging (MRI) agents act as reporters of the biological
environment where they are distributed by generating an MRI signal dependent on tissue
parameters such as pH, concentration of endogeneous ions, enzymes, etc.
1
Combining MRI
detection with complementary imaging techniques can provide additional, less ambiguous
information. Multimodal approaches are becoming more common in both biomedical research and
clinical imaging, and the development of responsive imaging agents with detection capabilities
has recently triggered increasing interest.
2-4
Lanthanide(III) ions (Ln
III
) have versatile magnetic
and luminescent properties and in the past, we and others have explored approaches for the creation
of bimodal imaging agents adapted for combined MRI and optical detection.
5-7
While the Gd
III
ion
with its S=7/2 electron spin and slow electronic relaxation is the most efficient relaxation agent
among all paramagnetic metal ions, the luminescence properties of several lanthanides of different
natures offer complementary advantages over organic dyes.
8
Their emission bands are much
narrower and cover UV, visible and near-infrared (NIR) spectral ranges. Ln
III
exhibit long
luminescence lifetimes (from s up to ms) thus enabling time-gated detection. Nevertheless, due
to the Laporte forbidden nature of most of the f-f transitions of lanthanide(III) ions, they are
characterized by very low absorption coefficients (< 10 M
-1
cm
-1
) that makes their direct excitation
inefficient reducing therefore the number of emitted photons. This problem can be circumvented
by locating in sufficiently close proximity Ln
III
ions and chromophoric moieties able to absorb the
excitation light and transfer the resulting energy to the Ln
III
(“antenna effect”).
9
The detection of neurotransmitters (NT) has been in the focus of recent efforts in molecular MRI.
10
Direct monitoring of neural activity by MRI via the detection of appropriate biomarkers is one of
the current challenges in neuroimaging. One strategy involves engineered proteins obtained in
directed evolution. In several mutation rounds, the affinity of paramagnetic metalloproteins could
be tuned for monoamine NT and these probes have been used in small animal studies to detect
dopamine or serotonin release.
11-12
In a different approach based on artificial NT receptors, we
have reported a series of MRI contrast agents that are responsive to zwitterionic NTs including

4
the ligands L
1
and L
2
(Scheme 1).
13-14
They consist of conjugates of Gd
III
DO3A-type chelates and
eighteen-membered mono- or triaza-crown ethers. These complexes act as ditopic receptors that
can accommodate zwitterionic, amino acid NTs in a dual binding mode. The binding occurs
between the positively charged Ln
III
chelate and the carboxylate function of the NTs, and between
the azacrown ether and the amine group of the neurotransmitters. The MRI response of the Gd
III
probe is based on the displacement of the inner-sphere water molecule(s) upon neurotransmitter
binding.
Several optical approaches have been also developed to sense neurotransmitters, involving both
small molecule and genetically encoded protein sensors,
15
but none of them was based on
lanthanide luminescence. In our previous studies, the interaction with the selected
neurotransmitters has been demonstrated to impact also the optical properties of the Eu
III
and Tb
III
complexes of the ligand L
1
and L
2
;
14
however, the phenyl group proved to be a poor sensitizer of
these luminescent Ln
III
ions. The luminescence of NIR emitting lanthanide cations is attractive in
respect to visible ones for application in biological systems as these possess only a low native
fluorescence in the NIR spectral range.
16
This advantage allows to obtain a better signal to noise
ratio and an enhanced detection sensitivity. Our goal here was to design a new chemical structure
by introducing a suitable lanthanide chromophoric sensitizer to generate an antenna effect
9
in the
ligand structure in order to develop a neurotransmitter-sensitive probe that would provide detection
capabilities both in MRI and optical imaging in the NIR domain, depending on the coordinated
Ln
III
ion. Y. Shiraishi et al.,
17
and J.A. Gareth Williams
18,19
et al. reported the sensitization of
characteristic emission of Eu
III
and Tb
III
ions in the visible range via the benzophenone (BP)
moeity. In addition, it has been found that Eu
III
complexes with the para-substituted BP derivative
exhibit significantly higher luminescence quantum yields compared to the corresponding ortho-
and meta-substituted complexes.
19
S. Pope et al. have also demonstrated the sensitization of
characteristic Nd
III
emission in the NIR range in a ternary cyclen complex with benzoylbenzoate.
20
The main electronic features of BP chromophores involve the n,π* character of the excited triplet
state and quantitative intersystem crossing efficiency with quantum yields for the formation of the
triplet state close to unity. Therefore, the BP chromophore has been incorporated in the Ln
III
chelate which also bears a mono-aza crown ether as illustrated in Scheme 1 (ligand L
3
).

Frequently asked questions

Q1. What are the contributions in "Toward mri and optical detection of zwitterionic neurotransmitters: near-infrared luminescent and magnetic properties of macrocyclic lanthanide(iii) complexes appended with a crown ether and a benzophenone chromophore" ?

