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Transportable hyperpolarized metabolites

Nature Communications 8, Article number: 13975 (2017)


Nuclear spin hyperpolarization of 13C-labelled metabolites by dissolution dynamic nuclear polarization can enhance the NMR signals of metabolites by several orders of magnitude, which has enabled in vivo metabolic imaging by MRI. However, because of the short lifetime of the hyperpolarized magnetization (typically o1 min), the polarization process must be carried out close to the point of use. Here we introduce a concept that markedly extends hyperpolarization lifetimes and enables the transportation of hyperpolarized metabolites. The hyperpolarized sample can thus be removed from the polarizer and stored or transported for use at remote MRI or NMR sites. We show that hyperpolarization in alanine and glycine survives 16 h storage and transport, maintaining overall polarization enhancements of up to three orders of magnitude.

Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification

Scientific Reports 6, 36938 (2016)


The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.108 for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high μM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells.

Investigation of Intrinsically Disordered Proteins through Exchange with Hyperpolarized Water

Angew. Chem. Int. Ed2017, 56, 389 –392


Hyperpolarized water can selectively enhance NMR signals of rapidly exchanging protons in osteopontin (OPN), a metastasis-associated intrinsically disordered protein (IDP), at near-physiological pH and temperature. The transfer of magnetization from hyperpolarized water is limited to solventexposed residues and therefore selectively enhances signals in 1H-15N correlation spectra. Binding to the polysaccharide heparin was found to induce the unfolding of preformed structural elements in OPN.

Dissolution dynamic nuclear polarization of deuterated molecules enhanced by cross-polarization

J. Chem. Phys., 2016, 145, 194203


We present novel means to hyperpolarize deuterium nuclei in 13CD2  groups at cryogenic temperatures. The method is based on cross-polarization from 1 H to 13 C and does not require any radio-frequency fields applied to the deuterium nuclei. After rapid dissolution, a new class of longlived spin states can be detected indirectly by 13C NMR in solution. These long-lived states result from a sextet-triplet imbalance (STI) that involves the two equivalent deuterons with spin I  = 1. An STI has similar properties as a triplet-singlet imbalance that can occur in systems with two equivalent I  = 1/2  spins. Although the lifetimes TSTI  are shorter than T1(Cz), they can exceed the life-time T1(Dz) of deuterium Zeeman magnetization by a factor of more than 20.


A Bis-Manganese(II)–DOTA Complex for Pulsed Dipolar Spectroscopy

ChemPhysChem 2016, 17, 2066 – 2078


High-spin gadolinium(III) and manganese(II) complexes have emerged as alternatives to standard nitroxide radical spin labels for measuring nanometric distances by using pulsed electron–electron double resonance (PELDOR or DEER) at high fields/frequencies. For certain complexes, particularly those with relatively small zero-field splitting (ZFS) and short distances between the two metal centers, the pseudosecular term of the dipolar coupling Hamiltonian is non-negligible. However, in general, the contribution from this term during conventional data analysis is masked by the flexibility of the molecule of interest and/or the long tethers connecting them to the spin labels. The efficient synthesis of a model system consisting of two [Mn(dota)]2- (MnDOTA; DOTA4-=1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate) directly connected to the ends of a central rodlike oligo(phenylene–ethynylene) (OPE) spacer is reported. The rigidity of the OPE is confirmed by Q-band PELDOR measurements on a bis-nitroxide analogue. The MnII-MnII distance distribution profile determined by W-band PELDOR is in reasonable agreement with one simulated by using a simple rotamer analysis. The small degree of flexibility arising from the linking MnDOTA arm appears to outweigh the contribution from the pseudosecular term at this interspin distance. This study illustrates the potential of MnDOTA-based spin labels for measuring fairly short nanometer distances, and also presents an interesting candidate for in-depth studies of pulsed dipolar spectroscopy methods on MnII-MnII systems.