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Resp. Pr. Gérard Audran]

Research Field of CRAB Team

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Axis 1: Nitroxides as Contrast Agents in Overhauser-enhanced Magnetic Resonance Imaging (Pr. G. AUDRAN).

Lungs inflammatory diseases such as COPD are the cause of 2.5 M death worldwide. These diseases have in common a high influx of neutrophils that secrete proteases responsible for the progressive loss of lungs function. To date there is no method able to assess lung enzyme activity in vivo. Such a method would allow an early diagnostic of any protease/inhibitor imbalance long before the detection of pulmonary lesions by anatomical imaging methods. Lungs function could thus be preserved with protease inhibitors. We propose a new proteolytic activity imaging method for the lungs using Overhauser-enhanced MRI (OMRI). We will take advantage of a world unique prototype device and of recently synthesized contrast agents. Expected contrasts are 600 to 1200% in vivo according to preliminary results, well above current MRI contrasts reagents (20 to 30% for gadolinium chelates). Method validation will be conducted with mouse models of emphysema and cystic fibrosis by using nitroxides.

- Enzymatically Shifting Nitroxides for EPR spectroscopy and Overhauser-Enhanced Magnetic Resonance Imaging Angew. Chem. Int. Ed. 2015, in press.

Axis 2: Nitroxides as spin-labels of proteins (Pr. MARQUE).

Tertiary and quaternary structures of proteins are of the highest importance in biology as highlighted by the PRION disease. Most of the time, tertiary and quaternary structures are investigated in sold state and thus only a little is known on there dynamics. By luck, EPR is a versatile and efficient technique to investigate dynamics of proteins. However, it requires developing new spins labels, e.g. nitroxides, which can either be grafted on new amino-acid residues or exhibit different EPR patterns than the conventional nitroxides used. Our team already got successes in these two sub-axes, which are currently under investigation: Mannich reaction as procedure to label tyrosine.
A three-component Mannich-type reaction extends the scope of site-directed spin labeling by selectively labeling the unique tyrosine residue of CP12 protein (see picture), as was confirmed by mass spectrometry. EPR spectroscopy of the labeled protein showed a very high mobility of the probe, which remained very mobile after complex formation with GAPDH.

Angew. Chem. Int. Ed. 2011, 50, 39, 9108-9111.

Six-lines EPR feature nitroxides: Site Directed Spin Labeling (SDSL) combined with EPR spectroscopy is a very powerful approach to investigate structural transitions in proteins in particular flexible or even disordered ones. To overcome the limitation due to the weak diversity of nitroxide label EPR spectral shapes, we designed a new spin label based on a β-phosphorylated nitroxide giving six-line spectra. It was grafted at four different positions of a model disordered protein able to undergo an induced α-helical folding and its characterization by EPR spectroscopy. Taken together the results demonstrate that the new phosphorylated label gives a very distinguishable signature which is able to report from subtle to larger structural transitions, as efficiently as the classical spin label.

- PhysChemChemPhys 2014, 16, 4202-4209.

Axis 3: Materials Sciences (Pr. MARQUE).
Two aims of the team in this axis are:

- the investigation of the intimacy of the materials using new techniques of EPR. The task of the team is to develop nitroxides highly sensitive to changes ion their environments. A first probes exhibiting a change of 25 G in phosphorus hyperfine coupling constant from n-hexane to water has been recently prepared. Its potential has been highlight by the titration of 0.1% water in THF.

- the investigation of the \gamma-irradiation on the polymers used for the preparation of bags suitable for biological liquids. Collaboration and grant with SARTORIUS Stedim company.

Axis 4: Alkoxyamines as Theranostic Agents (Pr. G.AUDRAN).

Since years, the development of new anti-cancer drugs relies on the concept that combines a high selectivity associated with a high activity. In this project, we propose to develop a new approach which dissociates selectivity and activity. That is, a high selectivity of the prodrug must be ensured using the specific enzymatic activity of cancer cells releasing an activated drug exhibiting a random and non-selective high activity. In this case, it will generate biological disorders triggering the apoptosis/necrosis mechanisms or immune processes leading to cell death.
In our opinion, free radicals are the most suitable family of intermediates affording highly reactive species reacting randomly. Indeed, excess radicals in cancer cells lead to membrane, protein and lipid modifications that ultimately trigger cancer cells death. Thus, increasing the amount of radicals in cancer cells may therefore be applied as a therapeutic approach, as long as they are generated at the right place and at the right time. However, the success of this approach relies on very drastic requirements that include carefully controlled generation of radicals in tumour environment, controlled kinetics of their generation, and low cytotoxicity of the radical prodrugs.
In our opinion, alkoxyamines R1–ONR2R3 are the most suitable molecules for such an aim as they can undergo homolysis to release two free radicals:
i) a transient and highly reactive alkyl radical R1•, usable for therapeutic by generation of biological disorders and ultimately cellular death. For this purpose, a stable alkoxyamine will be biologically activated by enzymatic hydrolysis into a highly labile alkoxyamine which can undergo spontaneous homolysis into the two radicals.
ii) a stable and persistent nitroxide R2R3NO•, which can be used for the biological monitoring of the effect of the drug using either Electron Paramagnetic Resonance Imaging or Overhauser-enhanced MRI techniques (vide supra). Consequently, alkoxyamines exhibit also the potential as theranostic agents. This aspect is very important to monitor in situ the effect of the drug on living mice as models, and at later stages, to adapt at the best the cure to the patient.

