Fullerenes, peptides and radicals

this Thesis concerns the synthesis, characterization and investigation of two series of compounds which are characterized by the presence of a fullerene moiety and / or of a nitroxide function covalently linked at the extremity of a rigid peptide. The structured peptide sustains a strong oriented dipole moment located along the main axes of the molecules and its direction is opposite in the two series, named 2+ and 2-. The dipole orientation was expected to cause opposite effects, for example, on the redox properties of the moieties located at its extremity. The syntheses required a considerable amount of experimental work and the method chosen to functionalize the fullerene was the Maggini-Prato reaction. We could synthesize the following compounds: the fullerene-peptides 2+ and 2-, the fullerene-peptides-reference 2+ and 2- and the peptides-reference 2+ and 2-. Besides other two fullerene-peptides were synthesized using the precious endofullerene that bears a hydrogen molecule entrapped in its cage. Characterization in solution (Mass Spectrometry, FT-IR absorption spectroscopy, 1H NMR spectroscopy) proves the identity of compounds under investigation. It also provides evidence of the presence of structured peptides in both series 2- and 2+. 1H NMR spectra of systems bearing the nitroxide function were acquired after quenching of the free radical with diphenylhydrazine. Cyclic voltammetries clearly show that the first reduction peak of fullerene is anticipated in the 2+ series with respect to the 2- series. This experimental outcome is in full agreement with the effect caused by the peptide macrodipole. An even more accentuated deviation is found on the oxidation side (TOAC oxidation) where the potential shifts are once again on the direction expected for an effect of the helix dipole moment, namely, easier reduction and easier oxidation for the 2+ series while more difficult reduction and more difficult oxidation for the 2- series. It is thus possible to state that in these compounds the helix dipole moment plays an important role on the tuning of the potentials of the active redox groups (the nitroxide function and the fullerene). Thank to the unique rigidity of the peptide spacer, an accurate estimation of the distance is obtainable and this parameter is crucial for several investigations where the interactions between the fullerene (or the hydrogen molecule encapsulated inside its cage) and the nitroxide are distance-dependent. Some of the compounds were thus studied by TR EPR (Time-Resolved Electron Paramagnetic Resonance) to investigate the coupling between the doublet ground state of the nitroxide and the triplet state of the fullerene generated after photo-excitation and by 1H NMR spectroscopy to obtain the nuclear spin relaxations and the ortho / para conversion rates of the encapsulated hydrogen

Gd(III) complexes for electron-electron dipolar spectroscopy : effects of deuteration, pH and zero field splitting

Spectral parameters of Gd(III) complexes are intimately linked to the performance of the Gd(III)-nitroxide or Gd(III)-Gd(III) double electron-electron resonance (DEER or PELDOR) techniques, as well as to that of relaxation induced dipolar modulation enhancement (RIDME) spectroscopy with Gd(III) ions. These techniques are of interest for applications in structural biology, since they can selectively detect site-to-site distances in biomolecules or biomolecular complexes in the nanometer range. Here we report relaxation properties, echo detected EPR spectra, as well as the magnitude of the echo reduction effect in Gd(III)-nitroxide DEER for a series of Gadolinium(III) complexes with chelating agents derived from tetraazacyclododecane. We observed that solvent deuteration does not only lengthen the relaxation times of Gd(III) centers but also weakens the DEER echo reduction effect. Both of these phenomena lead to an improved signal-to-noise ratios or, alternatively, longer accessible distance range in pulse EPR measurements. The presented data enrich the knowledge on paramagnetic Gd(III) chelate complexes in frozen solutions, and can help optimize the experimental conditions for most types of the pulse measurements of the electron-electron dipolar interactions

