Patrick Berthault

Conformation of the Oligosaccharide Chain of GM1 Ganglioside in a Carbohydrate-Enriched Surface

The solution structure of ganglioside G(M1) carbohydrate moiety at the surface of a 102-kDa lipid-modified-G(M1) micelle is investigated by high-resolution 1H-NMR in H2O. The micellar surface can be considered a cluster-like lateral distribution of the gangliosides, each single monomer being anchored in a carbohydrate-enriched model membrane matrix. 1H NOESY measurements at short mixing times reveal a rigid trisaccharide core -beta-GalNAc-(1-4)-[alpha-Neu5Ac-(2-3)]-beta-Gal- and a more flexible beta-Gal-(1-3)-beta-GalNAc- terminal glycosidic bond. In the lipid-modified G(M1) ganglioside micellar system, there is no evidence that intermolecular side-by-side carbohydrate interactions modulate, or alter in any way, the head-group spatial arrangement. Possible intermonomer interactions at the level of the branched trisaccharide portion were further investigated on mixed micelles of natural N-glycolyl- and N-acetylneuraminic acid containing G(M1) in D2O, taking advantage of the different NMR features of N-glycolyl- and N-acetylneuraminic acids, which allow discrimination between sialic acid ring proton signals. Measurements of the water/ganglioside-OH proton chemical exchange rates suggest hydroxyl group involvement at position 8 of sialic acid in strong intramolecular interaction processes.

Cell uptake of a biosensor detected by hyperpolarized 129Xe NMR: The transferrin case

For detection of biological events in vitro, sensors using hyperpolarized (129)Xe NMR can become a powerful tool, provided the approach can bridge the gap in sensitivity. Here we propose constructs based on the non-selective grafting of cryptophane precursors on holo-transferrin. This biological system was chosen because there are many receptors on the cell surface, and endocytosis further increases this density. The study of these biosensors with K562 cell suspensions via fluorescence microscopy and (129)Xe NMR indicates a strong interaction, as well as interesting features such as the capacity of xenon to enter the cryptophane even when the biosensor is endocytosed, while keeping a high level of polarization. Despite a lack of specificity for transferrin receptors, undoubtedly due to the hydrophobic character of the cryptophane moiety that attracts the biosensor into the cell membrane, these biosensors allow the first in-cell probing of biological events using hyperpolarized xenon.

A Water-Soluble Xe@cryptophane-111 Complex Exhibits Very High Thermodynamic Stability and a Peculiar 129Xe NMR Chemical Shift

The known xenon-binding (±)-cryptophane-111 (1) has been functionalized with six [(η(5)-C(5)Me(5))Ru(II)](+) ([Cp*Ru](+)) moieties to give, in 89% yield, the first water-soluble cryptophane-111 derivative, namely [(Cp*Ru)(6)1]Cl(6) ([2]Cl(6)). [2]Cl(6) exhibits a very high affinity for xenon in water, with a binding constant of 2.9(2) × 10(4) M(-1) as measured by hyperpolarized (129)Xe NMR spectroscopy. The (129)Xe NMR chemical shift of the aqueous Xe@[2](6+) species (308 ppm) resonates over 275 ppm downfield of the parent Xe@1 species in (CDCl(2))(2) and greatly broadens the practical (129)Xe NMR chemical shift range made available by xenon-binding molecular hosts. Single crystal structures of [2][CF(3)SO(3)](6)·xsolvent and 0.75H(2)O@1·2CHCl(3) reveal the ability of the cryptophane-111 core to adapt its conformation to guests.

Off-resonance rf fields in heteronuclear NMR: Application to the study of slow motions

The advantages of using off-resonance rf fields in heteronuclear self-relaxation experiments are explored on a fully 15N-enriched protein. It is firstly shown that in the absence of slow motions the longitudinal and transverse 15N self-relaxation rate values derived with this method are in agreement with the ones measured by the classical inversion-recovery and Carr–Purcell–Meiboom–Gill (CPMG) sequences, respectively. Secondly, by comparing the 15N transverse self-relaxation rates obtained by the proposed off-resonance sequence and by the CPMG sequence, 11 residues out of the 61 of toxin α are shown to exhibit a chemical exchange phenomenon in water on a time scale ranging from 1 µs to 100 ms. By varying the effective field amplitude, chemical exchange processes involving these residues are measured and the corresponding correlation times are evaluated without having assumed any motion model. Similar, though less precise, results are given by the analysis of the 15N off-resonance self-relaxation rates on the basis of the Lipari–Szabo model to describe the fast internal dynamics of toxin α.