Thus, alkoxyamines are small molecules, with low molecular weights, which does not present the drawbacks of nanoparticles (cytotoxicity, accumulation…), mainly used in theranostic, whereas they exhibit the equivalent physical/spectroscopic properties of nanoparticles and the biological activity of drugs.

- Org. Biomol. Chem. 2014, 12, 719-723.
- Mol. Pharmaceutics 2014, 11, 2412-2419.
- Chem. Commun. 2014, 50, 59, 7921-7928.

Axis 5: New initiators for polymerization (Prof. AUDRAN, Prof. MARQUE).

Alkoxyamines R1R2NO—R3 can undergo homolysis of the NO—C bond to release an alkyl radical and a nitroxide. They are well known in polymerization as radical initiator/controlling agent in Nitroxide-Mediated Polymerization (NMP). Particularly, our group has developed structure-reactivity relationships (SRR) to predict the homolysis rate (kd), activation energy (Ea) and half-life time (t1/2) of an alkoxyamine. Recently we also described the concept of smart alkoxyamines (see red dashed arrow), i.e. stable alkoxyamines (t1/2 (37 ºC) higher than several months) that can be chemically-activated and transformed into labile alkoxyamines (t1/2 smaller than few minutes) which spontaneously undergo homolysis.
We have recently described “semi-smart alkoxyamines” (see green arrow). Indeed, we recently investigated various types of chemical activation of an alkoxyamine carrying a pyridinyl ring, and observed a clear cut of the half-life time from t1/2 = 9 months in t-BuPh at 20 °C to t1/2 = 45 min in water at 37 °C (i.e. physiological conditions) by methylation. It showed clearly that smart alkoxyamines have potential applications as new initiators for polymerization, and that the homolysis rate can be readily controlled and tuned to comply with polymerization requirements.
Thus, using alkoxyamines as initiators requires the optimization of smart alkoxyamines: stable enough to be easily handled at room temperature (t1/2 > several months) and capable of being activated into highly labile species (t1/2 < few minutes) which release free radicals.

We also synthetize new radical initiators for a use in industrial processes (Collaboration with ARKEMA).

Axis 6: Molecular Modeling (Dr. V. Roubaud, Prof. M. Santelli).

Our team is focused on the activation of the C–ON bond homolysis by protonation, alkylation, benzylation, acylation, oxidation and complexation with a Lewis acid of the nitrogen atom of different pyridin fragments of (N-(2-methylpropyl)-N-(1-diethylphosphono-2,2-dimethyl-propyl)-N-oxyl) SG1-based alkoxyamines. The effect of the quaternization at the meta position was weak compared to ortho et para positions. The effects of quaternization at ortho, meta and para positions were investigated through natural bond orbital and Mulliken charges, HOMO–LUMO interactions in the starting materials and the radical stabilization energy of the released 1-pyridylmethyl radicals using DFT calculations with the B3LYP/6-31G(d) and UBMK/6-311+G(3df,2p)//R(O)B3LYP/6-31G(d) methods, respectively.

- J. Org. Chem. 2013, 78, 9914-9920.
- Org. Biomol. Chem. 2013, 11, 7738-7750.
We have studied the energetics of cascade reactions between dioxygen and polyunsaturated fatty acids like linolenic acid or arachidonic acid, via pentadienyl radical, in order to determine their thermodynamic parameters and reveal the key steps. We studied first the biosynthesis of cyclopentenones, precursors of jasmonic acid or prostanoids and, as models, then we calculated the structure of various intermediates of the biosynthesis of prostaglandins, prostacyclins and thromboxanes.

This work is now carried out on theoretical calculations concerning some others biological radical reactions.

- Tetrahedron 2014, 70, 8606–8613.
- Tetrahedron 2015, 71, 6920–6927.

Axis 7: Fundamental Chemistry (Prof. AUDRAN, Prof. MARQUE)

In this axis, our team investigates carefully the solvent effect on nitroxides, the thermal decomposition of nitroxides and alkoxyamines, the rate constants of homolysis, the EPR features of radicals in various media and different conditions.

- Chem. Rev. 2014, 114, 5011-5056.


Prof E. Thiaudière et Dr. P. Mellet (Bordeaux),
Prof. D. Braguer et Dr. M. Carré (Marseille),
Prof. B. Guigliarelli et Prof. V. Belle (Marseille),
Prof. E. Bagryanskaya et Dr. V. Tormyshev (Russie),
ARKEMA Company


2009-2013: ANR NITROMRI.
2009-2012: ANR JCJC D2R2.
2009-2013: ANR SPINFOLD.
2010-2011: CNRS/RAS people exchange program: grant ASR 23961.
2011-2014: ANR JCJC FRaPE.
2011-2015: ANR JCJC SonRadIs.
2013-2016: Industrial grant from SARTORIUS.
2010-2011: CNRS/RAS people exchange program: grant ASR 23961.
2013-2017: JCJC NewRap ANR.
2015-2016: AMIDEX TAG.
2015-2016: AMIDEX DYNACCO.
2015-2016: ARC GlioAlkox.
2015-2019: ANR NAR.
2015-2018: Russian Science Foundation: 15-13-2020.