Shape Persistence of Polyproline II Helical Oligoprolines

Oligoprolines are commonly used as molecular scaffolds. Past studies on the persistence length of their secondary structure, the polyproline II (PPII) helix, and on the fraction of backbone cis amide bonds have provided conflicting results. We resolved this debate by studying a series of spin-labeled proline octadecamers with EPR spectroscopy. Distance distributions between an N-terminal GdIII-DOTA (DOTA=1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) label and a nitroxide label at one of five evenly spaced backbone sites allowed us to discriminate between the flexibility of the PPII helix and the cis amide contributions. An upper limit of 2% cis amide bonds per residue was found in a 7:3 (v/v) water/glycerol mixture, whereas cis amides were not observed in trifluoroethanol. Extrapolation of Monte Carlo models from the glass transition to ambient temperature predicts a persistence length of ≈3-3.5 nm in both solvents. The method is generally applicable to any type of oligomer for which the persistence length is of interest

Orthogonal Spin Labeling and Gd(III)–Nitroxide Distance Measurements on Bacteriophage T4-Lysozyme

We present the first example of chemo-selective site-specific spin labeling of a monomeric protein with two spectroscopically orthogonal spin labels: a Gadolinium (III) chelate complex and a nitroxide radical. A detailed analysis of the performance of two commercially available Gd(III) ligands in the Gd(III)-nitroxide pulse double electron-electron resonance (DEER or PELDOR) experiment is reported. A modification of the flip angle of the pump pulse in the Gd(III)-nitroxide DEER experiment is proposed to optimize sensitivity

Model-free extraction of spin label position distributions from pseudocontact shift data

A significant problem with paramagnetic tags attached to proteins and nucleic acids is their conformational mobility. Each tag is statistically distributed within a volume between 5 and 10 Angstroms across; structural biology conclusions from NMR and EPR work are necessarily diluted by this uncertainty. The problem is solved in electron spin resonance, but remains open in the other major branch of paramagnetic resonance – pseudocontact shift (PCS) NMR spectroscopy, where structural biologists have so far been reluctantly using the point paramagnetic centre approximation. Here we describe a new method for extracting probability densities of lanthanide tags from PCS data. The method relies on Tikhonov-regularised 3D reconstruction and opens a new window into biomolecular structure and dynamics because it explores a very different range of conditions from those accessible to double electron resonance work on paramagnetic tags: a room-temperature solution rather than a glass at cryogenic temperatures. The method is illustrated using four different Tm3+ DOTA-M8 tagged mutants of human carbonic anhydrase II; the results are in good agreement with rotamer library and DEER data. The wealth of high-quality pseudocontact shift data accumulated by the biological magnetic resonance community over the last 30 years, and so far only processed using point models, could now become a major source of useful information on conformational distributions of paramagnetic tags in biomolecules

Corrigendum to: Shape Persistence of Polyproline II Helical Oligoprolines (Chem. Eur. J., (2015), 21, 10.1002/chem.201501190)

It has been brought to author’s attention that lowest line of subplots in Figure 3 was erroneously swapped with the upper line of subplots in Figure 4. Figure captions in the article are correct for the figures given below. (Figure Presented.)

Effect of Orientation of the Peptide-Bridge Dipole Moment on the Properties of Fullerene\u2013Peptide\u2013Radical Systems

We synthesized two series of compounds in which a nitroxide radical and a fullerene C-60 moiety were kept separated by a 3(10)-helical peptide bridge containing two intramolecular C=O center dot center dot center dot H-N hydrogen bonds. The direction of the resulting molecular dipole moment could be reversed by switching the position of fullerene and nitroxide with respect to the peptide nitrogen and carbon termini. The resulting fullerene-peptide-radical systems were compared to the behaviors of otherwise identical peptides but lacking either C-60 or the free radical moiety. Electrochemical analysis and chemical nitroxide reduction experiments show that the dipole moment of the helix significantly affects the redox properties of both electroactive groups. Besides providing evidence of a folded helical conformation for the peptide bridge, IR and NMR results highlight a strong effect of peptide orientation on the spectral patterns, pointing to a specific interaction of one of the helical orientations with the C-60 moiety. Time-resolved EPR spectra show not only that for both systems triplet quenching by nitroxide induces spin polarization of the radical spin sublevels, but also that the coupling interaction can be either weak or strong depending on the orientation of the peptide dipole. As opposed to the concept of dyads, the molecules investigated are thus better described as fullerene-peptide-radical systems to stress the active role of the bridge as an important ingredient capable of tuning the system's physicochemical properties