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

Magnetization transfer from laser‐polarized xenon to protons located in the hydrophobic cavity of the wheat nonspecific lipid transfer protein

Nonspecific lipid transfer protein from wheat is studied by liquid‐state NMR in the presence of xenon. The gas–protein interaction is indicated by the dependence of the protein proton chemical shifts on the xenon pressure and formally confirmed by the first observation of magnetization transfer from laser‐polarized xenon to the protein protons. Twenty‐six heteronuclear nOes have allowed the characterization of four interaction sites inside the wheat ns‐LTP cavity. Their locations are in agreement with the variations of the chemical shifts under xenon pressure and with solvation simulations. The richness of the information obtained by the noble gas with a nuclear polarization multiplied by ∼12,000 makes this approach based on dipolar cross‐relaxation with laser‐polarized xenon promising for probing protein hydrophobic pockets at ambient pressure.

Elucidation of the self-assembly pathway of lanreotide octapeptide into beta-sheet nanotubes: role of two stable intermediates.

Nanofabrication by molecular self-assembly involves the design of molecules and self-assembly strategies so that shape and chemical complementarities drive the units to organize spontaneously into the desired structures. The power of self-assembly makes it the ubiquitous strategy of living organized matter and provides a powerful tool to chemists. However, a challenging issue in the self-assembly of complex supramolecular structures is to understand how kinetically efficient pathways emerge from the multitude of possible transition states and routes. Unfortunately, very few systems provide an intelligible structure and formation mechanism on which new models can be developed. Here, we elucidate the molecular and supramolecular self-assembly mechanism of synthetic octapeptide into nanotubes in equilibrium conditions. Their complex hierarchical self-assembly has recently been described at the mesoscopic level, and we show now that this system uniquely exhibits three assembly stages and three intermediates: (i) a peptide dimer is evidenced by both analytical centrifugation and NMR translational diffusion experiments; (ii) an open ribbon and (iii) an unstable helical ribbon are both visualized by transmission electron microscopy and characterized by small angle X-ray scattering. Interestingly, the structural features of two stable intermediates are related to the final nanotube organization as they set, respectively, the nanotube wall thickness and the final wall curvature radius. We propose that a specific self-assembly pathway is selected by the existence of such preorganized and stable intermediates so that a unique final molecular organization is kinetically favored. Our findings suggests that the rational design of oligopeptides can encode both molecular- and macro-scale morphological characteristics of their higher-order assemblies, thus opening the way to ultrahigh resolution peptide scaffold engineering.

NMR study of a LewisX pentasaccharide derivative: solution structure and interaction with cations

The structure and conformation of the synthetic pentasaccharide Gal(beta 1-4){Fuc(alpha 1-3)}GlcNAc(beta 1-3)Gal(beta 1-4)Glc-beta OMe of the Lewis(X) family has been determined by NMR spectroscopy in dimethyl sulfoxide and methanol. In these solvents, the binding constants with calcium have been evaluated as 9.5 and 29.6 M-1, respectively. Study of the interaction sites has been achieved through the use of paramagnetic divalent cations and distance triangulation methods. Two regions have been found, the first one in the vicinity of the fucose unit, the second one closer to the lactose part.

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.

Effect of pH and Counterions on the Encapsulation Properties of Xenon in Water-Soluble Cryptophanes

In the (129)Xe NMR-based biosensing approach in which the hyperpolarized noble gas is transported to biological receptors for a sensitive molecular imaging, cryptophanes are excellent xenon host systems. However to avoid formation of self-organized systems, these hydrophobic cage molecules can be rendered water soluble by introduction of ionic groups. We show that the sensitivity of xenon to its local environment and the presence of these ionic functions can lead to interesting properties. For a first water-soluble cryptophane derivative, we show that a precise monitoring of the local pH can be performed. For a second cryptophane, the presence of ionic groups close to the cryptophane cavity modifies the xenon binding constant and in-out exchange rate. The latter allows the tuning of physical properties of xenon-cryptophane interactions without resorting to a change of the cavity size. These results open new perspectives on the influence of chemical modifications of cryptophanes for optimizing the biosensor properties.