Oukhatar et al. this paper proposed an approach for the detection of Zwitterionic Neurotransmitters ( NTs ) using near-infrared luminescent and magnetic properties of Macrocyclic Lanthanide ( III ) complexes with a Crown Ether and a Benzophenone Chromophore. 

Q2. What are the future works in "Toward mri and optical detection of zwitterionic neurotransmitters: near-infrared luminescent and magnetic properties of macrocyclic lanthanide(iii) complexes appended with a crown ether and a benzophenone chromophore" ?

This is an encouraging effect that opens possibilities for development of potent imaging probes suitable for neurotransmitter sensing by means of MR as well as optical ( e. g. NIR ) imaging. 

Q3. What is the common method used to promote the binding of HSA to MRI contrast agents?

Hydrophobic residues have been typically used to promote the binding between MRI contrast agents and HSA.32 Benzophenone has been reported to bind HSA with an association constant of Ka = 8.5×10 4 M-1, as determined by spectroscopic methods. 

Q4. What is the effect of the presence of HSA on the relaxivity of the complex?

In thepresence of human serum albumin, the relaxivity increases at intermediate fields due to slower rotation; however, this relaxivity gain is limited by a decrease in the GdIII hydration number. 

Q5. What is the advantage of using a bimodal responsive probe?

In general, the combination of MRI and in vivo NIR optical imaging using bimodal responsiveagents can further advance molecular imaging towards more specific and powerful detection of biomarkers. 

Q6. What are the advantages of lanthanide complexes?

Thanks to their versatile magnetic and luminescence features, lanthanide complexes have gaineda central position in biomedical imaging as MRI contrast agents and optical imaging probes. 

Q7. What is the effect of glutamate on the relaxivity of the GdL3 system?

In addition, when present in a largeexcess, glutamate becomes a competitor of HSA and will replace the protein carboxylates in the coordination sphere of the metal in GdL3, further contributing to the overall relaxivity decrease. 

Q8. What is the main electronic feature of a BP chromophores?

The main electronic features of BP chromophores involve the n,π* character of the excited tripletstate and quantitative intersystem crossing efficiency with quantum yields for the formation of the triplet state close to unity. 

Q9. What is the effect of the carboxylate function of the neurotransmitters?

In the presence ofzwitterionic neurotransmitters, the inner sphere water molecule is replaced by the carboxylate function of the neurotransmitters in the GdIII complex, leading to a decrease of the longitudinal relaxivity from 6.7 mM-1s-1 to 2-2.5 mM-1s-1 (300 MHz and 37 °C). 

Q10. What is the spectral range of the lanthanide cations?

The luminescence of NIR emitting lanthanide cations is attractive inrespect to visible ones for application in biological systems as these possess only a low native fluorescence in the NIR spectral range. 

Q11. What is the effect of the binding of free carboxylate functions in the side chains of G?

the authors can conclude that the binding of free carboxylate functions in the side chains of HSA to the GdIII also participates to the GdL3 – protein interactionand contributes to the affinity. 

Q12. What is the effect of the BP derivative on the luminescence of the ni?

In addition, it has been found that EuIII complexes with the para-substituted BP derivativeexhibit significantly higher luminescence quantum yields compared to the corresponding orthoand meta-substituted complexes. 

Q13. Why are lanthanide(III) ions characterized by low absorption coefficients?

dueto the Laporte forbidden nature of most of the f-f transitions of lanthanide(III) ions, they are characterized by very low absorption coefficients (< 10 M-1 cm-1) that makes their direct excitationinefficient reducing therefore the number of emitted photons. 

Q14. What is the q value of GdL3 in the presence of HSA?

The q value determined under these conditions is 0.1±0.2, indicating the replacement of the inner-sphere water molecule of GdL3 by the amino acidside chain residues of the HSA. 

Q15. What is the effect of the introduction of the BP moiety on the neurotransmitters?

it can be concluded thatthe introduction of the BP moiety leads to a loss of selectivity for neurotransmitters over carbonate. 

Q16. What are the main assumptions for the estimation of hydration numbers from solvent vibrations?

26-27 Their use, however, is restricted by a number of assumptions, in particular, that thequenching by solvent vibrations is the main deactivation process and that all the other non-radiative paths are the same in H2O and D2O. 

Q17. What is the simplest way to describe the rotational dynamics?

The rotational dynamics could be only described by applying the Lipari-Szabo approachthat separates local and global motions, characterized by the local and global rotational correlation times, lH 298 and gH 298, respectively, and a model independent order parameter, S2. 

Q18. What is the effect of the phenyl group on the optical properties of the NT?

In their previous studies, the interaction with the selected neurotransmitters has been demonstrated to impact also the optical properties of the EuIII and TbIII complexes of the ligand L1 and L2;14 however, the phenyl group proved to be a poor sensitizer of these luminescent LnIII ions.