Nanoscale solvent organization in metal-organic framework ZIF-8 probed by EPR of flexible ß-phosphorylated nitroxides.

Metal-organic frameworks (MOFs) draw increasing attention as nanoenvironments for chemical reactions, especially in the field of catalysis. Knowing the specifics of MOF cavities is decisive in many of these cases; yet, obtaining them in situ remains very challenging. We report the first direct assessment of the apparent polarity and solvent organization inside MOF cavities using a dedicated structurally flexible spin probe. A stable ß-phosphorylated nitroxide radical was incorporated into the cavities of a prospective MOF ZIF-8 in trace amounts. The spectroscopic properties of this probe depend on local polarity, structuredness, stiffness and cohesive pressure and can be precisely monitored by Electron Paramagnetic Resonance (EPR) spectroscopy. Using this approach, we have demonstrated experimentally that the cavities of bare ZIF-8 are sensed by guest molecules as highly non-polar inside. When various alcohols fill the cavities, remarkable self-organization of solvent molecules is observed leading to a higher apparent polarity in MOFs compared to the corresponding bulk alcohols. Accounting for such nanoorganization phenomena can be crucial for optimization of chemical reactions in MOFs, and the proposed methodology provides unique routes to study MOF cavities inside in situ, thus aiding in their various applications.

Enzymatic activity monitoring through dynamic nuclear polarization in Earth magnetic field.

Cost-effective and portable MRI systems operating at Earth-field would be helpful in poorly accessible areas or in developing nations. Furthermore Earth-field MRI can provide new contrasts opening the way to the observation of pathologies at the biochemical level. However low-field MRI suffers from a dramatic lack in detection sensitivity even worsened for molecular imaging purposes where biochemical specificity requires detection of dilute compounds. In a preliminary spectroscopic approach, it is proposed here to detect protease-driven hydrolysis of a nitroxide probe thanks to electron-nucleus Overhauser enhancement in a home-made double resonance system in Earth-field. The combination of the Overhauser effect and the specific enzymatic modification of the probe provides a smart contrast reporting the enzymatic activity. The nitroxide probe is a six-line nitroxide which lines are shifted according to its substrate/product state, which requires quantum mechanical calculations to predict EPR line frequencies and Overhauser enhancements at Earth field. The NMR system is equipped with a 13-mT prepolarization coil, a 153-MHz EPR coil and a 2-kHz NMR coil. Either prepolarized NMR or DNP-NMR without prepolarization provide NMR spectra within 3 min. The frequency dependence of Overhauser enhancement was in agreement with theoretical calculations. Protease-mediated catalysis of the nitroxide probe could only be measured through the Overhauser effect with 5 min time resolution. Future developments shall open the way for the design of new low-field DNP-MRI systems.

Magnetic Resonance Imaging of Protease-Mediated Lung Tissue Inflammation and Injury.

Pulmonary inflammation usually involves strong neutrophil recruitment with a marked release of proteases such as neutrophil elastase (NE). Noninvasive in vivo assessment of unregulated elastase activity in the lungs would provide a valuable diagnostic tool. Here, it is proposed to use Overhauser-enhanced magnetic resonance imaging (OMRI) in mice where inflammation was induced by the instillation of lipopolysaccharide (LPS). OMRI contrast in the lungs was generated by a dedicated NE free radical substrate. The free radical decayed more rapidly in LPS-treated mouse lungs than in control mice, indicating the occurrence of increased proteolysis under inflammation. Preclinical detection of abnormal proteolysis opens the way for new diagnosis modality and antiprotease testing in vivo.

Shifting-Nitroxides to Investigate Enzymatic Hydrolysis of Fatty Acids by Lipases Using Electron Paramagnetic Resonance in Turbid Media.

While optical methods are not efficient enough for the easy, fast, and efficient detection of enzymatic activity in turbid media, the properties of the electron paramagnetic resonance (EPR) technique make it suitable for use in such media. Nitroxides which exhibit a change in their EPR hyperfine coupling constants upon enzymatic activity and are selective to lipases were developed under the name of shifting-nitroxides. Several fatty acids, exhibiting saturated and unsaturated chains of various lengths, were coupled with the shifting-nitroxide via an enol ester link and tested against several lipases. As the solubility of fatty acids is low in HEPES buffer, experiments were performed in turbid aqueous solution. Almost all labeled fatty acids were hydrolyzed by Candida rugosa lipase, and more selectivity is observed with Porcine Pancreas lipase type II. No activity was observed for lipase AK Amano 20, Candida antartica lipase B, and Mucor miehei lipase.