Naoko Kotera

A Sensitive Zinc-Activated 129Xe MRI Probe†

The divalent zinc cation, Zn, is an indispensable and ubiquitous element of the body. As the second most abundant transition-metal ion in mammalian tissues, it is involved in many physiological and pathological processes. Zinc plays a vital role not only when bound to metalloproteins, but also in the form of mobile pools. A slight excess or lack of zinc ions can be connected to serious human afflictions, including heart disease, diabetes, cancer, and neurodegeneration such as Alzheimer s disease. Today, only two noninvasive techniques, optical imaging and magnetic resonance imaging (MRI), have the potential to offer real-time monitoring of the Zn distribution in different tissues of the body. However, optical methods suffer from limited penetration depth, which makes them unsuitable for global analysis of relatively large and opaque specimens, such as live animals. On the other hand, MRI is a particularly powerful modality used clinically for anatomic imaging and provides three-dimensional images with excellent resolution. However, conventional molecular MRI techniques that rely on the observation of water protons and require the introduction of contrast agents still suffer from reduced sensitivity and often lack selectivity. A few studies based on gadolinium complexes have been reported for Zn imaging. Nevertheless, to our knowledge, the detection threshold of free Zn ions is 30 mm, a value slightly above the total Zn concentration of 20 mm in blood. Therefore, the development of more sensitive methods is of crucial importance. Herein we propose the use of hyperpolarized Xe nuclear magnetic resonance (NMR) spectroscopy for the sensitive detection of Zn ions. To achieve this goal, the noble gas is encapsulated in dedicated host systems bearing a ligand that chelates the Zn ions. Cryptophanes, aromatic cage molecules made of cyclotriveratrylene groups, are perfectly suited to this purpose as 1) they can easily be rendered water-soluble, 2) the noble gas has a high affinity for their cavity, 3) when xenon is encapsulated, it takes a specific NMR frequency, and 4) xenon exchange in and out of the cavity insures a continuous refreshment of the Xe@cryptophane environment in hyperpolarization. Such a Xe biosensing approach has already been employed for detection of various biological systems, including enzymes and nucleic acids. Also, the first in-cell probing of biological events has been achieved: the endocytosis of transferrin could be detected by using Xe NMR spectroscopy. All these NMR spectroscopy studies based on the use of hyperpolarized xenon and molecular hosts are characterized by a high sensitivity. However, metal detection is a difficult challenge, which has never been achieved using such an approach. We aimed to design a responsive agent in which the chemical shift of encapsulated xenon would significantly vary when Zn ions are chelated to it. In this manner, a sensitive spectroscopic imaging based on this resonance-frequency variation can be envisioned. For this purpose, we designed sensor 1, which is made of three parts (Scheme 1): the cryptophane core hosting xenon, the spacer, and the chelating moiety. A short spacer was chosen to place the chelating moiety near the cryptophane cavity. As a zinc-chelating group we chose nitrilotriacetic acid (NTA), which is easily prepared from l-lysine. Sensor 1 was synthesized from cryptophane 2, which possesses six carboxylate groups ensuring solubility in water at physiological pH value. We were able to activate only one carboxylate group by esterification with N-hydroxysuccinimide. Then, the primary amino group of the enantiopure unit bearing the NTA moiety (l)-3 was directly coupled to this activated ester to form a chemically stable amide linkage. Thus, the use of host 2 in its racemic form (chirality is due to the helicity of the cryptophane) led to two diastereomers, which will be noted 1P and 1M (see the Supporting Information for the nomenclature). Compound 1 was obtained as a 50:50 diastereomeric mixture (1P + 1M), with a chemical purity higher than 95% after HPLC. For this sensor, the xenon binding constant is assumed to be in the same range as that of compound 2, that is, 6000m . As cryptophane 2 has already been shown to behave as a pH sensor, the present Xe NMR spectroscopy study was conducted in a phosphate buffer exempt of other ions, at pH 7.4. At this pH value, the affinity of the NTA group for Zn ions is well-documented (logK1> 10). [16] In the absence [*] N. Kotera, Dr. L. Delacour, Dr. T. Traor , D. A. Buisson, Dr. F. Taran, S. Coudert, Dr. B. Rousseau CEA Saclay, SCBM, iBiTec-S, Building 547, PC # 108 91191 Gif sur Yvette (France) E-mail: bernard.rousseau@cea.fr N. Tassali, E. L once, Dr. C. Boutin, Dr. P. Berthault CEA Saclay, IRAMIS, SIS2M, UMR CEA/CNRS 3299 Laboratoire Structure et Dynamique par R sonance Magn tique 91191 Gif sur Yvette (France) E-mail: patrick.berthault@cea.fr

