David-Alexandre Buisson

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

Control of peptide nanotube diameter by chemical modifications of an aromatic residue involved in a single close contact

Supramolecular self-assembly is an attractive pathway for bottom-up synthesis of novel nanomaterials. In particular, this approach allows the spontaneous formation of structures of well-defined shapes and monodisperse characteristic sizes. Because nanotechnology mainly relies on size-dependent physical phenomena, the control of monodispersity is required, but the possibility of tuning the size is also essential. For self-assembling systems, shape, size, and monodispersity are mainly settled by the chemical structure of the building block. Attempts to change the size notably by chemical modification usually end up with the loss of self-assembly. Here, we generated a library of 17 peptides forming nanotubes of monodisperse diameter ranging from 10 to 36 nm. A structural model taking into account close contacts explains how a modification of a few Å of a single aromatic residue induces a fourfold increase in nanotube diameter. The application of such a strategy is demonstrated by the formation of silica nanotubes of various diameters.

N-Methyldihydroquinazolinone Derivatives of Retro-2 with Enhanced Efficacy against Shiga Toxin

The Retro-2 molecule protects cells against Shiga toxins by specifically blocking retrograde transport from early endosomes to the trans-Golgi network. A SAR study has been carried out to identify more potent compounds. Cyclization and modifications of Retro-2 led to a compound with roughly 100-fold improvement of the EC50 against Shiga toxin cytotoxicity measured in a cell protein synthesis assay. We also demonstrated that only one enantiomer of the dihydroquinazolinone reported herein is bioactive.

(S)-N-Methyldihydroquinazolinones are the Active Enantiomers of Retro-2 Derived Compounds against Toxins

This study reports the synthesis, chromatographic separation, and pharmacological evaluation of the two enantiomers of a new compound, named Retro-2.1, active against toxins by inhibiting intracellular trafficking via the retrograde route. The absolute configuration of the bioactive enantiomer has been assigned from X-ray diffraction to the (S)-enantiomer. To date, (S)-Retro-2.1 is the most potent molecule to counteract the cytotoxic potential of ricin and Shiga toxin, with EC50 values of 23 and 54 nM, respectively.

Structural role of counterions adsorbed on self-assembled peptide nanotubes.

Among noncovalent forces, electrostatic ones are the strongest and possess a rather long-range action. For these reasons, charges and counterions play a prominent role in self-assembly processes in water and therefore in many biological systems. However, the complexity of the biological media often hinders a detailed understanding of all the electrostatic-related events. In this context, we have studied the role of charges and counterions in the self-assembly of lanreotide, a cationic octapeptide. This peptide spontaneously forms monodisperse nanotubes (NTs) above a critical concentration when solubilized in pure water. Free from any screening buffer, we assessed the interactions between the different peptide oligomers and counterions in solutions, above and below the critical assembly concentration. Our results provide explanations for the selection of a dimeric building block instead of a monomeric one. Indeed, the apparent charge of the dimers is lower than that of the monomers because of strong chemisorption. This phenomenon has two consequences: (i) the dimer-dimer interaction is less repulsive than the monomer-monomer one and (ii) the lowered charge of the dimeric building block weakens the electrostatic repulsion from the positively charged NT walls. Moreover, additional counterion condensation (physisorption) occurs on the NT wall. We furthermore show that the counterions interacting with the NTs play a structural role as they tune the NTs diameter. We demonstrate by a simple model that counterions adsorption sites located on the inner face of the NT walls are responsible for this size control.

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.

Synthesis of orthogonally protected azahistidine: application to the synthesis of a GHK analogue.

The synthesis of various orthogonally protected azahistidine derivatives are obtained via 1,3-dipolar cycloaddition reactions. The newly obtained amino-acids can be selectively deprotected either at the side chain or at the N-terminus of the amino acid and should thus allow the use of these derivatives in (solid phase) peptide synthesis.

Efficient tritiation of the translocator protein (18 kDa) selective ligand DPA‐714

DPA-714 (N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide) is a recently discovered fluorinated ligand of the translocator protein 18 kDa (TSPO). Labelled with the short-lived positron emitter fluorine-18, this structure is today the radioligand of reference for in vivo imaging of microglia activation and neuroinflammatory processes with positron emission tomography. In the present work, an isotopically tritium-labelled version was developed ([(3) H]DPA-714), in order to access high resolution in vitro and ex vivo microscopic autoradiography studies, repeated and long-lasting receptor binding studies and in vivo pharmacokinetic determination at late time points. Briefly, DPA-714 as reference, and its 3,5-dibrominated derivative as precursor for labelling, were both prepared from DPA-713 in nonoptimized 32% (two steps) and 10% (three steps) yields, respectively. Reductive debromination using deuterium gas and Pd/C as catalyst in methanol, performed at the micromolar scale, confirmed the regioselective introduction of two deuterium atoms at the meta positions of the phenyl ring. Tritiodebromination was analogously performed using no-carrier tritium gas. HPLC purification provided >96% radiochemically pure [(3) H]DPA-714 (7 GBq) with a 2.1 TBq/mmol specific radioactivity. Interestingly, additional hydrogen-for-tritium exchanges were also observed at the 5-methyl and 7-methyl positions of the pyrazolo[1,5-a]pyrimidine, opening novel perspectives in the labelling of compounds featuring this heterocyclic core.

Synthesis, Chiral Separation, Absolute Configuration Assignment, and Biological Activity of Enantiomers of Retro‐1 as Potent Inhibitors of Shiga Toxin

The Shiga toxin (Stx) family is composed of related protein toxins produced by the bacteria Shigella dysenteriae and certain pathogenic strains of E. coli. No effective therapies for Stx intoxication have been developed yet. However, inhibitors that act on the intracellular trafficking of these toxins may provide new options for the development of therapeutic strategies. This study reports the synthesis, chromatographic separation, and pharmacological evaluation of the two enantiomers of Retro‐1, a compound active against Stx and other such protein toxins. Retro‐1 works by inhibiting retrograde transport of these toxins inside cells. In vitro experiments proved that the configuration of the stereocenter at position 5 is not crucial for the activity of this compound. X‐ray diffraction data revealed (S)‐Retro‐1 to be slightly more active than (R)‐Retro‐1.

Directing peptide crystallization through curvature control of nanotubes

In the absence of efficient crystallization methods, the molecular structures of fibrous assemblies have so far remained rather elusive. In this paper, we present a rational method to crystallize the lanreotide octapeptide by modification of a residue involved in a close contact.