Smart Detection of Toxic Metal Ions, Pb2+ and Cd2+, Using a 129Xe NMR-Based Sensor

An approach for sensitive magnetic resonance detection of metal cations is proposed. Combining the use of hyperpolarized (129)Xe NMR and of a cage-molecule functionalized by a ligand able to chelate different cations, we show that simultaneous detection of lead, zinc, and cadmium ions at nanomolar concentration is possible in short time, thanks to fast MRI sequences based on the HyperCEST scheme.

Synthesis of a Functionalizable Water-Soluble Cryptophane-111

The development of optimized xenon host systems is of crucial importance for the success of molecular imaging using hyperpolarized (129)Xe MRI. Cryptophane-111 is a promising candidate because of its encapsulation properties. The synthesis of cryptophane-111-based biosensors requires both water-solubilizing and chemically activatable groups. An expeditious synthesis of a water-soluble and functionalizable cryptophane-111 is described.

Design and Synthesis of New Cryptophanes with Intermediate Cavity Sizes

The development of molecular imaging using hyperpolarized xenon MRI needs highly optimized biosensors. Cryptophane-111 and cryptophane-222 are promising candidates that show complementary encapsulation properties although they only differ by the length of the three alkane linkers joining two cyclotriphenolene units. Cryptophanes containing both methoxy and ethoxy linkers have never been synthesized. Here we synthesize two new cages with intermediate internal volumes, in two steps from cyclotriphenolene.

“Clickable” Hydrosoluble PEGylated Cryptophane as a Universal Platform for 129Xe Magnetic Resonance Imaging Biosensors

We describe the synthesis of a highly water-soluble cryptophane 1 that can be seen as a universal platform for the construction of (129)Xe magnetic resonance imaging (MRI)-based biosensors. Compound 1 is easily functionalized by Huisgen cycloaddition and exhibits excellent xenon-encapsulation properties. In addition, 1 is nontoxic at the concentrations typically used for hyperpolarized (129)Xe MRI.

Comparative study of affinity and selectivity of ligands targeting abasic and mismatch sites in DNA using a fluorescence-melting assay

Recently, several families of small-molecule ligands have been developed to selectively target DNA pairing defects, such as abasic sites and mismatched base pairs, with the aim to interfere with the DNA repair and the template function of the DNA. However, the affinity and selectivity (with respect to well-matched DNA) of these ligands has barely been evaluated in a systematic way. Herein, we report a comparative study of binding affinity and selectivity of a representative panel of 16 ligands targeting abasic sites and a T-T mismatch in DNA, using a fluorescence-monitored melting assay. We demonstrate that bisintercalator-type macrocyclic ligands are characterized by moderate affinity but exceptionally high selectivity with respect to well-matched DNA, whereas other reported ligands show either modest selectivity or rather low affinity in identical conditions.

Copper(II)‐Controlled Molecular Glue for Mismatched DNA

Isothermal hybridization of two DNA strands bearing three thymine‐thymine (T:T) mismatches can be brought about in the presence of a stoichiometric amount of a bis‐naphthalene macrocycle, 2,7‐BisNP‐NH. This process can be reverted by addition of a CuII salt due to formation of a dinuclear metal complex which does not bind to DNA. Subsequent sequestration of CuII releases the macrocycle and restores the hybridization state of DNA strands, thus allowing implementation of a fast fluorescent two‐state DNA switch.